Přírodovědecká fakulta, Masarykova univerzita
MODERNÍ POSTUPY A METODY V NÁDOROVÉ DIAGNOSTICE Dizertační práce
Vojtěch Adam
Brno 2009
Přírodovědecká fakulta, Masarykova univerzita
MODERNÍ POSTUPY A METODY V NÁDOROVÉ DIAGNOSTICE Dizertační práce
Vojtěch Adam
Brno 2009
Školitel:
1
Doc. Ing. René Kizek, Ph.D.1
Ústav chemie a biochemie, Agronomická fakulta, Mendelova zemědělská a lesnická
univerzita, Zemědělská 1, 613 00 Brno
2
Myslet lze jen rozumem, ale chápat jen srdcem. Joseph Addison
3
PODĚKOVÁNÍ Všechny mé „univerzitní“ a mnoho mých nejdůležitějších životních kroků, které stojí nejen za tvorbou této práce, jsou velkou zásluhou člověka, kterého mohu s velkou úctou nazývat svým kamarádem. Reni, děkuji. Mé poděkování patří dále Libušce Trnkové, která mi umožnila nahlédnout pod pokličku biofyzikální chemie a nejen té. Děkuji Tomáši Eckschlagerovi za trpělivost, kterou se mnou měl a za pomoc, kterou se mi u něj vždy dostalo. Můj velký dík patří paní Prof. RNDr. Miroslavě Beklové, CSc. za šanci, kterou mi dala, a velkou podporu, kterou mi poskytla. Na tomto místě si můj speciální dík zaslouží pan Prof. Ing. Ladislav Zeman, CSc. za vše, co pro mě v životě udělal. Z mých kolegů, které jsem měl tu čest poznat, bych moc rád poděkoval Jitce Petrlové, Soni Křížkové, Davidu Potěšilovi, Ondrovi Blaštíkovi, Michalu Svobodovi, Ivu Fabrikovi, Vaškovi Diopanovi, Dalimu Húskovi a Ondrovi Zítkovi. Všichni jmenovaní a další bývalí i nynější členové skupiny Molekulární biochemie a bioelektrochemie vytvářeli a vytvářejí prostředí, do kterého se člověk těší a rád v něm pracuje. Můj srdečný dík si za všechnu pomoc, podporu a oporu zaslouží Olinka Kryštofová. Haničko Zimová, má lásko, bez Tvého úsměvu, pohlazení a sluníčka ve tváři bych stěží bojoval se životem, jak s ním bojuji. Patří Ti z celého srdce mé „děkuji“. Miluji Tě. Všem mým přátelům děkuji za velikou trpělivost, kterou se mnou stále mají. Na posledních řádcích mi dovolte moc poděkovat své mamince a tatínkovi, protože tu pro mě vždy byli a jsou. Jsou to Ti nejlepší rodiče, jaké si může člověk přát. 4
OBSAH I.
ÚVOD.......................................................................................... 10
II. TEORETICKÁ ČÁST ................................................................. 11 Zhoubné nádorové onemocnění ............................................................................... 11 1.1 Vznik nádorového onemocnění ....................................................................... 11 1.2 Nádorová onemocnění v České republice ....................................................... 12 2. Diagnostika nádorových onemocnění ...................................................................... 12 2.1 Zobrazovací rentgenové metody ...................................................................... 12 2.2 Zobrazovací nerentgenové metody .................................................................. 13 2.3 Endoskopie....................................................................................................... 13 2.4 Hematologické a biochemické vyšetření ......................................................... 13 2.5 Nádorové markery ........................................................................................... 14 2.5.1 Nádorové antigeny ....................................................................................... 14 Karcinoembryonální antigen................................................................................ 15 Alfa-fetoprotein ................................................................................................... 15 Beta a gama-fetoprotein ....................................................................................... 15 CA 15-3................................................................................................................ 16 CA 125 ................................................................................................................. 16 CA19-9................................................................................................................. 16 CA 242 ................................................................................................................. 17 Specifický membránový antigen prostaty............................................................ 17 Antigen kmenových buněk prostaty .................................................................... 17 Antigen karcinomu močového měchýře .............................................................. 17 Produkty degradace fibrin/fibrinogenu ................................................................ 18 Beta-2-mikroglobulin........................................................................................... 18 2.5.2 Rodina lidských kalikreinů .......................................................................... 18 Kalikrein 1 ........................................................................................................... 18 Kalikrein 2 ........................................................................................................... 19 Kalikrein 3 neboli specifický antigen prostaty .................................................... 19 2.5.3 Hormony ...................................................................................................... 19 Hormony jako přirozené produkty přidružených orgánů nebo produkty abnormální syntézy v důsledku neregulovaného metabolismu nádorové buňky. 20 Hormony jako nepřirozené produkty přidružených orgánů ................................. 21 2.5.4 Enzymy ........................................................................................................ 22 Kyselá prostatická fosfatáza ................................................................................ 22 Neuron-specifická enoláza ................................................................................... 22 Galaktosyl transferáza II ...................................................................................... 22 Napsin A .............................................................................................................. 23 Katepsin ............................................................................................................... 23 Matrixové metaloproteinázy ................................................................................ 24 Aktivátor plazminogen-urokinázového typu ....................................................... 27 Tumor M2-pyruvát kináza ................................................................................... 27 Laktátdehydrogenáza ........................................................................................... 27 Prostasin ............................................................................................................... 28 nm23-H1 .............................................................................................................. 28 YKL-40 ................................................................................................................ 28 1.
5
2.5.5 Cytokeratiny ................................................................................................. 29 2.5.6 Cytokiny a cytokinové receptory ................................................................. 29 HER-2 .................................................................................................................. 29 Interleukin 6 ......................................................................................................... 29 Receptor interleukinu 2 ........................................................................................ 30 Transformující růstový faktor beta ...................................................................... 30 Receptor epidermálního růstového faktoru .......................................................... 31 CD30 .................................................................................................................... 31 2.5.7 Signální molekuly ........................................................................................ 31 Ras ........................................................................................................................ 31 trk ......................................................................................................................... 32 myc ....................................................................................................................... 32 Jaderný heterogenní ribonukleoprotein ................................................................ 32 2.5.8 Nádorové markery růstu nádorů: buněčný cyklus a proliferace .................. 33 Telomerázy........................................................................................................... 33 Jaderné matrixové proteiny .................................................................................. 33 Proteiny teplotního šoku ...................................................................................... 33 Mikrosatelity ........................................................................................................ 34 DNA ploidie ......................................................................................................... 34 Stav buněk v S fázi............................................................................................... 34 Protein p16 ........................................................................................................... 35 Protein p53 ........................................................................................................... 35 2.5.9 Nádorové markery a angiogeneze ................................................................ 35 Vaskulární endoteliální růstový faktor ................................................................. 35 Thrombospondin .................................................................................................. 36 Chemokiny ........................................................................................................... 36 Oxidu dusnatý ...................................................................................................... 37 2.5.10 Nádorové markery invazivity a metastazování ........................................ 38 Adhezní molekuly a nádorová invazivita............................................................. 38 Markery metastazování ........................................................................................ 39 2.5.11 Souhrn ...................................................................................................... 40 Metalothioneiny ................................................................................................... 42 3. Elektrochemické metody.......................................................................................... 45 3.1 Brdičkova reakce.............................................................................................. 46 3.2 H-pík ................................................................................................................ 48
III. CÍLE PRÁCE .............................................................................. 49 IV. EXPERIMENTÁLNÍ ČÁST ....................................................... 50 A. Literární rešerše o vztahu metalothioneinu a nádorových onemocnění ...................... 52 B. Literární rešerše o možnostech detekce metalothioneinu............................................ 69 C. Vliv kovů přirozeně obsažených ve struktuře metalothioneinu na jeho stanovení ..... 98 D. Elektrochemické stanovení metalothioneinu v krevních vzorcích pacientů s nádorem prsu ................................................................................................................................. 108 E. Vliv cisplatiny na hladinu metalothioneinu u buněčných linií, laboratorních krys a pacientů s nádorem v oblasti hlavu a krku ..................................................................... 122 F. Studium interakce želatinázy B s kolagenem ............................................................ 135
V. KOMENTÁŘ K PUBLIKACÍM .............................................. 142
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Literární rešerše o vztahu metalothioneinu a nádorových onemocnění ................ 142 Literární rešerše o možnostech detekce metalothioneinu ...................................... 143 Vliv kovů přirozeně obsažených ve struktuře metalothioneinu na jeho stanovení 144 6.1 Elektrochemické chování metalothioneinu .................................................... 144 6.2 Vliv EDTA na strukturu metalothioneinu ..................................................... 145 7. Elektrochemické stanovení metalothioneinu v krevních vzorcích pacientů s nádorem prsu ............................................................................................................... 147 8. Vliv cisplatiny na hladinu metalothioneinu u buněčných linií, laboratorních krys a pacientů s nádorem v oblasti hlavu a krku ..................................................................... 149 8.1 Změny hladiny metalothioneinu u pacientů se zhoubnými nádory v oblasti hlavy a krku léčenými cisplatinou ............................................................................. 150 8.2 Změny hladiny metalothioneinu u pacientů s retinoblastomem léčenými karboplatinou ............................................................................................................. 151 9. Studium interakce želatinázy B s kolagenem ........................................................ 152
4. 5. 6.
VI. ZÁVĚR ...................................................................................... 154 VII. SHRNUTÍ .................................................................................. 155 VIII. SUMMARY ............................................................................ 157 IX. SEZNAM POUŽITÉ LITERATURY ....................................... 159
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SEZNAM ZKRATEK ACS
Čistota chemikálií („chemicals meet the specifications of the American Chemical Society”)
ACTH
adrenokortikotropní hormon („AdrenoCorticoTropic |Hormone“)
ADH
antidiuretický hormon („AntiDiuretic Hormone“)
AdTS
adsoptivní přenosová rozpouštěcí technika („Adsorptive Transfer Stripping Technique“)
AFP
alfa-fetoprotein („Alpha-FetoProtein“)
β2M
beta-2-mikroglobulin („Beta 2-Microglobulin“)
BTA
antigen karcinomu močového měchýře („Bladder Tumor Antigen“)
Cdk
cyklin-dependentní kinázy („Cyclin-dependent kinases“)
CEA
karcinoembryonální antigen („CarcinoEmbryonic Antigen“)
CK
cytokeratiny („Cytokeratins“)
CT
kalcitonin („Calcitonin“)
DNA
deoxyribonukleová kyselina („DeoxyriboNucleic Acid“)
DPV
diferenční pulsní volumetrie („Differential Pulse Voltammetry“)
EDTA
Ethylendiamin-N,N,N`,N`-tetraoctová
kyselina
(„EthyleneDiamine
Tetraacetic Acid“) EGFR
receptor epidermálního růstového faktoru („Epidermal Growth Factor Receptor“)
HCG
lidský choriový gonadotropin („Hormone Chorionic Gonadotropin“)
HGF
růstový faktor hepatocytů („Hepatocyte Growth Factor“)
HMDE
visící rtuťová kapková elektroda („Hanging Mercury Drop Electrode“)
hnRNP
jaderný
heterogenní
ribonukleoprotein
(„Heterogeneous
Nuclear
Ribonucleoprotein“) HSP
proteiny teplotního šoku („Heat Shock Proteins“)
ICAM-1
intercelulární adhezní molekula-1 („Intercellular Adhesion Molecule-1“)
IL2R
receptor interleukinu 2 („Interleukin2 Receptor“)
IL6
interleukin 6 („Interleukin6“)
KLK
kalikreiny („Kallikreins“)
LDH
laktátdehydrogenáza („Lactic DeHydrogenase“)
LPA
lysofosfatidová kyselina („LysoPhosphatidic Acid“)
NGF
nervový růstový faktor („Nerve Growth Factor“) 8
MMP
matrixové metaloproteinázy („Matrix Metalloproteinases“)
NMP
jaderné matrixové proteiny („Nuclear Matrix Proteins“)
NNE
ne-neuronová enoláza („Non-Neuronal Enolase“)
NO
oxid dusnatý („Nitric Oxide“)
NOR
Národní onkologický registr („National Oncologic Registry“)
NOS
NO syntézy („Nitric Oxide Synthase“)
NSE
neuron-specifická enoláza („Neuron Specific Enolase“)
MT
metalothioneiny („Metallothioneins“)
M2-PK
tumor M2-pyruvát kináza („Tumor M2-Pyruvate Kinase“)
PAP
kyselá prostatická fosfatáza („Prostatic Acid Phosphatase“)
PCR
polymerázová řetězová reakce („Polymerase Chain Reaction“)
ProGRP
gastrin-vylučující peptid („Pro-Gastrin-Releasing Peptide“)
PSA
specifický antigen prostaty („Prostate Specific Antigen“)
PSCA
antigen kmenových buněk prostaty („Prostate Stem Cell Antigen“)
PSMA
specifický membránový antigen prostaty („Prostate-Specific Membrane Antigen“)
RNA
ribonukleová kyselina („RiboNucleic Acid“)
TGF-β
transformující růstový faktor beta („Transforming Growth Factor beta“)
TSP
thrombospondin („ThromboSPondin“)
uPA
aktivátor plazminogen-urokinázového typu („urokinase-type Plasminogen Activator“)
VCAM-1
buněčná adhezní molekula-1 („Vascular Cell Adhesion Molecule-1“)
VEGF
vaskulární endoteliální růstový faktor („Vascular Endothelial Growth Factor“)
9
I.
ÚVOD Dnešní doba nám nabízí mnoho různých formulářů, šablon a tabulek plných čísel
a dalších ukazatelů, podle kterých hodnotíme vše, co člověk činí. Známe tedy postupy umožňující hodnocení vesmírného projektu stejně, jako existuje jednotná šablona pro popis odrůdy červených růží, které nám kvetou před domem. V té velké změti čísel se ovšem někdy ztrácí to „něco“, co nám umožnilo před mnoha tisíci lety ovládnout oheň, postavit příbytek, zkrotit první zvíře. Dovolte mi to „něco“ nazvat faktorem lidskosti. Tento faktor, tedy další pokus o kategorizaci, je naštěstí krátký na všechny možné tabulky a počty, a především nás žene dál. Ať už slovo dál znamená tmavý pokoj v domě u babičky, kam se jako děti bojíme vstoupit, nebo je to náruč vesmíru, který lidstvo odjakživa fascinuje. Postup vpřed však neznamená pouze hledání nových technologií, odkrývání bílých míst na mapě nebo nalezení vypínače pokrytého pavučinou ve zmíněném pokoji u babičky. Tento faktor lidskosti obsahuje také touhu pozvednout společnost o stupínek, i když jen malý, výše. V této Sysifovské snaze se ukrývá něco, co nás oddělilo v dobách pravěkých od vývojových linií, které zanikly v toku času, a to starost o nemocné a staré. Již pravěcí obyvatelé naší planety si uvědomili kouzlo faktu, že když se jim přihodí cokoliv špatného, vždy se o ně jejich kmen v čele s šamanem postará. Dnes jsme vyměnili šamany za lékaře a vědce, kteří se stále pokoušejí míchat lepší „lektvary“, hledat lepší „berličky“, testovat složitější „zaklínadla“, ale hlavně se stále starat o staré a nemocné tak, jak tomu je již po tisíce let. Všichni lidé, kteří oplývají vysokou mírou faktoru lidskosti, jsou hnacím motorem společnosti. Proto bych rád na tomto místě a pomocí písmen, která se skládají ve slova, pomyslně smekl jeden virtuální klobouk před všemi, kteří posunuli, posouvají a budou posouvat lidskou společnost o stupínek výše.
10
II.
TEORETICKÁ ČÁST
1.
Zhoubné nádorové onemocnění Zhoubné
nádorové
onemocnění
je
chorobný
stav
charakterizovaný
nekoordinovaným růstem abnormálních buněk s jejich postupným šířením do okolních tkání, průnikem do mízního a krevního systému a postižením vzdálených orgánů (metastazování). Růst masy nádorových buněk probíhá autonomně bez projevu regulačních zásahů organismu, na úkor energetických a nutričních potřeb jeho zdravých buněk. Nádorové onemocnění nelze chápat jako jedinou jednotku. Jedná se o souhrnné označení celé skupiny onemocnění s rozdílným klinickým průběhem [1]. 1.1
Vznik nádorového onemocnění Vznik nádorového bujení je vícestupňový multietiologický proces, který je
výsledkem působení jak vnitřních, tak zevních faktorů (proces kancerogeneze). Počátek nádorové přeměny se odehrává na molekulární úrovni. Přeměna normální buňky na nádorovou vzniká v důsledku změn genetické informace (mutaci) na úrovni genomové deoxyribonukleové kyseliny (DNA). Mutace vznikají nejčastěji následkem působení zevních faktorů na DNA v buněčném jádře. Mezi tyto zevní faktory patří především ionizující záření, kancerogeny (látky podporujících vznik nádorového procesu) a některé viry. Kromě působení zevních faktorů mohou být mutace přenášeny z generace na generaci. Další možností vzniku mutací je jejich spontánní tvorba například při replikaci DNA v průběhu buněčného cyklu. V průběhu života organismu dochází k neustálému obnovování většiny buněčných populací, při kterých dceřiné buňky vznikají z buněk mateřských buněčným dělením a nadbytečné, poškozené nebo přestárlé buňky jsou sekvestrovány iniciací programované buněčné smrti. Tento proces provází malá, ale vzhledem k celkovému počtu buněk organismu početně významná pravděpodobnost vzniku mutací v DNA (10-5 až 10-10 na jednu buňku za generaci). Vyvolané genetické změny se nemusí funkčně projevit. Častěji však, zvláště při jejich kumulaci, dochází k zániku buňky v důsledku poškození důležitých regulačních nebo strukturních genů. Pouze ve velmi nízkém procentu vznikne v buňce kombinace mutací v kritických genech, která způsobí selhání kontrolních mechanizmů buněčné proliferace nebo buněčné smrti (apoptózy). Následné dělení takto změněné progenitorové buňky, postrádající adekvátní reakce na zevní i vnitřní regulační mechanizmy, způsobí vývoj klonu buněk s maligním
11
potenciálem. U většiny nádoru se riziko jejich vzniku s prodlužujícím věkem zvyšuje. Je to dáno delším obdobím působení faktoru zevního prostředí a postupně se snižující schopností opravovat mutace v DNA [1].
1.2
Nádorová onemocnění v České republice Nádorová onemocnění „soupeří“ už několik let s kardiovaskulárními chorobami
o nejčastější příčinu úmrtí v rozvinutých zemích. Díky řadě různých faktorů, ať již zdravějšímu zdravotnímu stylu, prevenci, anebo zdokonalování léčebných metod, se počet úmrtí z důvodu selhání srdce snižuje a tím pádem se dostávají do popředí nádorová onemocnění, u kterých je v posledních letech trend ze světového hlediska opačný. Situace v České republice je podle Národního onkologického registru (NOR) následující. V roce 2006 bylo do NORu nově nahlášeno celkem 71 913 případů zhoubných novotvarů a novotvarů in situ, z toho 36 682 případů u mužů a 35 231 případů u žen. Absolutní počet nově zjištěných nádorů u mužů poprvé od roku 1990 meziročně velice nepatrně klesl, a to o méně než 1 %. U žen absolutní počet nově zjištěných nádorů pokračuje v dlouhodobém stoupajícím trendu. Také věkově standardizovaná incidence u mužů klesla. V roce 2006 připadalo 674,4 případů zhoubných novotvarů na 100 tis. mužů, zatímco v roce 2005 byla hodnota standardizované incidence 689,4 případů na 100 tis. mužů. U žen hodnota standardizované míry incidence meziročně mírně vzrostla (499,1 případů na 100 tis. žen v roce 2005 a 501,4 případů na 100 tis. žen v roce 2006) [2].
2.
Diagnostika nádorových onemocnění Jednou z možností, jak snížit počet úmrtí pacientů trpících nádorovým
onemocněním, je onemocnění včasně diagnostikovat. Pro diagnostické účely je možné využít řadu postupů a technik založených na různých principech. 2.1
Zobrazovací rentgenové metody Rentgenové záření je forma elektromagnetického záření o vlnových délkách
10 nanometrů až 100 pikometrů. Je možné jej využít pro snímkování hrudníku, skeletu, jednotlivých orgánů a také měkkých tkání, kde nachází uplatnění dnes nejpoužívanější zobrazovací rentgenová metoda zvaná výpočtová tomografie (computed tomography) [1].
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2.2
Zobrazovací nerentgenové metody Tyto metody využívají různých fyzikálně-chemických principů pro zobrazení
měkkých tkání. Jedním z běžných zástupců technik této oblasti je sonografie, která využívá ultrazvuku odraženého od tkání. Robustnější zobrazovací technikou je magnetická rezonance, která je používána pro zobrazení vnitřních orgánů pomocí silného magnetického pole a elektromagnetického vlnění s vysokou frekvencí. Nenese tedy žádná rizika způsobená zářením jako např. rentgenové zobrazovací metody [3]. Pozitronová emisní tomografie je moderní zobrazovací technikou používanou v lékařské praxi od počátku devadesátých let minulého století. Pacientovi je před vyšetřením podáno radiofarmakum s velmi krátkým poločasem rozpadu (uhlík-11, dusík13, kyslík-15, fluor-18), které je schopno při svém rozpadu produkovat pozitrony. Pozitron se krátce po svém vzniku anihiluje s elektronem. Tato reakce produkuje dva fotony, které jsou snímány pomocí koincidenčního detektoru. Z velkého množství (až několik set tisíc) takových záchytů pak lze výpočetním algoritmem rekonstruovat tomografický obraz vyšetřovaného [4]. 2.3
Endoskopie Je to metoda umožňující prohlédnutí vnitřních tělesných dutin nebo dutých orgánů.
V dnešní době se pro tento účel využívá tří typů endoskopů a to endoskopická zrcátka, rigidní endoskopy a flexibilní endoskopy, které již využívají optická vlákna a studené světlo [1]. 2.4
Hematologické a biochemické vyšetření Hematologie zahrnuje vyšetření krevního obrazu, včetně diferenciálního rozpočtu,
sedimentace, stavu krevních destiček, krvácivosti, srážlivosti, diatéz a dalších. Mikroskopií speciálně barvených hematologických preparátů (krevní nátěr na sklíčku) lze sledovat morfologii jednotlivých typů leukocytů a jejich počty. Oproti tomu biochemické vyšetření zahrnuje stanovení koncentrace prvků vyskytujících se v krvi v relativně vysokých koncentracích jako např. Na, K, Cl, Ca, P, Fe, popřípadě aktivity enzymů, jako jsou alaninaminotransferáza, aspartátaminotransferáza, alkalická fosfatáza a řada dalších [1].
13
2.5
Nádorové markery Biochemické markery v onkologii jsou molekuly, jejichž hladina je v krvi, moči
nebo tělesných tkání zvýšena či snížena u pacientů s nádorovým onemocněním oproti obvyklému množství u zdravé populace. Nádorový marker může být produkován nádorem samotným, okolní zdravou tkání v reakci na přítomnost nádoru nebo metastázami [5]. Jsou různé typy molekulárních nádorových markerů zahrnující DNA, mRNA, proteiny, antigeny, hormony, které jsme schopni kvantitativně nebo kvalitativně detekovat vhodnými metodami. Pro detekci nádorových markerů se v dnešní době používá immunohistochemie, kvantitativní imunologické testy, polymerázová řetězová reakce (PCR), western a northern blot, microarrays (genomické a proteomické) a hmotnostní spektrometrie. Pomocí nádorových markerů lze získat řadu informací o průběhu onemocnění, druhu zasažené tkáně či reakci na léčbu. Nádorové markery nejsou v onkologické praxi používány samostatně pro určení přesné diagnózy, protože většinu těchto markerů lze nalézt ve zvýšené hladině i u pacientů s benigním nádorem. Dalším omezením v použití nádorových markerů je fakt, že jen velmi málo z nich přímo souvisí s konkrétním typem nádorového onemocnění. Další překážkou v použití markerů jako jasného důkazu přítomnosti malignity je, že ne každý nádor způsobí zvýšení či snížení hladiny nádorového markeru v časných stadiích nádorového onemocnění. I přes jmenovaná omezení jsou nádorové markery velmi užitečné pro klinické účely zejména z pohledu plošného vyšetřování populace, jako jedno z nejméně invazivních vyšetření pacienta s podezřením na nádorové onemocnění a také jako možnost sledovat průběh léčby [5].
2.5.1 Nádorové antigeny Nádorové antigeny zahrnují látky, které jsou produkovány nádorovými buňkami, a jejichž přítomnost spouští imunitní reakci v podobě monoklonální protilátky nebo polyklonální antiséra u zdravých buněk. Tato skupina zahrnuje také tzv. onkofetální antigeny, které jsou přítomny v embryu nebo plodu ve vysoké koncentraci, která se v průběhu dospělosti snižuje. Vznikem nádorového bujení se koncentrace těchto onkofetálních antigenů zvyšuje.
14
Karcinoembryonální antigen Karcinoembryonální antigen (CEA) je protein, nacházející se u mnoha typů buněk, ale také souvisí s nádorovým onemocněním a vývojem plodu. Tento antigen byl poprvé objeven u pacientů s adenokarcinomem tlustého střeva v roce 1965 [6]. Z biochemického pohledu to je glykoprotein o molekulové hmotnosti 180 kDa, který je přítomen na buněčné membráně nádorových buněk, z nichž může být uvolněn do krve. CEA představuje heterogenní skupinu molekul, které se skládají z jednoho polypeptidového řetězce s různými sacharidovými složkami. Poměr bílkovin k sacharidům v molekulách CEA se pohybuje od 1:1 do 1:5 v závislosti na typu nádorového onemocnění. CEA je primárně metabolizován játry s poločasem rozpadu mezi jedním až osmi dny. Jaterní onemocnění mohou změnit poločas rozpadu tohoto proteinu a uměle tak zvyšovat sérové koncentrace CEA. Přestože byl CEA poprvé zjištěn u nádorů tlustého střeva, abnormální hladiny CEA nejsou specifické ani pro toto onemocnění, ani pro maligní nádorové onemocnění obecně. Zvýšené hladiny CEA se vyskytují u mnoha druhů nádorových onemocnění jako např. prsu, plic, slinivky, žaludku a vaječníků [7,8]. Jeho hladina může být také zvýšena díky kouření, zánětu průdušek, zánětlivému střevnímu onemocnění, chronickému plicnímu onemocnění, rozedmě plic, zánětu žaludku, zánětu slinivky břišní, polypům tlustého střeva a konečníku, divertikulitidě, Crohnově chorobě, benigní hyperplazii prostaty a ledvin [5]. V důsledku toho chybí sérové hladině CEA specifičnost a jeho detekce nemůže nahradit patologické diagnózy.
Alfa-fetoprotein Alfa-fetoprotein (AFP) je onkofetální protein, který byl poprvé detekován v roce 1963 v séru myší trpících nádorem jater [9]. AFP je tvořen jedním polypeptidovým řetězcem o molekulové hmotnosti ~ 70 kDa. Přibližně 4 % z této molekuly se skládají ze sacharidů převážně tvořených sialovou kyselinou. AFP obsažený běžně ve fetálním séru je syntetizován v játrech a trávicím traktu, kde sdílí sekvenční homologii s albuminem [10]. Jeho koncentrace dosahuje nejvyšší hodnoty 3 mg/ml ve 12. týdnu těhotenství. Po porodu jeho koncentrace rychle klesá až na hladinu 20 ng/ml, která je běžná u zdravé populace. Beta a gama-fetoprotein Beta a gama-fetoproteiny se řadí do skupiny fetoproteinů s molekulovou hmotností 55 kDa a mohou být nalezeny ve střevech, séru a mozkové tkáni lidského plodu. Močové
15
fetoproteiny se zdají být užitečným nádorovým markerem pro tumory zažívacího a urogenitálního systému [5].
CA 15-3 Mnoho monoklonálních protilátek vzniklo proti mucinům přítomným na nádorových buňkách. Mnohé z těchto protilátek jsou namířeny proti epiteliálnímu sialomucinu, který je dnes nazýván episialin [11,12]. Episialin je jedním z hlavních sialovaných glykoproteinů na povrchu většiny typů nádorových buněk. Je syntetizován jako
transmembránová
molekula
s
poměrně
velkou
extracelulární
doménou
a cytoplazmatickou doménou skládající se z 69 aminokyselin. Extracelulární doména, která se skládá z řady téměř totožně se opakujících 20 aminokyselin, může být uvolněna z buňky. Tato doména je nejčastěji detekovatelná u pacientů trpících karcinomem prsu. Pro detekci extracelulární domény byla testována řada monoklonálních protilátek. Jedním z nejpoužívanějších testů je tzv. CA 15-3 test, který využívá monoklonálních protilátek 115D8 a DF3 [13].
CA 125 CA 125 je antigen přítomný na 80 % buněk epiteliálních ovariálních karcinomů [14]. Je definován monoklonální protilátkou (OC 125), která byla vytvořena imunizací myších linií lidskými buňkami odvozených z ovariálního karcinomu [15]. CA 125 je silně glykosylovaný protein o vysoké molekulové hmotnosti [16]. Molekulární charakteristiky CA 125 (> 200 kDa) jsou poměrně heterogenní, v závislosti na zdroji, kultivačních podmínkách a analyzovaném typu buněk [17]. CA19-9 CA19-9 je monoklonální protilátka vytvořená proti buněčné linii karcinomu tlustého
střeva
k
detekci
monosialoganglyosidu
nalezeného
u
pacientů
s gastrointestinálním adenokarcinomem [18]. Jeho zvýšená hladina byla detekována u 21-42 % případů karcinomu žaludku, 20-40 % nádorů tlustého střeva a 71-93 % nádorů slinivky [5].
16
CA 242 S rozvojem nádoru spojený antigen CA 242, definovaný monoklonální protilátkou C 242, byl nejprve studován imunohistochemicky [19,20]. Antigenní determinantou protilátky C 242 je sialovaný sacharid, příbuzný, ale chemicky odlišný od nádorových antigenů CA 19-9 a CA 50 [21]. Specifický membránový antigen prostaty Specifický membránový antigen prostaty (PSMA) je glykoprotein přítomný na povrchu buněk, který je produkován epiteliálními buňkami prostaty. PSMA-specifické monoklonální protilátky byly využity pro charakterizaci biologické funkce a in vivo biodistribuce tohoto proteinu. PSMA je atraktivním cílem pro monoklonální protilátky používané v zobrazovacích metodách nebo léčebných postupech karcinomů prostaty, protože jeho tvorba je relativně omezena na zdravé epiteliální buňky prostaty a u nádorových buněk včetně pokročilých stádií je zvýšena [22]. Antigen kmenových buněk prostaty Antigen
kmenových
buněk
prostaty
(PSCA)
je
glykoprotein
kódovaný
123 aminokyselinami, který je ukotven k buněčné membráně glykosylfosfatidylinositolovou kotvou [23]. Pomocí kvantitativní PCR (RT-PCR) a imunohistochemických studií bylo prokázáno, že ve zdravé tkáni je tento protein omezeně distribuován, zatímco v nádorových buňkách prostaty je jeho tvorba zvýšena. Antigen karcinomu močového měchýře První generace testu antigenu karcinomu močového měchýře (BTA) je tzv. Bard BTA test, který se skládal z latexové aglutinace pro kvalitativní detekci antigenu v moči. Antigen se skládá z komplexů bazální membrány, které byly izolovány z moči pacientů s karcinomem močového měchýře a následně charakterizovány. Jedná se o vysoko molekulární komplex vzniklý z proteolytické degradace, který obsahuje proteinové fragmenty s molekulovou hmotností v rozmezí od 16 do 165 kDa. Tyto proteiny obsahují kolagen typu IV, fibronektin, laminin a proteoglykany. V dnešní době je k dispozici Bard BTA stat Test druhé generace [24].
17
Produkty degradace fibrin/fibrinogenu V moči
zdravých
osob
chybějí
nebo
jsou
přítomny
produkty
degradace
fibrin/fibrinogenu ve velmi nízké koncentraci. Zvýšení jejich hladiny je pozorováno u pacientů s karcinomem močového měchýře, ale také v případě nespecifických zánětlivých onemocnění močových cest [25,26]. Beta-2-mikroglobulin Beta-2-mikroglobulin (β2M) se skládá z polypeptidového řetězce tvořeného 99 aminokyselinovými zbytky (molekulová hmotnost 11,8 kDa) majícími strukturu skládaného listu, která je stabilizována jednou disulfidovou vazbou mezi Cys25 a Cys80 [27]. β2M existuje na povrchu éměř t všech buněk jako součást lehkého řetězce hlavního histokompatibilního komplexu. Sérový β2M je citliv ý marker nádorové masy u mnohočetného myelomu a v menší míře v lymfomu [28,29]. 2.5.2 Rodina lidských kalikreinů Donedávna se předpokládalo, že se rodina lidských tkáňových kalikreinů (KLK) skládá ze tří genů. KLK1 (kódující lidský kalikrein 1 (hK1) neboli pankreatický/ledvinový kalikrein), KLK2 (kódující hK2, dříve známý jako lidský žlázový kalikrein 1) a KLK3 (kódující hK3 neboli specifický antigen prostaty (PSA)). KLK2 a KLK3 mají důležitou funkci v diagnostice karcinomu prostaty a potenciálně i prsu. Yousef et al. objevili nové kalikreinu podobné geny KLK4 – KLK15 [30]. Všechny geny se významně shodují v několika bodech, jako jsou lokace na stejném chromozomálním lokusu (19q13.4), signifikantní homologie jak na úrovni nukleotidů, tak i proteinů, a také mají podobnou organizaci genomu. Navíc je většina z genů regulována steroidními hormony. Nedávné výsledky naznačují, že několik z těchto kalikreinových genů je spojeno se zhoubným bujením [31]. Mnoho členů lidské rodiny kalikreinových genů je regulováno v případě tumorů vaječníku [32]. Kalikrein 1 Mezi ostatními členy rodiny lidských kalikreinů je jediný enzym s účinnou kininogenázovou aktivitou a to kalikrein hK1 [33]. Kalikrein/kininový systém je zapojen do mnoha patologických procesů, včetně zánětu, hypertenze, onemocnění ledvin, pankreatitidy a karcinogeneze. Bylo prokázáno, že kromě své hlavní činnosti,
18
kininogenásové aktivity, může hK1 štěpit pro-inzulin, lipoprotein o nízké hustotě, prekurzor síňového natriuretického faktoru, prorenin, vazoaktivní intestinální peptid, prokolagenázu a angiotenzinogen [33].
Kalikrein 2 hK2, také pojmenovaný jako lidský žlázový kalikrein, je protein specifický pro prostatu, který má z hlediska aminokyselinového složení 80% shodu s PSA (hK3) a 62% shodu s častěji se vyskytujícím hK1 [34]. hK2 se nachází v krvi pacientů v různých relativních poměrech k PSA, což svědčí o možném významu tohoto ukazatele u nádoru prostaty [35]. Zjištění, že hK2 štěpí pro-PSA za účelem vytvoření enzymaticky aktivního PSA ukazuje na fyziologickou úlohu hK2 v regulaci PSA [36].
Kalikrein 3 neboli specifický antigen prostaty Specifický
antigen
prostaty
(PSA)
je
prostatický
sekreční
glykoprotein
o molekulární hmotnosti 33 kDa. Je to serinová esteráza s trypsin- anebo chymotrypsinpodobnou aktivitou. Existují různé cirkulující formy PSA a to i) PSA kovalentně a ireverzibilně vázaný v komplexu s alfa1-chymotrypsinem, ii) PSA vázaný k beta2makroglobinu, anebo iii) nekomplexovaný PSA, který zůstává volně v séru [37]. Poločas rozpadu komplexovaného PSA je 2-3 dny u volného PSA se doba výrazně zkracuje a to na 2-3 hodiny. PSA je produkován epitelem prostaty, jeho funkcí je velmi rychlá hydrolýza seminogelinu I a II, a stejně tak i fibronektinu. Výsledkem je zkapalnění do konzistence gelu, který obklopuje spermie a umožňuje jim plnou pohyblivost [38]. Sérový PSA je momentálně nejužitečnějším biologickým markerem nádorů prostaty [39,40]. Na druhou stranu je třeba zmínit, že se PSA nachází i ve zdravém epitelu prostaty a séru, a jeho hladina se přirozeně mění s věkem [41,42]. 2.5.3 Hormony Hormony, které jsou používány jako nádorové markery, mohou být v závislosti na jejich původu klasifikovány do dvou skupin a to i) hormony jako přirozené produkty přidružených orgánů nebo produkty abnormální syntézy v důsledku neregulovaného metabolismu nádorové buňky a ii) hormony jako nepřirozené produkty přidružených orgánů.
19
Hormony jako přirozené produkty přidružených orgánů nebo produkty abnormální syntézy v důsledku neregulovaného metabolismu nádorové buňky Kalcitonin (CT) je hypokalcemický faktor sekretovaný C buňkami štítné žlázy. Poločas rozpadu sérového CT je 12 minut a normální hladiny jsou <0,1 ng/ml. CT produkuje medulární karcinom štítné žlázy, a jeho detekce může signalizovat přítomnost tumoru, ale také účinnost léčby pacientů trpících tímto nádorovým onemocněním [43]. Katecholaminy.
Nejčastěji
jsou
analyzovány
obsahy
katecholaminů
vanilylmandlové kyseliny a homovanilkové kyseliny, které jsou metabolity noradrenalinu a dopaminu. Hladiny těchto metabolitů mohou být přesně stanoveny přímo ze vzorku moče. Pro detekci katecholaminů jsou více přesné metody plynové chromatografie oproti kolorimetrickým metodám, které omezují pacienta v konzumaci čaje, kávy, ovoce a vanilky po dobu 72 hodin před odběrem vzorků moče. Biochemická detekce močových metabolitů katecholaminů zvyšuje pravděpodobnost výskytu feochromocytomu [44]. Lidský choriový gonadotropin (HCG). HCG je hormon, glykoprotein, který se skládá
ze
dvou
polypeptidových
řetězců,
které
nejsou
kovalentně
spojeny.
Alfa-podjednotka je společná pro několik glykoproteinových hormonů sekretovaných přední hypofýzou. Beta-řetězec dává tomuto hormonu strukturní a funkční identitu a navíc poskytuje základ pro radioimunoanalýzu tohoto hormonu. Nicméně existuje jistá příbuznost mezi beta-podjednotkou HCG a lidským luteinizačním hormonem, která může vyvolat imunitní zkříženou-reaktivitu („cross-reactivity“) mezi těmito dvěma hormony. Poločas rozpadu cirkulujícího HCG je 12-20 hodin. Hormon je obvykle produkován syncytiotrofoblastickými buňkami placenty a jeho hladina se zvyšuje v těhotenství. V nádorové diagnostice se nejvýznamněji uplatňuje u gestační trofoblastické nemoci a u diagnostiky zárodečných buněk nádorů [45-47]. Hladina HCG je občas zvýšená i u jiných typů tumorů včetně nádorů prsu, plic, trávicího ústrojí. Pro potvrzení jeho úlohy jako nádorového markeru těchto onemocnění není dostupné dostatečné množství klinických studií. Thyreoglobulin, jodoglykoprotein o molekulové hmotnosti 339 kDa je proteinovým prekurzorem hormonů štítné žlázy vylučovaný folikulárními buňkami štítné žlázy. Kvartérní struktura nativního thyreoglobulinu se skládá ze dvou podjednotek o stejné velikosti [48].
20
Gastrin-vylučující
peptid
(ProGRP).
GRP
je
peptid
skládající
se
z 27 aminokyselinových zbytků. Je imunoreaktivní ve fetálních a novorozeneckých plicích a malobuněčném karcinomu plic [49]. Bylo prokázáno, že exprese pro-GRP mRNA je úzce spjata se syntézou pro-GRP proteinu, který se uvolňuje do krve. Předpokládá se, že GRP má funkci autokrinního růstového faktoru nádorových buněk u pacientů trpících malobuněčným karcinomem plic z důvodu zvýšené exprese jeho receptoru [50,51]. ProGRP je v současné době používán jako sérový nádorový marker specifický pro malobuněčný karcinom plic [52]. Hormony jako nepřirozené produkty přidružených orgánů Adrenokortikotropní hormon (ACTH) je nejčastěji pozorovaný mimoděložní hormon produkovaný nádory. Ektopická produkce ACTH byla poprvé zaznamenána u pacientů trpících malobuněčným karcinomem plic. Následně bylo zjištěno, že produkce ACTH je spojena s jinými maligními onemocněními včetně adenokarcinomu a spinocelulárního karcinomu plic, karcinoidy, nádorů buněk ostrůvků pankreatu, karcinomu prsu, karcinomu tlustého střeva, feochromocytomu, tymomu, medulárního karcinomu štítné žlázy a karcinomu pohlavních žláz [53]. Zvýšené hladiny ACTH byly pozorovány také u dalších onemocnění, například u chronické obstrukční plicní choroby, obezity, hypertenze a cukrovky [54]. Studie dostupné v dnešní době ukazují na omezenou použitelnost ACTH pro plošné vyšetření, pro určení stagingu nebo monitorování odpovědi na protinádorovou terapii. Antidiuretický hormon. Malobuněčný karcinom plic je maligní onemocnění nejčastěji spojené s ektopickou sekrecí antidiuretického hormonu (ADH). Sekrece ADH může být detekována biochemicky [55]. K dalším zhoubným onemocněním, u kterých byla zjištěna ektopická sekrece ADH, patří karcinom pankreatu, bronchiální karcinoidní nádory, karcinom kůry nadledvin, thymom, karcinom močového měchýře a prostaty. K benigním příčinám produkce ADH patří plicní onemocnění, onemocnění centrálního nervového systému, aplikace anestetik či požití drog. Kvůli nedostatečné specifičnosti není ADH vhodný jako plošný marker karcinomu a monitorování odpovědi na léčbu.
21
2.5.4 Enzymy Tato skupina nádorových markerů zahrnuje enzymy, které jsou nativní v normální tkáni a jejich struktura se mění v tkáni nádorové, anebo ty, které mají spojitost se změnami v metabolismu a jsou specifické pro nádorovou tkáň. Kyselá prostatická fosfatáza Kyselá prostatická fosfatáza (PAP) je glykoprotein s molekulovou hmotností 100 kDa, který se skládá ze dvou totožných podjednotek. Existuje několik izoenzymů, které se liší v poměru jednotlivých sacharidů [56]. Vysoké koncentrace tohoto enzymu lze nalézt ve zdravé prostatické tkáni stejně jako v primárních a metastatických nádorech. Kyselé fosfatázy jsou skupinou enzymů, které jsou rovněž v nižších koncentracích přítomny v kostech, ledvinách, játrech, slezině a střevu. Hladina PAP je taktéž zvýšena v procesech jako je osteoporóza, hypertyreóza, chirurgická léčba prostaty, katetrizace močového ústrojí a benigní hyperplazie prostaty. Kromě karcinomu prostaty patří k ostatním maligním onemocněním se zvýšenou hladinou kyselé fosfatázy také mnohočetný myelom, osteogenní sarkom a kostní metastázy [5]. Neuron-specifická enoláza Enoláza je enzym se třemi imunologicky odlišnými podjednotkami alfa, beta a gama. V běžné lidské tkáni existují tři izoenzymy enolázy. Alfa-alfa-izoenzym přítomný v gliálních buňkách a řadě dalších tkání se označuje jako ne-neuronová enoláza (NNE). Beta-Beta- enoláza se nachází převážně ve svalu, a u gama-gama izoenzymu, také známého jako neuron-specifická enoláza (NSE), byl prokázán výskyt v neuronech a neuroendokrinních buňkách. Bylo zjištěno, že neurony během diferenciace přechází od produkce NNE k NSE. Tento proces vysvětluje přítomnost NSE v ne-neuronových a neneuroendokrinních tumorech. NSE je imunohistochemický marker pro nádory centrálního nervového systému a neuroblastomu [57]. Zvýšené hladiny v séru se vyskytují zřídka, s výjimkou pacientů s neuroblastomem a malobuněčného plicního karcinomu [58,59]. Galaktosyl transferáza II Zvýšená hladina galaktosyl transferázy II, izoenzymu galaktosyl transferázy, byla zaznamenána u různých typů malignit, které byly převážně gastrointestinálního původu. U karcinomu tlustého střeva koreluje její hladina v séru s rozsahem a progresí onemocnění
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[60]. Při rozlišení benigních nebo maligních forem abnormalit slinivky se ukazuje samotná detekce transferázy nebo kombinace se zobrazovacími technikami více citlivá a specifická než detekce CEA anebo použití zobrazovacích technik samotných [61]. Galaktosyl transferáza II může být také užitečná jako marker nádorů pohlavních žláz [5]. Napsin A Napsiny jsou ''Nové asparagové proteinázy pepsinové rodiny''. Je dobře zdokumentováno, že v lidském těle je produkováno pět asparagových proteináz. Pepsin, gastricsin a renin jsou sekrečními enzymy, jejichž fyziologické funkce nejsou přesně definované, a navíc jsou spojovány s patologickými stavy včetně karcinogeneze [62]. Další dvě asparagové proteinázy, katepsin D a katepsin E, jsou přítomny v intracelulárních prostorech a jsou snadno odlišitelné díky svojí molekulární struktuře a cytomorfologickým úsekům [63]. Pronapsin A je gen exprimovaný především v plicích a ledvinách. Předpokládá se, že jeho translačních produkt je plně funkčně glykosylovaným prekurzorem pro asparagovou proteinázu obsahující motiv Arg-Asp-Gly a dalších 18 zbytků na svém C-konci. Gen Pronapsinu B je přepisován výhradně v buňkách související s imunitním systémem [64]. Napsin byl detekována hybridizací in situ u plicních buněk typu II jak ve zdravé plicní tkáni, tak v tkáni adenokarcinomu [65].
Katepsin Asparagová proteáza známá jako katepsin D a E a cysteinové proteázy jako katepsin B, se podílejí na progresi zvířecích a lidských nádorů [66,67]. Tyto enzymy se přímo podílejí na degradaci mimobuněčné hmoty anebo mohou nepřímo aktivovat jiné proteázy, které pak rozkládají mimobuněčnou hmotu. Invaze nádorových buněk zahrnuje místní proteolýzu a také lokalizaci aktivního katepsinu B na invazivním okraji nádoru, což je důležité pro odbourávání okolní mimobuněčné hmoty. Katepsin B může rozkládat proteiny extracelulární hmoty laminin, fibronektin a kolagen typu IV, také může aktivovat prekurzorovou formu aktivátoru plazminogenu urokinázy [66,68]. Zvýšená sekrece prokatepsinu B nebo katepsinu B byla pozorována v lidských buněčných liniích kolorektálního karcinomu [69] a hematomu [70], stejně jako v lidských buněčných liniích tumoru prsa [71].
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Matrixové metaloproteinázy Matrixové metaloproteinázy (MMP, Obr. 1), jenž mohou být také označovány jako matrixiny, jsou početnou skupinou zinek-dependentních proteinů, které mají za úkol štěpení a přestavování jednotlivých součástí pojivové tkáně jako je kolagen, elastin, želatina a kasein. Jejich výskyt byl prokázán jak u obratlovců, tak u bezobratlých a dokonce byly nalezeny i v rostlinách. Jejich fylogenetický původ je přisuzován nižším organismům, a to konkrétně Bacteroides fragilis [72].
Obrázek 1. Struktura želatinázy A. Převzato z www.rscb.org.
MMP byly objeveny v roce 1962 u obratlovců, kdy vědci Jerome Gross a Charles M. Lapiere studovali degradaci helikálního trojvláknového kolagenu při metamorfóze ocasu pulce [73]. Tato degradace byla možná pomocí enzymu, jenž byl pojmenován jako intersticiální kolagenáza. V roce 1968 byl tento enzym poprvé izolován z lidské kůže ve formě zymogenu (neaktivní forma). Později byl objeven jak u bezobratlých, tak u rostlin. Až v roce 1990 se zjistilo, že udržení enzymu v inaktivní formě má na svědomí 24
mechanismus cysteinového sepnutí, objevující se v literatuře s anglickým názvem „Cysteine switch“ [74]. Po dokončení sekvenace genomu člověka bylo stanoveno 24 různých genů, kódujících množinu veškerých lidských MMP [75]. Rodina MMP se mezi svými členy vyznačuje až 40% shodou v primární struktuře. Je známo přibližně 20 typů MMP, které jsou klasifikovány podle presyntetického úseku na chromozomech a substrátové specifity. K jejich označení se používají čísla MMP-1 až MMP-28, ale některé číslice v této řadě nejsou k pojmenování MMP využity [75]. MMP jsou homologní proteiny, podle mechanismů štěpení substrátu je můžeme je rozdělit do šesti kategorií: kolagenázy, stromelysiny, matrilysiny, želatinázy, membránové metaloproteinázy a jiné MMP. Jsou syntetizovány ve formě neaktivních preproenzymů. Tato inaktivní forma zabraňuje MMP štěpit esenciální složky buňky. MMP jsou tedy z buňky většinou sekretovány jako neaktivní proenzymy kromě membránově vázaných MMP (anglicky membrane-type MMP). Samotná struktura molekuly proenzymu se člení do tří základních domén: N-terminálního propeptidu, katalytické domény a C-terminální části molekuly [76]. N-terminální propeptid zajišťuje enzymovou latenci proenzymu. Obsahuje asi 80 aminokyselin, z nichž nejvýznamnější funkční roli zastává cystein, jehož postranní řetězce interagují s katalytickým zinkovým atomem přes thiolovou skupinu a utváří takzvané cysteinové sepnutí. V propeptidu je přítomná vysoce konzervativní sekvence (Pro-Arg-Cys-Gly-X-Pro-Asp, kde X značí jakoukoliv aminokyselinu), jejímž odstraněním dojde k aktivaci zymogenu [77]. Katalytická doména se skládá z pětipramenného β-listu, tří α-helixů a spojovacích smyček. Je tvořená 170-ti aminokyselinami a obsahuje zinek-vazebný motiv (His-Glu-XXHis-XX-Gly-XX-His, kde X značí jakoukoliv aminokyselinu) s upevněným methioninem, který vytváří unikátní strukturu – methioninový ohyb. Katalytická doména obsahuje dva zinečnaté kationty a 2-3 vápenaté kationty. První Zn2+ je přítomen v aktivním místě a účastní se přímo katalytických procesů, druhý Zn2+ (také nazývaný strukturní) a současně i Ca2+ jsou vzdáleny od Zn2+ v katalytickém místě přibližně 12 nm [78]. C-terminální doména vykazuje strukturní podobu s proteiny tzv. hemopexinové rodiny (proteiny obsažené v séru). Má relativně velký povrch pro případnou interakci s jinými proteiny. Vypadá jako disk elipsoidního tvaru se 4 listovými β-strukturami připomínající vrtuli, každý list “vrtule“ se skládá ze 4 antiparalelních β-pramenů a α-helixu. První a čtvrtý list jsou spojeny disulfidickým můstkem [79]. Katalytická
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a C-terminální domény, jak bylo publikováno u kolagenázy-1, jsou zabaleny jako entity v krystalu spojujícím se flexibilním peptidovým linkerem, neboli závěsem, který je volně připojen [79]. Tento závěs má extrémně variabilní délku, u kolagenáz obsahuje jen 16 aminokyselinových zbytků, MMP-15 má naopak 65 takovýchto zbytků. Přesná funkce závěsu (bohatého na prolin), který spojuje katalytickou a hemopexinovou doménu, není zcela objasněna. Pro správnou funkci musí být MMP aktivována. MMP jsou vylučovány z buňky v neaktivní podobě jako zymogeny. Extracelulární aktivace enzymu zahrnuje dva kroky. První je iniciační štěpení propeptidu MMP proteázou, destabilizace propeptidových vazebných interakcí a přerušení koordinační vazby cysteinu a zinečnatého kationu tzv. mechanizmem cysteinového sepnutí [80]. Druhým krokem je finální odštěpení propeptidu, obvykle zajištěné jinou MMP. Výsledkem je maturovaný enzym. V mnoha případech se MMP nemohou účastnit reakce, dokud poslední část propeptidu není odstraněna. Aktivace MMP je stále intenzivně studovanou otázkou a jednou z řady možností je účast intracelulárního proteinu metalothioneinu [81,82]. Většina MMP je schopna degradovat jak majoritní, tak i minoritní složky extracelulární matrix. Až na výjimky, kterými jsou MMP-11 a 23, má většina MMP širokou substrátovou specifitu. MMP nezpracovávají pouze složky extracelulární matrix, ale mohou například působit jako aktivátory molekul. Například MMP-2,-3,-7 štěpí dekorin, který je důležitým rezervoárem transformujícího růstové faktoru β. Tím se do tkání může uvolňovat růstový faktor, jenž iniciuje další procesy, které nemusí přímo souviset s degradací extracelulární matrix [83]. Kromě fyziologických procesů, jako jsou apoptóza, děložní cyklus, embryonální vývoj, ovulace anebo zánět, se vliv MMP promítá také do množství patologických procesů, jako jsou artritida, Alzheimerova choroba, ateroskleróza, cévní onemocnění, gastritický vřed, choroby centrální nervové soustavy, jaterní cirhóza a nádorové onemocnění. Není tedy překvapením, že MMP mohou být studovány z několika různých pohledů jako markery
některých
nádorových,
neurodegenerativních,
imunitních
a
také
kardiovaskulárních onemocnění [84]. MMP se z pohledu studia jejich vlivu na člověka v oblasti nádorových onemocnění dostávají do popředí zájmu na začátku devadesátých let, kdy jsou studovány v souvislosti s karcinomem žaludku a tlustého střeva [85] anebo prostaty [86]. Možnosti izolace a studia enzymové aktivity MMP byly publikovány o rok později [87]. Ve stejném roce bylo
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zjištěno, že buňky stromatu jsou schopny syntetizovat MMP za účelem degradovat spolu s neoplastickými epiteliálními buňkami základní membrány, což je typický znak pro invazivní nádorové bujení [88]. Proto v dalších letech problém MMP v souvislosti s nádory začal být intenzivněji studován [89]. V roce 1995 byl popsán výskyt MMP membránového typu u karcinomu tračníku, hrudníku, krku a hlavy [90]. V průběhu rakovinného bujení [91], a zejména při tvorbě metastáz [92], jsou MMP zodpovědné za přestavbu tkáně v těsném okolí proliferujících buněk zhoubného novotvaru a umožňují tak rozrůstání novotvaru do okolní tkáně. Například MMP-9 je studována v souvislosti s nádorem plic [93,94]. Je publikována řada prací, které jsou zaměřeny na lokální inhibici MMP pomocí inhibitorů [93,95], za účelem zpomalit nebo zastavit progresivní bujení nádoru [96].
Aktivátor plazminogen-urokinázového typu Aktivátor plazminogen-urokinázového typu (uPA) má dvě hlavní strukturální domény, C-terminální s proteinázovou aktivitou, který generuje plazmin z plazminogenu a N-terminální fragment, který se váže na specifický receptor na povrch buněk zvaný uPAR [97]. uPAR je zakotvený glykosylfosfatidylinositolový glykoprotein (50-60 kDa) na povrchu různých typů buněk. Tento receptor může být vázán nebo štěpen urokinázou. Rozštěpený receptor představuje protein o velikosti 35 kDa. uPA je serinová proteáza fibrinolýzy a předpokládá se, že hraje klíčovou roli v extracelulární proteolýze a usnadňuje migraci rakovinných buněk při invazivním šíření a metastazování [5]. Tumor M2-pyruvát kináza Tumor M2-pyruvát kináza (M2-PK) je izoformou pyruvát kinázy a je exprimována v několika krokovém vzniku nádoru [98]. Formování nádoru je obvykle spojeno se zvýšením procesu aerobní glykolýzy. Prvním krokem v průběhu karcinogeneze je ztráta tkáňově-specifického izoenzymu L-PK v játrech a ledvinách a M1-PK v mozku a svalech, následuje exprese M2-PK isoenzymu [99,100].
Laktátdehydrogenáza Laktátdehydrogenáza (LDH) je tetramer o molekulové hmotnosti 140 kD. U savců existují tři monomerní jednotky a to: M (svaly, také nazývaný A) a H (srdce, také nazývaný B), a typ X (také nazývaný C) nacházející se pouze v spermiích. Oba M a H
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monomery váží asi 35 kDa. M monomer efektivně katalyzuje produkci laktátu z pyruvátu, ale tuto funkci LDH postupně ztrácí ve chvíli, kdy počet H monomerních jednotek převyšuje M [101]. Prostasin Prostasin je protein skládající se z 343 aminokyselinových zbytků, kde 32 aminokyselin tvoří signální peptid a 311 aminokyselin proprostasin. Proprostasin je štěpený mezi Arg12 a Ile13, přičemž vzniká 12 aminokyselin dlouhý lehký řetězec a 299 aminokyselin dlouhý těžký řetězec, které jsou spojeny prostřednictvím disulfidového můstku. Sekvence aminokyselin z těžkého řetězce je z 34-42% identická s lidským acrosinem, plazmovým kalikreinem a hepsinem. mRNA prostasinu byla detekována v epiteliálních buňkách prostaty u člověka a je exprimována v prostatě, játrech, slinných žlázách, ledvinách, plicích, slinivce, tlustém střevě, průduškách, renálních proximálních tubulárních buňkách, a buňkách karcinomu prostaty. Prostasin vykazuje trypsinu podobnou enzymatickou aktivitu, která může být inhibována řadou látek. Fyziologické funkce prostasinu nejsou zcela objasněny. Bylo prokázáno, že exprese prostasinu je výrazně snížena u nádorů prostaty s vyšším gradingem a je zcela inhibována ve vysoce invazivních lidských a myších buněčných liniích prostaty [102]. Transfekce dvou nádorových lidských prostatických buněčných linií DU-145 a PC-3 lidskou cDNA prostasinu snižuje in vitro invazivitu buněk, což svědčí o supresorové úloze prostasinu v případě pronikání nádorových buněk do okolní tkáně [5]. nm23-H1 nm23 proteiny jsou zapojeny do regulace nádorových metastáz a mají aktivitu nukleosiddifosfát kináz, díky které katalyzují ATP-dependentní syntézu nukleosid difosfátů [103]. U lidí bylo objeveno pět nm23 isotypů (nm23-H1, nm23-H2, DR-nm23, nm23-H4 a nm23-H5). Byl prokázán inverzní vztah mezi metastatickým potenciálem a hladinou nm23-H1 u různých typů nádorových onemocnění [103]. YKL-40 Glykoprotein YKL-40, pojmenovaný podle svých tří N-terminální aminokyselin a molekulové hmotnosti je savčím členem rodiny chitinázových proteinů ovšem bez chitinázové aktivity. Funkce YKL-40 je neznámá, ale předpokládá se, že hraje důležitou
28
roli ve fyziologii kostní tkáně. YKL-40 je syntetizován in vitro buněčnou linií osteosarkomu MG-63, lidskými synoviocyty a chondrocyty [104-106]. Zvýšené sérové hladiny YKL-40 byly dány do souvislosti se špatnou prognózou u kolorektálního karcinomu [107], u opakovaného metastatického karcinomu prsu [108], u primárního a recidivujícího ovariální karcinomu [109] a pokročilého renálního karcinomu před léčbou. Zvýšená hladina YKL-40 v séru je dále spojována se špatnou prognózou u kolorektálního karcinomu a karcinomu prsu po operaci [110,111]. Sérové hladiny YKL-40 v rané fázi karcinomu vaječníků může předpovídat recidivu onemocnění [112]. Microarray analýza umožňuje detekovat YKL-40 gliomech, v němž koreluje s gradingem tumoru [113]. 2.5.5 Cytokeratiny Cytokeratiny (CK) jsou součást proteinů intermediárního filamenta, které jsou charakteristické biochemickou rozmanitostí, a jsou zastoupeny v epiteliální tkáni 20 různými proteiny označenými 1-20 [114]. Jejich molekulová hmotnost se pohybuje mezi 40 až 68 kDa [115]. CK 1 má nejvyšší molekulovou hmotnost, zatímco CK 19 má nejnižší molekulovou hmotnost. CK jsou rozděleny do dvou podskupin a to I a II, přičemž podskupinu II tvoří bazické až neutrální CK 1-8, zatímco podskupinu I kyselé CK 9-20. Je vždy exprimováno několik cytokeratinů zároveň, což charakterizuje typ epitelu a stupeň zrání, a diferenciaci uvnitř epitelu [116]. Různé komplexy CK byly pozorovány u karcinomu močového měchýře, prsu, plic a tlustého střeva [117-120]. 2.5.6 Cytokiny a cytokinové receptory HER-2 HER2 je glykoprotein o molekulové hmotnosti 185 kDa, který je obvykle exprimován v epitelu různých orgánů jako plic, močového měchýře, slinivky břišní a prostaty. Zvýšená exprese HER-2 ve tkáni a zvýšené sérové hladiny HER-2 byly pozorovány u karcinomu prsu, prostaty, vaječníků a plic [121-124]. Interleukin 6 Interleukin 6 (IL6) je cytokin o molekulové hmotnosti 21-28 kDa, který stimuluje růst myelomových a plazmocytomových buněk a buněk lymfomu, dále tlumí růst buněk myeloidní leukémie a buněk karcinomu prsu. Zjistilo se, že IL6 může fungovat jako autokrinní růstový faktor u malignit [125]. Hladina IL6 v séru je zvýšená u pacientů
29
s karcinomem prostaty a je tedy považován za významný prognostický marker [126]. IL-6 je také nezávislý negativní prognostický ukazatel přežití u pacientů s kolorektálním karcinomem [127]. Sérový IL-6 byl také označen za nezávislou negativní prognostickou proměnnou pro celkové přežití u pacientů s metastazujícím karcinomem prsu [128]. Receptor interleukinu 2 Receptor interleukinu 2 (IL2R) je transmembránový receptor. Byly popsány tři formy receptoru s různou afinitou pro interleukin 2. Vysokou afinitu má heterotrimerový receptor skládající se z alfa, beta a gama (c)-polypeptidických řetězců. Alfa řetězec o molekulové hmotnosti 55 kDa se může distribuovat z povrchu buňky v podobě 45 kDa IL-2Rα. Rozpustná IL2R (sIL2R) je signifikantně spojena s metastázami u melanom u, karcinomu tlustého střeva, žaludku, plic a prsu [129-133]. Zvýšení hladiny sIL2R v séru u pacientů trpících trichocelulární leukémií a karcinomem vaječníků naznačuje zvýšené riziko vzniku relapsu onemocnění [134,135]. Prognóza pacientů s vysokou hladinou sIL2R je výrazně horší v případě adenokarcinomu ledvin, ne-Hodgkinova lymfomu, melanomu, karcinomu žaludku, slinivky břišní, hlavy a krku, a malobuněčného plicního karcinomu [136-142]. Transformující růstový faktor beta Aktivní forma transformujícího růstového faktoru beta (TGF-β) je homodimer ze 112 aminokyselin o molekulové hmotnosti 25 kDa spojený disulfidickým můstkem s prekurzorem tvořeným 391 aminokyselinami [143]. TGF-β1 byl původně izolován jako jedna ze dvou složek (TGF-α a β), k et rá by mohla způsobit transformaci fenotypu u fibroblastů odvozených ze zdravé ledviny potkanů [144]. Následně vyšlo najevo, že TGF-β, na rozdíl od svého partnera mitogenního TGF -α, působí silně inhibičně na růst většiny buněčných linií. A dále bylo prokázáno, že TGF-β má protektivní roli proti karcinogenezi, přičemž genetická nebo epigenetická ztráta TGF-β signalizace vede k tvorbě a progresi nádoru. Na druhé straně četné příklady ukázaly, že TGF-β1 má nejen potenciál transformovat, ale může také řídit maligní progresi, invazivitu a metastazování jak in vitro, tak in vivo [145,146]. V raných fázích vzniku tumoru, kdy je nádor benigní, působí TGF-β přímo na potencionálně nádorové buňky, aby potlačil jejich bujení. Jak se nádor vyvíjí, genetické nebo biochemické změny umožňují TGF-β podněcovat progresi nádoru [5].
30
MIC-1 je odlišným členem TGF-β rodiny, která byla původně identifikována na základě zvýšené exprese spojené s aktivitou makrofágů [147]. Je znám pod řadou dalších jmen, jako je placentární TGF- β, růstový a diferenciační faktor 15/MIC-1, placentární kostní morfogenetický protein. Jeho funkce je stále neobjasněna, i přesto, že byla popsána jeho schopnost inhibovat tumor nekrotizující faktor alfa produkovaný makrofágy po stimulaci lipopolysacharidy. Dále je schopen indukce tvorby chrupavky v raných stádiích tvorby endochondrální kosti, inhibuje proliferaci základních hemopoietických kmenových buněk a působí jako neurotrofický faktor. Na rozdíl od TGF-β, není MIC -1 produkován cirkulujícími krevními destičkami nebo ostatními krevními buňkami ve větším množství, a proto může být spolehlivě detekován v séru nebo plasmě [148]. Receptor epidermálního růstového faktoru Receptor
epidermálního
růstového
faktoru
(EGFR)
je
transmembránový
glykoprotein, který je aktivován pomocí vazby EGF a TGF-α na vn ější doménu. Po aktivaci jsou fosforylovány intracelulární tyrosinkinásy a následně dochází k buněčné proliferaci, transformaci a dělení. Zvýšené množství EGFR a jeho ligandů bylo identifikováno jako běžná součást mnoha typů nádorových onemocnění, zahrnující karcinom prsu, vaječníku, plic, tlustého střeva, hlavy a krku [149]. V mnoha případech špatná aktivace EGFR, způsobená především změnou v amplifikaci genu a autokrinní stimulací, se zdá být důležitým faktorem v tumorigenezi a hybnou silou pro růst, který je typický pro rakovinné buňky. CD30 CD30 je 120 kDa molekula patřící do rodiny receptorů tumor nekrotizujícího faktoru a faktoru pro stimulaci nervového růstu. Funguje jako transmembránový receptror pro cytokiny a je exprimován Hodgkinsovými a Reed Sterbergovými buňkami [150]. 2.5.7 Signální molekuly Ras Ras je klíčový regulátor buněčného růstu v eukaryotických buňkách. Několik ras genů již bylo popsáno včetně K-ras, H-ras a N-ras. Ras geny kódují rodinu příbuzných proteinů, které mají molekulární hmotnost 21 kDA (p21) a silný transformující potenciál [151]. Všechny ras proteiny jsou malé GTPázy. Přenášejí extracelulární signály
31
indukované pomocí aktivace povrchových receptorů, signál pak pokračuje několika cestami až do jádra [152]. To vede k několika různým biologickým procesům, jako je proliferace, diferenciace a apoptóza. Ras proteiny u karcinomů tlustého střeva mohou mít klinický význam v před-operativní identifikaci agresivnějších tumorů a pro stanovení horší prognózy [153]. U pacientů, kteří byli léčeni s progresivním karcinomem tlustého střeva, může mutace K-Ras predikovat jejich přežití [154]. trk trk protoonkogen kóduje membránový protein tyrozin kinázu o velikosti 140 kDa (p140prototrk), který je exprimován pouze v nervové tkáni. Nervový růstový faktor (NGF) stimuluje fosforylaci p140prototrk v nervových buněčných linií a v embryonálních míšních uzlinách. Identifikace p140protokrk jako receptoru pro NGF značí, že se tento protein zapojuje do primárního přenosu signálu NGF [155]. myc Genová rodina myc obsahuje c-myc, N-myc a L-myc [156]. Tyto transkripční faktory jsou důležité jaderné onkogeny. Působí jako sekvenčně specifické transkripční faktory, které regulují důležité geny v normálně rostoucích a diferencujících buňkách. Špatná exprese těchto onkoproteinů je jedna z mnoha příčin vzniku nádorového onemocnění [157]. Jaderný heterogenní ribonukleoprotein V roce 1996 Zhou et. al. zjistili, že jaderný heterogenní ribonukleoprotein A2/B1 (hnRNP A2/B1), RNA vázající protein, je hlavní jaderný protein a jedna z hlavních složek jádrového komplexu hnRNP v živočišných buňkách. Jeho molekulová hmotnost je 31 000 kDa [158]. Předpokládá se, že hnRNP A2/B1 by mohl být do jisté míry zapojený do syntézy RNA a transportu mRNA z jádra do cytoplasmy, podobně, jako je tomu u jiných proteinů z rodiny hnRNP [159]. Sueoka et al. potvrdili, že hnRNP B1 je nový nádorový marker pro karcinom plic [160].
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2.5.8 Nádorové markery růstu nádorů: buněčný cyklus a proliferace Telomerázy Telomery jsou specializované struktury na konci chromosomů eukaryot, které hrají důležitou roli při ochraně a replikaci chromozomů [161]. Buněčné dělení vede k postupnému zkracování telomer, telomeráza (RNA-dependentní DNA polymeráza) kompenzuje tento problém přidáváním hexametrické repetice (TTAGGG)n na konce chromosomů.
Progresivní
zkracování
telomerických
repeticí
přispívá
k buněčné
senescenci. Reaktivace telomerázy a stabilizace telomer pak umožňuje nesmrtelnost nádorových buněk [162]. Jaderné matrixové proteiny Jaderné matrixové proteiny (NMP) jsou součástí vnitřního systému buněčného jádra. Je znáno, že hrají důležitou roli při replikaci a transkripci DNA, syntéze RNA a celkově při regulaci genové exprese. NMP-22 je protein, patřící do komplexu jaderného mitotického aparátu a tudíž se účastní separace chromatid do dceřiných buněk během buněčného dělení. Prokázalo se, že koncentrace NMP-22 je u karcinomu močového měchýře nejméně 25 krát vyšší, oproti hladinám detekovaných ve zdravé tkáni [163]. Proteiny teplotního šoku Mnoho stresových stimulů jako je teplo, napadení virem, oxidační poškození, těžké kovy apod. vedou u eukaryotických buněk ke zvýšení exprese tzv. proteinů teplotního šoku (HSP) [164]. HSP jsou na základě jejich molekulové hmotnosti klasifikovány do pěti hlavních rodin: HSP65, HSP70, HSP90 a HSP100 řazeno od nejnižší molekulové hmotnosti. Jednotlivé rodiny se dále skládají z několika skupin, z nichž každá má rozdílnou expresi. HSP73 se běžně exprimují v buněčném cyklu, zatímco HSP72 se ve velkém množství exprimuje teprve po tepelném šoku [165]. Fyziologickou funkcí HSP je syntéza a degradace proteinových komplexů a dále účast na transportu některých proteinů přes intracelulární membrány. Schopnost HSP se exprimovat i mimo intracelulární oblast, a to na povrchu nádorových buněk, naznačuje jejich možnou roli v navození imunitní reakce proti nádorovým buňkám [166]. Mezi rodinami HSP, HSP27 a HSP70, byla prokázána silná asociace s různými karcinomy. I přesto, že exprese HSP byla uznána jako prognostický marker, chybí dostatečný počet klinických dat a navíc výsledky jsou často protichůdné. Například exprese HSP70 u kolorektálního karcinomu a karcinomu prsu
33
korelovala
s nízkou
diferenciací
a
špatnou
prognózou
[167,168],
zatímco
u adenokarcinomu ledviny s prognózou dobrou [169]. U
karcinomu
jícnu
exprese
HSP
27
a
70
negativně
korelovala
s metastázami mízních uzlin a indikovala tak dobrou prognózu [170,171], stejně jako u karcinomu pankreatu a melanomu, ve kterém zvýšená exprese HSP70 korelovala se zlepšením stavu pacienta [172,173]. Silné imunozabarvení HSP70 korelovalo se zvětšením maligních nádorů děložního čípku a s vyšší úmrtností pacientů trpících karcinomem děložní sliznice a močového měchýře [174-176]. HSP70 by mohl být potenciální marker pro diagnostiku časných hepatocelulárních karcinomů z prekancerózních lézí nebo nekarcenogeních jater [177].
Mikrosatelity Pro mnohá nádorová onemocnění je charakteristické zvýšení chromozomálních abnormalit a ztráta heterozygozity [178]. Tyto genetické vlastnosti mohou být detekovány za využití analýzy vysoce polymorfních markerů, jakými jsou mikrosatelity [179]. Častý výskyt ztráty či změny délky mikrosatelitů byl popsán u mnohých karcinomů např. plic, spinocelulárního a kolorektálního karcinomu, karcinomu močového měchýře a dalších [180-182].
DNA ploidie Pomocí testu ploidie lze detekovat DNA v nádorových buňkách. Zjistilo se, že DNA ploidie má prognostický význam u pacientů trpících karcinomem prsu, tlustého střeva, nosohltanu a děložního čípku. Prognostický význam DNA ploidie se zvyšuje, pokud se zkombinuje s dalšími biologickými parametry [183]. Stav buněk v S fázi Poté, co má buňka zdvojený genetický materiál a je rozdělena procesem zvaným mitosa na dvě buňky dceřiné, stává se buď inaktivní, nebo může začít další buněčný cyklus. Analýza DNA průtokovým cytometrem ukázala, že u nádorů se ve větší míře vyskytuje frakce DNA, která je v S-fázi buněčného cyklu. Toto je pozorovatelné zejména u primárních lymfomů žaludku, prsu, tlustého střeva, nosohltanu a hrtanu [183-186].
34
Protein p16 Progrese prostřednictvím buněčného cyklu je řízen aktivitou cyklin-dependentních kináz (Cdk) [187]. Aktivní Cdk holoenzym se skládá z katalytické kinázové podjednotky spojené s regulačním cyklinem. D-cykliny podporují aktivitu Cdk4, nicméně buněčné cykliny D a Cdk4 jsou závislé na dalších signálních drahách [188]. Jednou z nich je interakce proteinů, jako je p21CIP1 (p21) a p27KIP1 (p27), s Cdk4 komplexem. p16 je specifický inhibitor D-typu Cdk [189]. Interakce p16 s Cdk4 způsobuje disociaci cyklinu D a tím dochází k inhibici cyklin D-Cdk4 komplexu. Aktuální studie předpokládají, že vznik neaktivního p16-Cdk4 komplexu je regulován nadbytkem obou proteinů, ačkoli fosforylace proteinu p16 může v tomto procesu také hrát roli [190]. Protein p53 p53 je jaderný fosfoprotein složený z 393 aminokyselin s pěti evolučními doménami. Je produktem tumorsupresorového genu, který je zmutován nebo inhibován, např. Mdm2 u více než 50 % nádorů. Normální produkt genu p53 má krátký poločas rozpadu, ale mutovaný proteinový produkt je mnohem stabilnější a lze ho detekovat imunohistochemicky. Za běžných podmínek protein p53 zastaví dělení buňky s poškozenou DNA do doby, než dojde k její opravě nebo spustí apoptózu poškozené buňky. Když se gen p53 poškodí nebo zmutuje, protein se stává nefunkčním a nemůže bránit rakovinovým buňkám v replikaci [191-193]. 2.5.9 Nádorové markery a angiogeneze Vaskulární endoteliální růstový faktor Vaskulární endoteliální růstový faktor (VEGF) byl původně identifikován v roce 1983 jako tumorem produkovaný faktor, který zvyšuje vaskulární permeabilitu [194,195]. VEGF je rozhodující pro normální vývoj embrya, kde má antiapoptické, mitogenní a propustnost zvyšující vlastnosti specifické pro cévní endotel. Četné izoformy VEGF jsou výsledkem alternativního sestřihu exonů příslušného genu [196]. Kromě různých izoforem byla popsána rodina VEGF, která souvisí s angiogenezí (VEGF-B, VEGF-C, VEGF-D, VEGF-E) a růstovým faktorem placenty. Jejich činnost závisí částečně na rozdílné afinitě ke třem signálním receptorům VEGF: VEGFR-1 (Flt-1), VEGFR-2 (flk-1/KDR) a VEGFR-3 (FLT4). VEGFR-1 se zdá být hlavní VEGF receptor pro angiogenezi [197]. Dále bylo ukázáno, že primární ložiska nádorů jsou schopna prostřednictvím VEGF-1
35
specificky indukovat MMP9 u před-metastatických plicních endoteliálních buněk a makrofágů, čímž podporují vznik plicních metastáz [198]. VEGF mRNA mohou produkovat buňky v reakci na hypoxii a zánět ale také nádorové buňky. To hraje významnou roli v rozvoji neovaskularizace jak u fyziologických, tak u patologických procesů. Cirkulující faktory angiogeneze, jako jsou βFGF, VEGF, růstového faktoru hepatocytů (HGF) a angiogeninu mohou být detekovány u různých druhů nádorových onemocnění a mohou být použity pro stanovení rizik metastazování a také pro včasné odhalení nádoru. U pacientů s prokázanou malignitou byly tyto růstové faktory ukázány jako užitečné pro určení prognózy, předpovědi reakce na léčbu a sledování jejího průběhu [199]. Vysoké hladiny VEGF byly nalezeny v korelaci s pokročilejšími stádii nebo horší prognózou u nádorů močového měchýře, mozku, prsu, tlustého střeva, plic, vaječníků, adenokarcinomu ledviny, spinocelulárního karcinomu v oblasti krku a neuroblastomu [200-208]. Úloha VEGF u hematologických malignit byla nedávno studována u periferního lymfomu T buněk, Hodgkinovy nemoci, mnohočetný myelomu a akutní myeloidní leukémie [209-211].
Thrombospondin Thrombospondin (TSP) je trimerní glykoprotein syntetizovaný a vylučovaný jak zdravými, tak maligními buňkami. Váže se na heparin, fibronektin, vitronektin a kolagen. Byly identifikovány tři TSP: TSP1, TSP2 a TSP3. TSP je jedním z inhibitorů angiogeneze. Produkce TSP1 je regulována proteinem p53. Mutace nebo ztráta z proteinu p53 je spojena se ztrátou produkce TSP1 a posunem rovnováhy směrem k stimulaci abiogeneze v melanomu, a karcinomu prsu a prostaty [212,213]. Snížená exprese TSP1 koreluje se špatnou prognózou u pacientů s různými nádorovými onemocněními, jako jsou malobuněčný plicní karcinom, melanom, karcinom slinivky břišní, děložního hrdla, prsu, močového měchýře, tlustého střeva a prostaty [213-220]. Chemokiny Studium procesů spojených s migrací leukocytů v organizmu ukázalo, že existuje celá skupina látek, které se uplatňují jako specializované chemoatraktanty. Byl pro ně zvolen název chemokiny. Do dnešní doby jich bylo u člověka identifikováno přibližně padesát [221]. Chemokiny mají velmi podobnou strukturu. Jsou to malé proteiny
36
s charakteristickou skupinou čtyř cysteinů. Cysteinové repetice jsou klíčem pro rozdělení chemokinů tří podrodin a to i) C-X-C (např. PBSF/SDF-1), jsou přítomny dva cysteiny, mezi něž je vmezeřena další aminokyselina α ( -chemokiny), ii) C-C (např. RANTES, MCP-1), první dva cysteiny přímo sousedí β ( -chemokiny), iii) C, cysteiny jsou obklopovány
jinými
aminokyselinami
bez
opakujícího
se
strukturního
motivu
(γ-chemokiny) [222]. Funkce chemokinů je spjata s vazbou na specifické receptory spojené s G proteiny. Byly naklonovány a charakterizovány čtyři CXC chemokinové receptory (CXCR-1-4) a pět CC chemokinových receptorů (CCR-1-5) [223]. Vazba chemokinů spouští G proteiny aktivující signalizační kaskádu v buňkách, ovlivňuje transport a umístění leukocytů. Konkrétní kombinace chemokinů, chemokinových receptorů a adhezních molekul tvoří prostorový a časový kód, který určuje, které podskupiny leukocytů mohou migrovat a jaký je jejich cíl [5]. Kromě své role při zánětu a infekci ovlivňují chemokiny další procesy, například hematopoézu, angiogeneze a růst nádoru. CXCR4 má rozhodující roli při metastazování nádoru prsu, prostaty a karcinomu tlustého střeva [224-226]. Systém SDF1 ligand/CXCR4 receptor hraje také roli v růstu nádoru prostřednictvím stimulace buněčné migrace u karcinomu pankreatu a ovaria [227,228]. Exprese RANTES a CCR7 koreluje s progresí onemocnění a metastazováním v případě karcinomu žaludku, plic a prsu [229-231]. Dále bylo ukázáno, že lze expresi RANTES nádorovými buňkami považovat za vhodný prediktivní marker karcinomu plic [232]. Další CC chemokin, MCP-1, je produkován nádorovými buňkami prsu a zvýšení jeho hladiny je významným ukazatelem špatné prognózy a předčasného relapsu onemocnění [233]. Hladina tohoto chemokinu v moči koreluje s rozvojem karcinomu močového měchýře [234]. Zatímco jeho sérová hladina klesá v souladu s progresí onemocnění karcinomu žaludku [235]. Oxidu dusnatý Oxid dusnatý (NO) představuje významnou signální molekulu. Podílí se na funkci nervového, kardiovaskulárního a imunitního systému. V kardiovaskulárním systému se uplatňuje při relaxaci hladkého svalstva, čímž ovlivňuje krevní tlak. V rámci imunitního systému je produkován aktivovanými makrofágy a spolu s ROS se podílí na zabíjení patogenů. NO je také produkován v mnoha částech centrálního nervového systému, kde může ovlivňovat jednak průtok krve lokálními cévami a také aktivitu samotných neuronů
37
[236]. V roce 1992 byl NO časopisem Science vyhlášen molekulou roku [237]. NO a jeho deriváty mohou přímo způsobit poškození DNA a mutace in vitro, a proto mohou hrát roli procesu karcinogeneze [238]. Bylo zjištěno, že NO a jeho deriváty přispívají k patogenezi několika druhů nádorových onemocnění včetně kracinomu mozku, prsu, močového měchýře, žaludku, slinivky, plic, tlustého střeva, prostaty a štítné žlázy a mnohočetného myelomu [5]. NO syntézy (NOS) tvoří rodinu enzymů zodpovědných za tvorbu NO z aminokyseliny L-arginin. Byly identifikovány geny pro tři izoformy NOS. Dvě z těchto izoforem, endoteliální NOS (typ III) a neuronální NOS (typ I), jsou závislé na vápníku a jsou konstitutivní. Cytokiny indukovatelná NOS (iNOS, typ II) je na vápníku nezávislá. Na druhou stranu je také známo, že NOS typu I a III mohou být indukovány a že nízké hladiny NOS typu II jsou v některých tkáních tvořeny konstitutivně. U některých typů nádorových onemocnění byla pozorována zvýšená exprese iNOS [5]. 2.5.10 Nádorové markery invazivity a metastazování Adhezní molekuly a nádorová invazivita Intercelulární adhezní molekuly hrají významnou roli v procesu invaze nádoru a rozvoje metastáz. Byly popsány čtyři hlavní třídy adhezních molekul. Selektiny, první třída adhezních molekul a zástupce rodiny lektinů, jsou transmurální proteiny zahrnuté v rané fázi adheze. Jsou rozděleny do tří skupin E, P a L, přičemž jejich označení souvisí s typem buněk (endotelové buňky, krevní destičky a leukocyty). Selektiny umožňují vytvořit slabé, nízko-afinitní vazby mezi endoteliálními buňkami a leukocyty. To vede k aktivaci leukocytových integrinů, které se váží ke členům rodiny imunoglobulinů, což je předpokladem pro řízenou migraci leukocytů na povrchu endoteliálních buněk. Zástupcem selektinů je galektin-3, u kterého byla pozorována zvýšená hladina u karcinomu štítné žlázy, jater, žaludku, a centrálního nervového systému [239-242]. Na druhou stranu byly publikovány práce, které ukazují na sníženou hladinu galektinu-3 u karcinomu vaječníků, dělohy a prsu [243-245]. Druhou třídu představují integriny, transmembránové proteiny skládající se ze dvou podjednotek α a β. Narušení tvorby integrinů může být zodpovědné za řadu aberantních buněčných aktivit v průběhu maligní transformace, růstu nádoru a progrese, metastatického šíření a apoptózy [5]. Hashida et al. pozorovali, že nízká exprese integrinu α3 je ukazatelem
38
špatné prognózy u pacientů s karcinomem tlustého střeva [246]. Integrin αVβ3 je významným prognostickým markerem relapsu u pacientů trpících karcinomem prsu [247]. Do třetí třídy jsou zařazeny imunoglobulinu podobné membránové proteiny, které jsou uspořádány v podobě skládaných listů. Jsou známy jako intercelulární adhezní molekuly a zahrnují intercelulární adhezní molekulu-1 (ICAM-1) a buněčnou adhezní molekulu-1 (VCAM-1). Cirkulující forma ICAM-1 o molekulové hmotnosti 82 kDa byla detekována u karcinomu prsu, žaludku, melanomu a Hodgkinovy choroby [139,248-250]. Selektin E, ICAM-1 a VCAM-1 mohou hrát roli v procesu adheze nádorové buňky k cévnímu endotelu, který předchází extravazaci buněk a rozvoji metastáz. Čtvrtou třídu tvoří kadheriny, které jsou mediátory interakcí mezi buňkami. Snížená exprese kadherinu E je spojena se špatnou prognózou a metastazováním u karcinomu plic, prostaty, prsu, močového měchýře, žaludku a tlustého střeva [251-256]. Kromě výše zmíněných byly identifikovány další adhezní molekuly včetně rodiny sialomucinů, CD44 a mesothelin. Bylo pozorováno, že je zvýšená exprese CD44 je spojena s progresí nádoru a jeho metastazováním u většiny nádorových onemocnění [257]. Výjimku tvoří karcinom prostaty a neuroblastom, kde je detekována nízká nebo žádná exprese CD44 [258-260]. Nicméně, úloha CD44 jako tumorového supresoru byla také diskutována, protože inhibice tohoto proteinu je spojena se zvýšením malignity a metastatického potenciálu u buněk karcinomu prostaty [258]. Kromě CD44, exprese mesothelinu se zvyšuje u dlaždicového karcinomu jícnu, plic, děložního hrdla a ovariálního karcinomu, přičemž předběžné studie prokázaly, že může být tento protein slibný cíl pro imunoterapii [261,262]. Boucharaba et al. pozorovali, že lysofosfatidová kyselina (LPA) hraje roli ve vývoji kostních metastáz pocházejících z karcinomu prsu [263]. Dále ukázali, že receptory LPA (LPA1, LPA2 a LPA3) jsou produkovány buňkami karcinomu prsu. Tyto buňky vyvolají uvolnění LPA z aktivovaných krevních destiček, což podporuje proliferaci nádorových buněk a sekreci IL-6 a IL-8 jako silných stimulátorů kostní resorpce. Markery metastazování S100A4. Rodina proteinů nesoucí označení S100 se ukázala být významnou skupinou látek podporujících invazivitu a metastazování řady lidských nádorů. Vápník vázající proteiny tvoří velkou skupinu, která se podílí na řadě funkcí, od kontroly buněčného cyklu a vývoje, přes diferenciaci buněk, až po aktivaci enzymů a regulaci
39
svalové kontrakce [264]. Jeden z významných zástupců této skupiny nese název S100A4. Je to protein skládající se ze 101 aminokyselin [265]. Přítomnost S100A4 byla detekována pouze u buněk vaječníků a prostaty, přičemž se nevyskytuje v tkáních prsu, tlustého střeva, štítné žlázy, plic, ledvin a slinivky břišní [266]. Několik studií potvrzuje roli S100A4 u invazivního růstu a metastazování různých druhů nádorových onemocnění [267]. Několik klinických studií prokázalo souvislost exprese anebo sérové hladiny S100A4 s agresivitou nebo metastazováním u pacientů s karcinomem močového měchýře, prostaty a plic [268-271]. Markery metastáz kostí. Kost je jedním z nejčastějších míst metastáz, zejména u karcinomu prostaty, prsu a plic. Detekce metastazování je důležitá, protože je určujícím faktorem pro rozhodnutí, zdali bude nejvhodnější chirurgický zákrok, radioterapie či chemoterapie. Diagnóza kostních metastáz obvykle se spoléhá na rentgen a kostní scintigrafii. Nicméně, kostní metastázy např. u primárních ložisek, jako jsou ledviny, melanom a mnohočetný myelom, nemusí být pomocí zmíněných technik detekovány [5]. Další možností jak detekovat kostní metastázy je prostřednictvím vhodných markerů, které souvisejí s tvorbou nové kosti anebo degradací a resopcí kosti stávající. Mezi intenzivně studované markery patří alkalická fosfatáza [272,273], osteokalcin [274,275], katepsin K [276], N-terminální propeptid prokolagenu typu 1 [277-282], C – Telopeptid kolagenu typu I [283-285] a další.
2.5.11 Souhrn V Tab. 1 je zobrazen souhrn více než šedesáti nádorových markerů, kde jsou sumarizovány typy nádorových onemocnění, u kterých je možné látky použít. Naneštěstí je ověřena a tím pádem používána v běžné klinické praxi jen malá část studovaných molekul, kam patří CEA, AFP, hCG, PSA, CA 15-3, CA 19-9, CA 125, S100A4, NSE, M2-PK, CYFRA 21-1, B2M, Kalcitonin [286]. Výzkum markerů nádorových onemocnění je dynamicky se rozvíjející se oblast, přičemž se hledají a ověřují stále nové a nové skupiny látek i jednotlivé peptidy, proteiny popř. nukleové kyseliny. Do této obrovské skupiny látek patří i proteiny s názvem metalothioneiny.
40
Tabulka 1 (1. část). Přehled nádorových markerů a onemocnění, u kterých mohou být využity. • • • • • • • • • • • • • • •
Marker Nádorové antigeny Karcinoembryonální antigen (CEA) Alfa-fetoprotein (AFP) Beta a gamma-fetoprotein CA 15-3 CA 125 CA19-9 Specifický membránový antigen prostaty (PSMA) Antigen kmenových buněk prostaty (PSCA) Antigen karcinomu močového měchýře (BTA) Beta-2-mikroglobulin (β2M) Rodina lidských kalikreinů Kalcitonin (CT) Lidský choriový gonadotropin (HCG) Gastrin-vylučující peptid (ProGRP) Adrenokortikotropní hormon (ACTH) Antidiuretický hormon (ADH)
Druh onemocnění
adenokarcinom tlustého střeva, karcinom prsu, plic, slinivky, žaludku a vaječníků karcinom jater, tumory zažívacího a urogenitálního systému karcinom prsu epiteliální karcinom ovarií karcinom tlustého střeva, žaludku, slinivky karcinom prostaty karcinom prostaty karcinom močového měchýře mnohočetný myelom a lymfom rakovina prostaty a prsu karcinom štítné žlázy gestační trofoblastická nemoc a zárodečné buňky nádorů malobuněčný karcinom plic, adenokarcinom, spinocelulární karcinom plic, prsu, tlustého střeva a pohlavních žláz, feochromocytom, tymom, medulárním karcinom štítné žlázy karcinom pankreatu, kůry nadledvin, močového měchýře, prostaty, bronchiální karcinoidní nádory,
Enzymy •
YKL-40
•
Kyselá prostatická fosfatáza (PAP)
•
Neuron-specifická enoláza (NSE)
• • •
Galaktosyl transferáza Katepsin Matrixové metaloproteinázy (MMP) Prostasin Cytokeratiny
•
• • • •
Cytokiny a cytokinové receptory HER2 Interleukin 6(IL6) Receptor interleukinu 2 (IL2R) Receptor epidermálního růstového faktoru (EGFR)
kolorektální karcinom, metastatický karcinom prsu, primární a recidivující ovariální karcinom karcinomu prostaty, mnohočetným myelom, osteogenní sarkom, kostní metastázy neuroblastom a malobuněčný plicní karcinom karcinom tlustého střeva, nádory pohlavních žláz kolorektální karcinom, hematom karcinom žaludku, tlustého střeva, hrudníku, hlavy a krku, prostaty, nádor plic, buňky karcinomu prostaty karcinom močového měchýře, prsu, plic a tlustého střeva karcinom prsu, prostaty, vaječníků a plic karcinom prostaty adenokarcinom ledvin, ne-Hodgkinův lymfom, melanom, karcinom žaludku, slinivky břišní, hlavy a krku, a malobuněčný plicní karcinom karcinom prsu, vaječníku, plic, tlustého střeva, hlavy a krku
41
Tabulka 1 (2. část). Přehled nádorových markerů a onemocnění, u kterých mohou být využity. • • • •
Marker Signální molekuly hnRNP B1 Ras Nádorové markery růstu nádorů Jaderné matrixové proteiny (NMP) Proteiny teplotního šoku (HSP)
•
Mikrosatelity
•
DNA ploidie
•
Stav buněk v S fázi
•
Nádorové markery a angiogeneze Vaskulární endoteliální růstový faktor (VEGF)
•
Thrombospondin (TSP)
•
Chemokiny
•
Nádorové markery invazivity Adhezní molekuly • • •
Integriny imunoglobulinu podobné membránové proteiny kadheriny
•
CD44
• LPA1, LPA2 a LPA3 Nádorové markery metastazování • S100
Druh onemocnění
karcinom tlustého střeva karcinom plic karcinom močového měchýře nádor děložního čípku, děložní sliznice a močového měchýře, časný hepatocelulární karcinom z prekancerózních lézí karcinom močového měchýře, plic, spinocelulární a kolorektální karcinom karcinom prsu, tlustého střeva, nosohltanu a děložního čípku primární lymfom žaludku, prsu, tlustého střeva, nosohltanu a hrtanu karcinom močového měchýře, mozku, prsu, tlustého střeva, plic, vaječníků, adenokarcinomu ledviny, spinocelulární karcinom v oblasti krku a neuroblastom malobuněčný plicní karcinom, melanom, karcinom slinivky břišní, děložního hrdla, prsu, močového měchýře, tlustého střeva a prostaty metastáze nádoru prsu, prostaty a karcinomu tlustého střeva, močového měchýře karcinom štítné žlázy, jater, žaludku, a centrálního nervového systému karcinom tlustého střeva, prsu karcinom prsu, žaludku, melanom, Hodgkinova choroba metastáze karcinomu plic, prostaty, prsu, močového měchýře, žaludku a tlustého střeva většina nádorových onemocnění, kromě karcinomu prostaty a neuroblastomu karcinom prsu karcinom močového měchýře, prostaty a plic
Metalothioneiny Metalothioneiny
(MT,
Obr.
2)
jsou
skupinou
jednořetězcových
protein
o molekulové hmotnosti od 2 do 13 kDa (u savců okolo 6,5 kDa). U savců byly identifikovány čtyři hlavní izoformy MT-1 až MT-4. Kromě toho bylo u člověka popsáno nejméně dalších třináct proteinů úzce souvisejících s [287,288]. Konkrétní funkční role
42
jednotlivých izoforem a jejich molekulární interakce jsou stále předmětem intenzivního výzkumu [289].
Obrázek 2. Struktura lidského metalothioneinu obsahujícího sedm atomů zinečnatých iontů (zeleně). Převzato z www.expasy.org.
MT tvoří rodinu proteinů s velkým stupněm homologie v primární struktuře, které byly popsány u bakterií, hub, rostlin a živočišných druhů [290]. Jsou to proteiny bohaté na cystein se zajímavou strukturní vlastností a tou je nepřítomnost aromatických aminokyselin. Obecně se MT skládají ze dvou vazebných doménα a β, přičemž každá doména obsahuje 20 zbytků cysteinu střídaných lysinem a argininem. Na sulfhydrylové skupiny cysteinů je možno navázat až 7 molů iontů kovů nesoucích náboj 2+ na jeden mol MT-1 a MT-2, přičemž molární poměr iontů kovů nesoucích náboj 1+ je dvanáct ku jedné. Přestože molekula metalothioneinu přirozeně obsahuje Zn2+ v obou vazebných doménách, mohou být tyto ionty nahrazeny jinými, která mají vyšší afinitu sulfhydrylovým skupinám a to Pb, Cu, Cd, Hg, Ag, Fe, Pt a Pd [287,291-297].
43
MT se nacházejí v cytoplazmě, lysozomech, mitochondriích a buněčných jádrech. MT-1 a MT-2 jsou všudypřítomné, zejména jsou obsaženy v játrech, slinivce, střevech a ledvinách, zatímco MT-3 se nachází pouze mozku a MT-4 v kůži [298]. MT-3 a MT-4 jsou produkovány konstitutivně, zatímco MT-1 a MT-2 jsou navíc vysoce inducibilní. Co se týče buněčného cyklu, byla nejvyšší koncentrace MT zjištěna v pozdní G1 a G1/S fázi [299]. Kromě intracelulárního prostoru bylo prokázáno, že jsou buňky in vitro schopny aktivně vylučovat MT-1 a MT-2, ačkoli mechanismus nebyl dostatečně prozkoumán [300]. Vysoká míra MT syntézy byla zjištěna v rychle proliferující tkáni [301]. MT se intenzivně účastní intracelulárního metabolismu a detoxikace iontů kovů [302], a také ochrany proti reaktivním kyslíkovým radikálům [303]. Ochranu proti toxickým účinkům iontů kovů zajišťují především MT-1 a MT-2, i když MT-3 hraje důležitou roli v homeostáze Zn u neuronů [269,304-307]. Mezi další významné biologické role MT patří jejich schopnost regulovat hladinu, činnost a buněčnou lokalizaci na transkripčního faktoru NF-κB [308-311]. Schéma možného vztahu metalothioneinu a transkripčního faktoru NF-κB je uk ázáno na
Obr. 3. Kovů zbavená molekula
metalothioneinu je schopna interakce s proteinem p53 [308,312,313].
Obrázek 3. Možný vztah metalothioneinu a transkripčního faktoru NF-κB. (a) Transkripční faktor NF-κB je aktivován po předání signálu IKK kaskádou. (b) Aktivovaný transkripční faktor přechází do jádra, kde může reagovat s molekulou MT nesoucí zinek. (c) Zinek je předán transkripčnímu faktoru NF-κB a ten seáže v na regul ační sekvence DNA. (d) Hladina zinku je regulována transkripčními faktory MTF-1, (e) který se váže na MRE na DNA.
44
3.
Elektrochemické metody Vysoce citlivé, rychlé a selektivní metody detekce anorganických i organických
látek ve složité biologické matrici jsou metody elektrochemické a elektroanalytické [314,315]. Podstatou elektrochemických metod je studium závislosti elektrochemického chování roztoků na jejich složení a koncentraci. Objektem zkoumání je elektrochemický článek – soustava, v níž je analyzovaný roztok v kontaktu s elektrodami. Elektrody zprostředkují jeho spojení s měřícím přístrojem, který sleduje některé z elektrických veličin (proud I, potenciál E, vodivost G, elektrický náboj Q, kapacitu C, aj.) [314-317]. Voltametrie je elektrochemická metoda založená na sledování intenzity proudu na velikosti proměnlivého vloženého napětí vkládaného mezi pracovní (polarizovatelnou) a referentní (nepolarizovatelnou) elektrodu – dvouelektrodové zapojení. Z důvodu eliminace rušivé vlivy plynoucích z dvouelektrodového zapojení (např. proudového zatížení referentní elektrody) se v dnešní době používá kromě pracovní a referentní i elektroda pomocná – tříelektrodové zapojení. Na pracovní elektrodě probíhá elektrodový proces, který sledujeme. Potenciál pracovní elektrody je kontrolovaný vůči elektrodě referentní s konstantním potenciálem. Vlastní referentní elektroda je od měřeného roztoku oddělena solným můstkem se zatavenou hustou skleněnou fritou. Proud protéká elektrodami pomocnou a pracovní. Metody, kde se stále využívá rtuťové kapkové elektrody, se nazývají polarografické [315,317]. Roztokem neprochází proud, pokud zkoumaný vzorek neobsahuje elektroaktivní látku, tzv. depolarizátor, který se v daném potenciálovém rozsahu oxiduje nebo redukuje. Přítomnost depolarizátoru se při určitém potenciálu projeví zvýšením proudu. Elektrodové děje (oxidace anebo redukce) způsobují změny jen ve velmi těsné blízkosti povrchu polarizovatelné elektrody. Samotný elektrodový proces zahrnuje děje, které jsou spojeny s transportem elektroaktivní látky (analytu) k elektrodě, vlastním elektrodovým dějem a vylučováním produktu na elektrodě, případně jeho transportem od elektrody [315,316,318]. Transport elektroaktivní látky je k elektrodě zprostředkován třemi pochody a to difúzí, migrací a konvekcí. Pro snížení detekčního limitu a zvýšení citlivosti elektrochemických metod vyvinul profesor Emil Paleček adsorptivní přenosovou rozpouštěcí techniku (AdTS), která může bát spojena s kteroukoliv elektrochemickou metodou. Její výhoda spočívá především pro látky schopné se akumulovat na povrch visící rtuťové kapkové elektrody (HMDE), protože poté je elektroda opláchnuta (snížení množství interference) a takto modifikovaná elektroda je vložena do měřící nádobky
45
s vhodným elektrolytem [319]. Více podrobností o základech elektrochemických metod lze nalézt v následujících publikacích [315,317,320]. Přesně více než sto lety se narodil profesor Rudolf Brdička, který v roce 1937 publikoval v časopise Nature svůj objev využití polarografie při diagnostice nádorového onemocnění [321]. Objevil citlivý polarografický signál způsobený přítomností proteinů, který vykazovalo krevní sérum [321,322]. Jeho vznik profesor Brdička připisoval katalytické aktivitě sulfhydrylových skupin v přítomných proteinech. Pozorovaný polarografický signál (tvořený charakteristickou vlnou závislosti proudu na potenciálu) byl vždy vyšší v případě analýzy zdravého krevního séra než v séru pacienta s nádorovým onemocněním. Jeho kolega, držitel Nobelovy ceny za chemii – profesor Jaroslav Heyrovský, publikoval o rok později v témže časopise článek, kde shrnul výsledky v oblasti Polarographic Research on Cancer a zároveň zde vyslovil myšlenku, že je to pouze počátek široce se rozvíjející se výzkumné oblasti [323]. S postupem času se elektrochemie z nádorové diagnostiky spíše vytrácela vytlačována moderními metodami analytické chemie a molekulární biologie.
3.1
Brdičkova reakce Jak je zmíněno výše, profesor Brdička při elektrochemickém zkoumání kobaltitých
komplexů zjistil, že pokud je do elektrolytu přidáno krevní sérum, pozorujeme katalytické vlny. Dlouhou dobu bylo nejasné, co se skrývá za podstatou pozorovaných elektrochemických signálů. Dr. Biserka Raspor navrhla na základě několikaletého výzkumu této problematiky pravděpodobné schéma reakce [324]. Ve stručnosti lze shrnout, že chemické jevy popsané níže se zakládají na interakci chloridu hexaamminokobaltitého komplexu ([Co(NH3)6]Cl3) s proteinem obsahujícím –SH skupinu. Jako elektrolytu je využíváno amonného pufru (NH3 + NH4Cl) s vysokým pH. Při elektrochemické analýze kobaltitého komplexu bez přítomnosti látky obsahující –SH skupinu dochází k redukci CoIII na CoII za vzniku [Co(NH3)6]2+. Produkt redukce (hexaamminkobaltnatý ion) je dále hydrolyzován podle následující reakce: [Co(NH3)6]2+ + 6 H2O → [Co(H2O)6]2+ + 6 NH3 První redukce kobaltu z oxidačního čísla III na II vytvoří polarografickou vlnu v potenciálu přibližně Ep = - 0,3 V. Druhá redukce, teď už hexaaquakobaltnatého
46
komplexu na čistý kobalt probíhá při potenciálu (-1,2 V). Jako výsledek redukce [Co(NH3)6]Cl3 v amonném pufru tedy vzniknou dvě polarografické vlny v potenciálu přibližně Ep = - 0,3 V (CoIII → CoII) a Ep = - 1,2 V (CoII → Co0). Pokud by ale byla přidána látka obsahující sulfhydrylové skupiny, reakce by po první redukci probíhala jinak. Na výsledném voltamogramu by místo jednoho elektrochemického signálu byly pozorovány signály čtyři. První signál je opět signál redukce kobaltitého komplexu na kobaltnatý. Druhý signál je signál redukce stabilního komplexu RS2Co vzniklého vytěsněním vody z aquakomplexu podle rovnice: [Co(H2O)6]2+ + R(SH)2 → RS2Co + 6 H2O + 2 H+ Průběh redukce by se dal popsat sumární rovnicí: RS2Co + 2 e- → Co0 + R(S-)2 Při vyšších koncentracích thiolových chelátů může být pozorován i pík Co1, který odpovídá redukci [Co(H2O)6]2+, a který se nalézá v kladnějším potenciálu než pík redukce RS2Co. Vodíkové ionty, vzniklé záměnou ligandů vody za sulfhydrylové skupiny, jsou absorbovány molekulami amoniaku za vzniku amonných iontů. Po redukci CoII na Co0 je skupina R(S-)2 okamžitě protonována NH4+ skupinou a sloučenina je obnovena a je schopna vázat další hexaquakobaltnaté ionty. Poslední dva signály pozorovatelné ve voltamogramu (Cat1 - Ep = - 1,35 V a Cat2 - Ep = - 1,48 V) s přídavkem sloučeniny obsahující –SH skupinu jsou katalytické povahy; Cat2 je pak zřejmě výsledkem redukce H+ iontů vzniklých z reakce mezi R(SH)2 a [Co(H2O)6]2+. Jedná se o katalytický jev, protože po zvýšení teploty byl pozorován úbytek signálu, což nasvědčuje tomu, že je závislý na povrchové reakci. Reakce R(SH)2 s [Co(H2O)6]2+ totiž již probíhá na povrchu elektrody a R(SH)2 je tudíž katalyzátorem vývoje vodíku z elektrolytu. Jelikož je celý proces řízen fyzikálně-chemickými aparáty, je nutno dodržovat stálé podmínky měření (pH elektrolytu, teplota, koncentrace amoniaku atd.), aby bylo možno dosáhnout dobré opakovatelnosti a reprodukovatelnosti měření [324-326]. Pánové Olafson a Sim zjistili, že analýza tepelně-denaturovaného vzorku pomocí Brdičkovy reakce je velmi vhodná pro detekci MT. Výška posledního signálu na voltamogramu Brdičkovy reakce s reálným vzorkem je závislá na koncentraci
47
metalothioneinu [327,328]. Elektrochemická detekce je využívána především v analýzách používajících metalothioneinu jako markeru znečištění životního prostředí [329-332]. Elektrochemické metody lze využít pro stanovení MT i u pacientů se zraněním mozku [333] nebo s nádorovým onemocněním [334-336]. 3.2
H-pík Při této metodě je opět je sledována evoluce vodíku katalyzovaná přítomností
proteinu. Charakteristikou této techniky je schopnost detekovat protein v dokonce subnanomolárním množství [337]. V základním elektrolytu ale není nutná přítomnost komplexu těžkého kovu (na rozdíl od Brdičkovy reakce) a z našich výsledků vyplývá, že pro analýzu je optimální 0.1 M H3BO3 + 0.05 M Na2B4O7 borátový pufr o pH = 8.0 [338], přičemž pH hraje vliv na výšku i polohu signálu a odezva kyseliny borité je zřejmě způsobena schopností (reakcí s vodou) sloužit jako donor protonů [339]. Vlivy pH je možno přisoudit pohyblivosti proteinu vzhledem k jeho pI. Katalytický proces při analýze MT je ovlivněn i obsahem kyslíku v elektrolytu; vyšší koncentrace je příznivější [339]. Signál je měřen pomocí chronopotenciometrie, tedy jako závislost derivace potenciálu na derivaci času dt/dE. První práce zaměřené na využití píku H v analýze proteinů byly publikovány před dvaceti lety [340,341]. Na katalytickém signálu se podílí velkou měrou – SH, ale i –NH2 skupiny a celý mechanismus reakce není zatím uspokojivě vysvětlen. Výsledkem analýzy MT je signál v potenciálové oblasti E = -1.7 V [342]. Metoda je velmi senzitivní, s jejím použitím byly detekovány femtomolární množství MT v malých objemech (5 µl) [343]. Nedávno bylo dokázáno, že lze touto metodou odlišit nativní a denaturovano formu proteinu [344,345]. Průběh reakce a tím i citlivost stanovení je závislý na mnoha parametrech, především pH a iontová síla elektrolytu, dále pak izoelektrický bod stanovovaného proteinu, v menší míře je signál ovlivněn také teplotou [339,342,343]. Bylo také zjištěno, že přídavek [Co(NH3)6]Cl3 do základního elektrolytu může zvýšit citlivost až o 30 % [341], nárůst výšky signálu je zřejmě způsoben formací komplexu sůl-protein. Metoda AdTS CPSA byl použita pro detekci MT v kvasince Yarrowia lipolytica vystavené působení Zn, Ni, Co a Cd [338] a v rybích parasitech Acanthocephalus anguillae [346].
48
III. CÍLE PRÁCE Předložená práce je orientována na možnosti využití elektrochemických technik v nádorové diagnostice. Jejím cílem je ukázat, že aplikace moderních elektrochemických metod při řešení určitých problémů v biologickém a biochemickém výzkumu může být velmi výhodná. Pozornost je zaměřena především na protein metalothionein a jeho roli jako potencionálního markeru v nádorové diagnostice. Pro tuto práci byly zvoleny následující dílčí cíle:
Připravit literární rešerši o metalothioneinu, jeho biologickém významu, předpokládaných úlohách v kancerogenezi a jeho možné detekci
Navrhnout nové a originální metodiky pro snadnou a spolehlivou detekci metalothioneinu jako potencionálního nádorového markeru
Ověřit hypotézu metalothioneinu jako nádorového markeru pomocí navržených technik na vhodných biologických vzorcích
Studovat
možnou
interakci
metaloproteinázami
49
metalothioneinu
s matrixovými
IV. EXPERIMENTÁLNÍ ČÁST Výsledková část předkládané dizertační práce je přiložena ve formě publikací v časopisech.
A.
Literární
rešerše
o vztahu
metalothioneinu
a nádorových
onemocnění Eckschlager, T., Adam, V., Hrabeta, J., Figova, K. and Kizek, R. (2009) Metallothioneins and cancer. Curr. Protein Pept. Sci., 10, 360-375. Impact Factor 3.011
B.
Literární rešerše o možnostech detekce metalothioneinu
Adam, V., Fabrik, I., Eckschlager, T., Stiborova, M., Trnkova, L. and Kizek, R. (2009) Metallothioneins as Target Molecules for Analytical Techniques. TRAC-Trends Anal. Chem., odesláno po revizi. Impact Factor 5.485
C.
Vliv kovů přirozeně obsažených ve struktuře metalothioneinu na jeho stanovení
Adam, V., Krizkova, S., Zitka, O., Trnkova, L., Petrlova, J., Beklova, M. and Kizek, R. (2007) A determination of apo-metallothionein using adsorptive transfer stripping technique in connection with differential pulse voltammetry. Electroanalysis, 19, 339-347. Impact Factor 2.949
D.
Elektrochemické stanovení metalothioneinu v krevních vzorcích pacientů s nádorem prsu
Adam, V., Baloun, J., Fabrik, I., Trnkova, L. and Kizek, R. (2008) An electrochemical detection of metallothioneins at the zeptomole level in nanolitre volumes. Sensors, 8, 2293-2305. Impact Factor 1.870
50
E.
Vliv cisplatiny na hladinu metalothioneinu u buněčných linií, laboratorních krys a pacientů s nádorem v oblasti hlavu a krku
Fabrik, I., Krizkova, S., Huska, D., Adam, V., Hubalek, J., Trnkova, L., Eckschlager, T., Kukacka, J., Prusa, R. and Kizek, R. (2008) Employment of electrochemical techniques for metallothionein determination in tumour cell lines and patients with a tumor disease. Electroanalysis, 20, 1521-1532. Impact Factor 2.901
F.
Studium interakce želatinázy B s kolagenem
Huska, D., Adam, V., Zitka, O., Kukacka, J., Prusa, R. and Kizek, R. (2009) Chronopotentiometric stripping analysis of gelatinase B, collagen and their interaction. Electroanalysis, 21, 536-541. Impact Factor 2.901
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A. Literární rešerše o vztahu metalothioneinu a nádorových onemocnění
52
Current Protein and Peptide Science, 2009, 10, 000-000
1
Metallothioneins and Cancer Tomas Eckschlager1, Vojtech Adam2, Jan Hrabeta1, Katarina Figova1 and Rene Kizek2,* 1
Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University, V Uvalu 84, CZ150 06 Prague 5, Czech Republic; 2Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic Abstract: Metallothioneins (MTs) are low molecular, cysteine-rich proteins that have naturally-occurring Zn2+ in both clusters. They may serve as a reservoir of metals for synthesis of apoenzymes and zinc-finger transcription regulators. MTs are also involved with several important proteins e.g. p53, NF-B, PKCl, and GTPase Rab3A. New biological roles for these proteins have been identified including those needed in the carcinogenic process. However, their use as a predictive marker remains controversial. Several reports have disclosed MTs expression as a prognostic factor for tumor progression and drug resistance in a variety of malignancies particularly breast, prostatic, ovarial, head and neck, non-small cell lung cancer, melanoma, and soft tissue sarcoma. The role of MTs as a tumor disease marker or as a cause of resistance in cancer treatment is reviewed and discussed. Moreover, we describe some analytical methods that were developed to detect MTs.
Keywords: Metallothioneins, cancer marker, cancer cell chemoresistance, detection. 1. INTRODUCTION Metallothioneins (MTs) are a group of low molecular weight (about 6.5 kDa) single-chain proteins. Four major isoforms (MT-1 through MT-4) have been identified in mammals. In addition at least thirteen known closely related MT proteins in humans have been described [1,2]. MTs genes are tightly linked, and at a minimum they consist of eleven MT-1 genes (MT-1A, -B, -E, -F, -G, -H, -I, -J, -K, -L, and -X) encoding functional or non-functional RNAs, and one gene for each of the other MTs isoforms (the MT-2 A gene, MT-3 gene, and MT-4 gene). The nomenclature for MTs isoforms has not been standardized until now [3]. A gene called MT-like 5 (MTL-5) that encodes a testis-specific MT-like protein called tesmin has been described in the q13 region of chromosome 11 [4]. Some biochemical properties of human MTs and tesmin are shown in Table 1. The specific functional roles of MTs isoforms and their molecular interactions are still unclear [5]. MTs are a family of proteins with a large degree of sequence homology, which have been described in bacteria, fungi, plants, and animal species [6]. They are found in cytoplasm, lysosomes, mitochondria and nuclei of cells. MT-1 and 2 have ubiquitous tissue distribution particularly in liver, pancreas, intestine, and kidney, whereas MT-3 is found in brain and MT-4 in skin [7]. MT-3 and -4 are constitutively expressed, whereas MT-1 and –2 are both constitutive and highly inducible. MTs function in intracellular metal metabolism and detoxification, and also protection against oxidants. Protection against metal toxicity is ensured mainly by MT-1 and MT-2, although MT-3 plays a role in Zn homeostasis in neurons [7,8]. The current accepted scheme for the
regulation of levels of heavy metal ions in a cell by metallothionein is shown in Fig. (1). There are no conclusive data on the functional significance of their distribution in different cellular compartments. The highest cytoplasmic concentration was found in the late G1 and G1/S cell cycle phase [9]. Depending on the cell cycle phase, cell differentiation or in the case of toxicity, MT-1 and MT-2 are rapidly translocated to the nucleus, as seen in oxidative stress and during early S-phase [3,10]. In addition, cells have been shown to actively secrete MT-1 and MT-2 in vitro, although there had been no known peptide signal for cellular export until now [11]. High rates of MT synthesis have been detected in rapidly proliferating tissues. This observation suggests an important role in both normal and neoplastic cell growth [12]. 2. BIOLOGICAL ROLES OF MTS 2.1. Molecular Regulation of Metallothionein Synthesis The genes for MTs are clustered and they are located on chromosome 16q12-22 in humans and on chromosome 8 in mice [13]. Gene transcription is initiated when zinc ions associates with the metal-regulatory transcription factor-1 (MTF-1). MTF-1 is the only known mediator of metal responsiveness of MTs [3,14,15]. MTF-1 binds to metal responsive elements (MREs) that regulate MTs expression. MREs are located in the promoter regions of MT genes [14] and are present in multiple copies in the promoter/enhancer regions of almost all metal-inducible MTs [16,17]. Due to the property of MTs being metal-inducible, homeostasis of heavy metal levels is probably their most important biological function. 2.2. Structure of Metallothioneins
*Address correspondence to this author at the Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic; Tel: +420-54513-3350; Fax: +420-5-4521-2044; E-mail:
[email protected] 1389-2037/09 $55.00+.00
The metal (M) binding domain of MTs consists of 20 cysteine residues juxtaposed with basic amino acids (lysine and arginine) arranged in two thiol-rich sites. The cysteine © 2009 Bentham Science Publishers Ltd.
2 Current Protein and Peptide Science, 2009, Vol. 10, No. 4
Eckschlager et al.
Fig. (1). Metallothionein and heavy metal ions. A heavy metal ion enters through a cytoplasmic membrane of a cell via ionic channels or special transporters (a). After the entering the cytoplasm, the ion interacts with complex of metal-regulatory transcription factor-1 (MTF-1) and metal synthesis inhibitor (MTI) (b). The ion binds to MTI. Due to this MTF-1 is released and can bind to a regulatory sequence of DNA called metal responsive element (MRE) (c). Then, the gene responsible for synthesis of metallothioneins is transcribed. The synthesized mRNA molecule is translated into MT (d). MT binds to the heavy metal ion. The metal-MT complex can be transported to the kidney, where the metal is excreted (e), or bound to DNA regulatory proteins, which are metal dependent (f).
sulphydryl groups bind 7 moles of divalent metal ions per mol of MT-1 or MT-2, while the molar ratio for monovalent metal ions (Cu and Ag) is twelve (Fig. 2). Although the naturally occurring protein has Zn2+ in both binding sites, this ion may be substituted for another metal ion that has a higher affinity for thiolate (Pb, Cu, Cd, Hg, Ag, Fe, Pt and Pd) [1,18-26]. 2.3. Other Biological Roles of MTs When MTs bind to zinc and copper, they may serve as reservoirs of metals for synthesis of apoenzymes and zincfinger transcription regulators [27-29]. Zinc is a cofactor for approximately three hundred enzymes and therefore is essential for cell growth. Its cellular levels, which are important for protein, nucleic acid, carbohydrate, and lipid metabolism, are regulated by specific transport proteins of which there are two families, the ZnT family and ZIP family. Proteins of the ZnT family transport Zn out of cells and into intracellular compartments from the cytoplasm, whereas ZIP proteins transport zinc into the cytoplasm from either outside the cell or from intracellular compartments [30]. Copper is an element essential for all organisms as part of metalloenzymes that are important in electron transfer, oxygen transport and oxygenation reactions. Copper has three principal carriers, ceruloplasmin, albumin and transcuprein [31]. MTs sequester metal ions when the latter are present in excess [32]. MTs interact with the p50 subunits of NF-B, kinase domain of PKCl, and GTPase Rab3A, and they can modulate the biological activity of p53 by zinc exchange. MT-1 and MT-2 regulate the level, activity and cellular location of the transcription factor NF-B [33-36]. NF-B is necessary to ensure cells protection from the apoptotic cascade induced
by TNF and other stimuli through activation of antiapoptotic genes and protooncogenes such as Bcl-2, c-myc and TRAF1. In addition apo-MT-1 (metal-free form of MT-1) but not MT-1 (MT-1 with metal ion) forms a complex with p53 [35,37,38]. Activation of p53 is an important factor that increases metal-dependent expression of MREs [39]. 2.4. Metallothionein as a Scavenger of Reactive Oxygen Species MTs can also serve as “maintainers” of the redox pool (Fig. 3). A binding of “free” copper ions by MTs also diminishes the amount of this metal available to generate free radicals [40]. The metal binding sites have a low redox potential and are easily oxidized by intracellular oxidants. Biological disulfides such as glutathione disulfide (GSSG) oxidize MTs with a concomitant release of zinc, while glutathione (GSH) reduces the oxidized protein to thionein, which then binds to available zinc. The GSH/GSSG redox pair can be efficiently coupled with MTs. This coupling could provide a very effective means of modulating oxidation and reduction [41]. In [41] the authors reported on the release of Zn from MTs by intracellular oxidants such as GSSG. However, these authors used non-physiologically high ratios of GSSG to GSH (3 mM to 1.5 mM), while intracellular concentrations of GSSG are much lower that those of GSH. Therefore, this phenomenon is worthy of further study. Moreover, nuclear MTs can protect cells against UV and ionic radiation [42-44] as well as against some cytotoxic alkylating agents including chemotherapeutics [45-48]. Moreover, they modulate nitric oxide and can inhibit apoptosis [49]. MTs stabilise lysosomes and decrease apoptosis following oxidative stress by inhibition of Fenton-type reactions and by ensuing per-
Metallothioneins and Cancer
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3
Fig. (2). Metallothionein structure. Model of two binding sites of metallothionein according to [133].
Fig. (3). Metallothionein scavenging of reactive oxygen species. Presence of redox metals, such as Cu and Fe, in a cell can produce reactive oxygen species (ROS) leading to damaging of DNA and cell structures (a). The cell protects itself using various molecules as scavengers of the radicals. One of the most crucial cell pathways to scavenge the radicals is the glutathione redox complex [235]. However, free SH moieties of MT can be also involved in the scavenging of ROS (b). Moreover, free heavy metal ions lead to activation of MTF-1 and, thus, to synthesis of MT (c). MT can both bind metal ions and scavenge ROS (d).
oxidation of lysosomal membranes [18]. The mechanism involves an antioxidant response element (ARE) in the promotor region, ARE binding transcription factors, MTF-1, transcription factors of the basic zipper type (Fos and Fra-1), NF-E2-related factor 2, and the upstream stimulatory factor family (USF, a basic helix–loop–helix–leucine zipper protein), although it is probable that other as yet unidentified proteins are involved in these mechanisms [50,51]. MTs are also released to the extracellular matrix [52]. Extracellular MTs has been shown to have significant modulatory effects
on cellular specific, non-specific and humoral specific immunity. However, mechanisms causing this effect are still unclear [53-55]. 2.5. Induction of Metallothionein by Biologically Active Molecules MTs are usually expressed at low levels, but they are inducible. Acute intake of metals induces MT synthesis in tissues, as well as administration of certain hormones or cytokines such as glucocorticoids, interleukin-1 or interleukin-6,
4 Current Protein and Peptide Science, 2009, Vol. 10, No. 4
interferon , tumor necrosis factor , erythropoetin and vitamin D3 [56-61]. Heat-shock proteins, reactive oxygen species, or endotoxin also induce MT synthesis. MTs are strongly chemotactic for inflammatory cells such as T cells [55]. MT-1 is expressed in endothelial cells at the site of angiogenesis. The down-regulation of MT-1 in endothelial cells inhibited in vitro proliferation, migration, and human umbilical vein endothelial cells network formation, as well as experimental angiogenesis in vivo [62]. MTs are induced in human skeletal muscles by exercise. Because they are antioxidants, they may protect contracting muscle fibres against cellular stress and injury [63]. 2.6. Metallothionein and Tissue Protection The neuroprotective function of MT-1 and MT-2 appears to be important. MTs-knockout mice show significantly enhanced brain tissue destruction, neuronal cell death, and clinical symptoms after cortical injury when compared with wild-type controls [64,65]. MT-1 overexpression after brain injury stimulates the astroglial responses including the expression of anti-inflammatory cytokines, growth factors, neurotrophins and their receptors, such as IL-10, fibroblast growth factor (FGF), FGF-receptor (FGF-R), transforming growth factor (TGF-), TGF--receptor (TGF--R), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), neurotrophins (NT)-3–5, brain-derived neurotrophic factor (BDNF), and glial-derived neurotrophic factor (GDNF) reduced inflammatory responses of macrophages and lymphocytes including significantly decreased levels of proinflammatory cytokines, matrix metalloproteinases (MMPs), and ROS [66-69]. MT-1 and MT-2 levels are inversely related to the degree of brain tissue damage after trauma, ischemia, experimental autoimmune encephalomyelitis, and neurodegenerative diseases. In injured brain, MT-1 and MT-2 inhibit macrophages, T lymphocytes and their production of interleukins, TNF-, matrix metalloproteinases, and ROS. In addition, MTs enhance cell cycle progression, mitosis and cell survival, while neuronal apoptosis is inhibited [70]. A receptor was identified, which mediates MTs transport into neurons - megalin. MTs stimulate regeneration of axons. This regeneration is dependent on megalinmediated MT uptake [71]. In addition low intracellular levels of MT-3 detected by RT PCR, immunohistochemistry and Western blotting were found in temporal cortex of brains of patients suffering from Alzheimer´s disease. These observations support the conclusion that loss of MT’s protective effects lead to an exacerbation of pathogenic processes [72]. Administration or induction of MTs protects the mucosa against experimental stress-induced gastric ulcers in rats. One may speculate that MTs or MT inducers might be a new therapeutic agent for gastric ulcers [73]. Experiments with MT-null mice showed that MTs in the gastric mucosa might play an important role in the protection against H. pyloriinduced gastric ulceration [74]. In vivo experiments showed that MTs protected the liver from degenerative changes of liver injury induced by ethanol. Zinc is directly involved in the protection of liver from alcoholic injury. Zinc has been shown to antagonize the catalytic properties of the redox-active metals and inhibit HO
Eckschlager et al.
and O2 formation in different systems. However, MTs are critical for maintaining high levels of zinc in the liver [75]. 3. TECHNIQUES THIONEINS
TO
DETECT
METALLO-
To detect levels of MTs in various biological samples such as human body liquids, cells and tissues effective, sensitive and easy to use analytical instruments are needed. The techniques and methods used for this purpose can be divided to several groups. The first one is based on quantification of metal ions bound in MT molecules. Then, the quantified amounts of the metal ions are proportional to MT amounts. It has been shown that the affinity of MTs to various metal ions differs as follows Hg(II) > Ag(I) ~ Cu(I) > Cd(II) > Zn(II) [76]. A mechanism of interaction of MTs with metal ions is not understood yet, but the state of knowledge in this field is sufficient for using this phenomenon to efficiently quantify the level of MTs in various samples of interest. The simplest method from the first group is the Cd-hem method, which belongs to the so called saturation techniques [76-80]. The method is based on different affinities of MT to metals, for example, Cd displaces Zn. The Ag saturation assay for the measurement of MT in tissue was developed as a modification of the Cd saturation assay, but Ag has a higher in vitro affinity for protein thiols than other metals that are commonly found in association with MTs. Its main purpose is to improve analytical accuracy when measuring MTs that contain a high proportion of Cu or other metals having a higher binding affinity for MT than Cd and the Ag saturation takes advantage of the higher affinity of Ag(I) than of Cu(I) to MT [77,81-83]. Moreover, it has been found that platinum group elements (palladium, platinum and rhodium) have considerable affinity to MTs but without any analytical usage [23,26]. The second group of methods quantifies the total content of MTs in the target sample according to content of metal ions bound in MTs. To detect such various metal ions, which could be bound to MT molecules, graphite furnace atomic absorption spectrometry (GF-AAS) or high performance liquid chromatography coupled with AAS or inductively coupled plasma mass spectrometry (ICP-MS) have been used by Szpunar et al. [84-94] and others [95-103]. The third group of methods is based on detecting of sulfhydryl moieties of MTs using their chemical labelling by the Ellman reagent. This chemical labelling enables the MTs to be detectable by UV spectrometry and fluorimetry [95-98]. However, without labelling, MTs can not be detected using spectrometry or fluorimetry. The fourth group of methods uses antibodies against MTs. Among these methods, Western blotting, immunohistochemistry, immunofluorescence, radio-immunoanalysis (RIA) and enzyme-linked immunosorbent assay (ELISA) are included. ELISA and RIA have high sensitivity for detection of MTs in tissues [99]. Therefore, they are commonly used for investigation of MT expression in tissues sections. Antibodies usually cross-react with MT-1 and MT-2, and cannot distinguish single isoforms. The advantage of immunohistochemistry and immunocytochemistry is possibility to detect the cellular location of MTs (cytoplasmic or nuclear) [100102].
Metallothioneins and Cancer
Separation techniques belong to the fifth group of methods, which enables one to distinguish certain isoforms of MTs. To separate single isoforms of MTs, gel-permeation chromatography using Sephadex G-75 is commonly used. Less is known about separation of MTs by ionic exchange chromatography or high performance liquid chromatography (HPLC). Nevertheless, it has been shown that single isoforms of MTs can be detected by HPLC-MS [103-108]. In comparison with chromatographic techniques, electrophoretic separation techniques, most of all capillary electrophoresis, belong to the most utilized techniques for determination of MTs [109-114]. Capillary zone electrophoresis has been developed in 1990 and since 1993 it has been utilized for MT determination [115]. There have been tested experimental and instrumental conditions (electrolytes, capillaries, detectors) for suitable separation and consequent detection of MTs [116,117]. The experimental data show that the most suitable electrolytes for analysis of MTs are borate-SDS at alkali pH in an uncoated capillary or Tris-HEPES buffer without detergents at neutral pH in a capillary coated by polyacrylamide [118-120]. Capillary electrophoresis coupled with mass spectrometry was several times employed successfully to detect single MT isoforms [111,113,121].
Fig. (4). Metallothionein and a cell resistance to a heavy metalbased cytostatic event. Scheme of potential interaction between essential metal such as zinc and a toxic one such as cadmium. Ions enter the cell membrane and lead to synthesis of MT via activation of MTF-1 (a). The synthesized MT binds both essential (Zn ) and toxic (Cd ) metal ions and, thus, ensures the detoxification of the toxic metal ions or the transport of essential metal ions to the place of their need (b). The toxic metal ion can replace the essential metal already bound to MT due to a higher affinity (c). Furthermore, the essential metals can obstruct ionic channels transporting the toxic metals to a cell (d).
The sixth group of methods is based on detection of mRNA. The family of human MTs gene have 17 sub-genes on chromosome 16 (13 sub-genes belong to MT-1, two to MT-2, one to MT-3 and one to MT-4) [1]. Levels of mRNA can be studied by RT-PCR, real time RT-PCR, Northern blotting and microarrays [122-128]. The main advantage of mRNA methods is possibility to detect different MT iso-
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5
forms. Detection of mRNA is different from detection of protein. The amount of MT protein can be different by MT mRNA. This is related to different induction and degradation times. Thus, both protein and mRNA can be useful determinations. The seventh group of methods is based on electroactivity of certain moieties of MTs. Among the broad range of electrochemical methods, cyclic voltammetry [129-133], differential pulse voltammetry [133-151] and chronopotentiometric stripping analysis [152-161] and others [162-168] have been employed for the detection of MTs. More than seventy years ago the Brdicka reaction using cobalt(III) complexes was first used to determine proteins containing sulphydryl moieties. The resulting electrochemical signals had characteristic shapes depending on the content of –SH moieties in the target molecule, the size of the molecule and their concentration [153]. Moreover, these signals are very temperature-dependent. It has been shown that temperature of a supporting electrolyte within 5 to 10°C enhances the signals of MTs for more than 50% when compared to those measured at room temperature [133,146]. To prevent interferences during electrochemical detection of biomacromolecules, Palecek and his colleagues introduced a technique called adsorptive transfer stripping [169], which can be coupled with well known electrochemical methods such as cyclic voltammetry, differential pulse voltammetry as well as with the Brdicka reaction. The main improvement is based on the fact that a measurement is not carried in the presece of a sample. After renewing the surface, the electrode is immersed into a solution (2-5 l). MTs accumulate on the surface of the electrode. Furthermore, the electrode is rinsed and immersed into a pure supporting electrolyte, in which no interfering substances are present (Fig. 5a). Typical DP Brdicka reaction voltammograms of MTs are shown in Fig. (5b). Signals of Cat1, Cat2 and Cat3 correspond to the reduction of hydrogen at the mercury electrode [170]. Another signal, which appeared at a potential about –1.0 V, is due to the reduction of the RS2 Co complex [133,170]. Another technique called peak H utilizes a signal of hydrogen evolution from a supporting electrolyte catalyzed by a protein [152,153,156,157]. This signal can be measured by chronopotentiometric stripping analysis (CPSA) through recording the inverted time derivative of potential (dE/dt)-1 as a function potential E [152]. CPSA represents one of the most sensitive methods for detection of peptides and proteins. Thanks to this signal, subattomolar concentrations of MTs can be detected [133]. The character and origin of the catalytic peak H is not clear yet. Free –SH moieties together with –NH2 ones are involved in the catalysis of hydrogen evolution at very negative potentials. Peak H is less temperature dependent compared to Brdicka signals, but there is a greater dependence on ionic strength of the supporting electrolyte and on the isoelectric point of the peptide or protein analysed. In spite of these ambiguities, peak H can be utilized for sensitive detection of many peptides and proteins. CPSA together with an adsorptive technique (Fig. 5a) was employed for determination of MTs in human body liquids and cell lines [171]. The advantage of this technique is low cost, a low variance coeficient, low detection limits, easy miniaturization and no interference. The method was tested to determine whether the most abundant thiol called glu-
6 Current Protein and Peptide Science, 2009, Vol. 10, No. 4
Eckschlager et al.
Fig. (5). Scheme of adsorptive transfer technique: (1) – Renewing of the surface of a working electrode; (2) adsorbing of metallothioneins onto the surface of the working electrode at open circuit; (3) rinsing of the working electrode in washing buffer; (4) immersing of the electrode to a supporting electrolyte and measurement. The whole electrochemical system is controlled by a personal computer (a). Typical DP voltammograms of the Brdicka supporting electrolyte, MTs (1 M) measured in the presence of the Brdicka electrolyte; (b) in inset: DP voltammogram of MTs (100 M) (for more details see in ref. no. [133]).
tathione could influence CPSA measurement. It was found that the influence of glutathione was negligible [171]. A disadvantage is that no automated analysis of large set of samples can be conducted. In the case of blood serum from blood donors, we obtained a linear dependence of the catalytic signal from standard additions of MTs. We found out that MT’s concentration was about 1.1 ± 0.1 M (n = 5) in the human blood serum samples. The detection limit (3 S/N) of MTs calculated for human blood serum was about 350 fmol. Moreover, the AdTS CPSA technique was used for the determination of MTs in cell lines and in human blood serum from a cisplatin-treated patient. The concentration of MTs determined in cell lines resistant to cisplatin was two times higher when compared to the concentration determined in cell lines sensitive to cisplatin (Fig. 6a,b). If we compare MT levels in a blood donor with those in a patient treated with cisplatin, we observe a marked increase in MT levels in the patient (Fig. 6c,d). 4. METALLOTHIONEINS AND CANCER In the last decade, several reports disclosed MT expression as a useful diagnostic factor for tumor progression and drug resistance in a variety of malignancies e.g. leukaemia, melanoma, breast, ovarian, renal, lung, pancreatic, gall bladder, esophageal, and basal cell carcinomas [154,172-184]. One may suggest that MTs may lead to a protection of tumor cells against apoptosis and support the metastatic behaviour of tumors and/or cancer cell proliferation. On the other hand in some other studies devoted to colorectal and bladder cancer and others, no significant correlation between MT expression and prognosis was observed [185,186].
Fig. (6). Changes in peak H height of Neuroblastoma cell line of malignant tumor. Two lines of cells were studied – UKF-NB4 (sensitive to cisplatin) and UKF-NB4CDDP (resistant to cisplatin). Moreover, the technique was used for determination of MTs in blood serum of a patient treated with cisplatin. The CPSAsupporting electrolyte contained 0.1 M H3BO3 + 0.05 M Na2B4O7. AdTS CPSA parameters were as follows: an initial potential of 0 V, an end potential of –1.85 V, temperature of 25°C and accumulation time of 120 s.
In spite of the fact that metals, their transporters and MTs participate in the carcinogenic process, their use as a potential marker for tumor differentiation, cell proliferation, or predictive marker remains controversial [187,188]. Serum
copper levels are upregulated in many human tumors and correlate with tumor burden and prognosis. Copper chelators reduce tumor growth and microvascular density in animal
Metallothioneins and Cancer
models and possible targets include copper-dependent enzymes, chaperones, and transporters [189]. Zinc has an important role in cellular processes and deregulated expression of zinc transporters could have remarkable consequences in the regulation of zinc, which could be pivotal in the initiation or progression of breast cancer. The zinc transporter LIV-1, which is member of the ZIP family, was found to be associated with the estrogen receptor and a variety of growth factors commonly associated with breast cancer [30]. In the next section of this review, we discuss changes in the level of MTs, their isoforms and/or MT mRNAs in various types of tumor diseases with regard to usage of these proteins as new markers and/or predictive factors for tumor diseases. 4.1. Breast Cancer In normal breast tissue, strong nuclear and cytoplasmic MT expression is detected in myoepithelial cells, but in epithelial cells lining the large ducts only rarely. Also in benign breast lesions, only myoepithelial cells express MTs [174,190]. Lobular cancer cells from in situ or invasive tumors showed weak to no expression of MTs as well. In contrast, a significant proportion (26 to 100%) of invasive ductal breast cancers exhibited MT expression, which was detected by immunohistochemistry [174,191]. MT-3 mRNA was detected using RT PCR in 73% of breast cancers, although this MT isoform is not expressed in normal breast tissue [192]. The high MT expression detected by immunohistochemistry was associated with a histological grade 3 and with estrogen receptor negativity but not with tumor size and the presence of lymph node metastasis (TNM staging) at the time of diagnosis. In breast cancer tissues none of biomarkers (p53, pRb, Bcl-2, type IV collagen, laminin, fibronectin, cathepsin D, CD44, matrix metalloproteinase-3, c-erbB2, EGFR) are associated with MT expression which was detected by immunohistochemistry [174]. Increased MT-1F and MT-2A mRNAs were found in high grade breast cancer. MT-1E mRNA expression was found in estrogen receptor-negative tumors, but there was no significant difference in MT-1E expression between progesterone receptor positivity or negativity [174,193]. Higher MT immunoexpression in breast cancers has been shown to be a sign of worse prognosis (shorter overall and shorter event free survival) [194,195]. Multivariate analysis, which were reported only in two of these studies, showed that MT expression detected by immunohistochemistry did not provide additional prognostic information because of a strong association of MT expression with other factors predicting poor prognosis (receptor negativity, tumor size and lymph node metastases, high mitotic index, etc.) [174,196,197]. Therefore, another research of MTs using more detailed analysis of MT isoforms and cellular localization evaluated by multivariate analysis is necessary for interpretation of its predictive significance. 4.2. Reproductive System Prostate. A difference in location of MT was found between benign prostatic hypertrophy and prostatic adenocarcinoma. MTs are detected by immunohistochemistry in the nuclei of epithelial cells in a benign lesion, whereas in the adenocarcinoma MTs occurred mainly in the cytoplasm [27].
Current Protein and Peptide Science, 2009, Vol. 10, No. 4
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One may speculate that immunohistochemistry using an anti MT antibody may be used in diagnostics of prostatic tumors. Expression of MT-1X mRNA is downregulated in advanced prostate cancer, while in normal human prostate MT-1X mRNA is detected [198]. The independent predictive value of MT immunostaining and a positive correlation with the Gleason score (grading) and Ki-67 expression were found in a recent study [199]. Ovarian. In ovarian epithelial tumors MT expression is a sign of malignancy. There was found a significant correlation between the expression of MTs and benign tumors, borderline tumors, and cancer cases. In cancer a difference is observed between grades 1 and 3. In diagnostic problems MT immunohistochemistry may help to distinguish between benign, borderline and malignant tumors [200,201]. In another study MT expression detected by immunohistochemistry was negatively associated with survival when all patients were analyzed. However, no significant association was observed when subgroups of patients with histological grades of 1, 2 or 3 carcinomas were analyzed [202]. Endometrium. MT expression in normal endometrium is inversely correlated with PCNA (Proliferating Cell Nuclear Antigen) scores, oestrogen, and progesterone receptor as shown by an immunohistochemical study. In benign hyperplastic lesions, MT expression is detected only in a few cases, while in a group of carcinomas, it is observed in approximately 20% of the cases. In carcinomas, MT expression is positively correlated with grade, Ki-67 detected by MIB1 antibody, and p53 expression, but inversely with the progesterone receptor. These data suggest that MT expression immunohistochemically detected is under hormonal control in normal endometrium and could be used as a marker of progression of endometrial tumors [203]. 4.3. Respiratory System Oral tumors. A high MT labelling index predicts shorter survival in oral squamous cell carcinoma. In multivariate analysis the MT labelling index (percentage of immunohistochemically labelled cells among 500 cells) and clinical stage are independent prognostic factors [204]. The total MT immunostain was significantly higher in moderate dysplasia grade of oral leukoplakia when compared with normal oral mucosa and mild dysplasia. MT location exhibited a mosaic pattern and was predominantly in compartments cytoplasmatic and nuclear simultaneously. The authors suggested that MTs may be a marker of dysplasia and may play a role in oral carcinogenesis [205]. In laryngeal cancer there is a significantly augmented expression of MTs as demonstrated by immunohistochemistry when compared to a group of benign lesions. MT expression was not related to prognosis, but MT examination may provide a tool for detection of malignant transformation of benign laryngeal epithelial lesions [206]. Lung. Immunostaining of MTs was evident in squamous cell lung carcinoma and adenocarcinoma, but it was absent in all eleven cases of small-cell lung carcinoma [207]. Shorter survival of patients with high expression of MTs pointed to a prognostic significance of the protein in nonsmall cell lung cancers. Positive correlations are disclosed
8 Current Protein and Peptide Science, 2009, Vol. 10, No. 4
Table 1.
Eckschlager et al.
Biochemical Properties of Human Metallothioneins
Name1
No. of Amino Acids
Molecular Weight (Da)
Theoretical pI*
Swiss-Prot no.2
MT 1A
61
6133.2
8.38
P04731
MT 1B
61
6115.3
8.47
P07438
MT 1E
61
6014.1
8.38
P04732
MT 1F
61
6086.2
8.23
P04733
MT 1G
61
6070.2
8.38
P13640
MT 1H
61
6039.2
8.49
P80294
MT 1I
61
6040.2
8.38
P80295
MT 1K
62
6141.3
8.38
P80296
MT 1L
61
6068.2
8.38
P80297
MT 1R
61
6062.2
8.38
Q93083
MT 2
61
6042.1
8.23
P02795
MT 3
68
6926.9
4.79
P25713
MT 4
62
6418.7
8.38
P47944
8.23
Q9Y4I5
MTL5**
299 1
33110.0 2
*
**
ExPASy Proteomics Server – http://www.expasy.ch; http://www.expasy.org/sprot/, isoelectric point; tesmin.
between MT expression and grade and also between MT expression and the expression of Ki-67 proliferation associated antigen in non-small cell lung cancers [172]. MT overexpression is an independent predictive factor of shorter survival in patients with small cell lung cancers undergoing chemotherapy when compared with patients with tumors exhibiting low MT expression [208].
finding that MT-3 expression is minimal in normal bladder suggests that MT-3 detected by Western blotting, immunohistochemistry or at the mRNA level by RT PCR might be developed into an effective biomarker for bladder cancer [192].
There are statistically significant differences between age groups or between asbestos exposure times and MT expression in diffuse malignant pleural mesothelioma. The patients with positive MT immunostaining have longer exposure times and are older than those having negative immunostaining. The differences between survival of the patients are not statistically significant in terms of the immunohistochemical results for p53, p21 and MTs [209].
MT-3 mRNA detected by RT PCR is frequently underexpressed in oesophageal squamous cell carcinoma. Its promoter has a heterogeneous pattern of methylation in oesophageal cancer cell lines and is associated with a reduction in gene expression. MT-3 expression is frequently downregulated also in clinical samples of oesophageal carcinoma, especially in tumors with a methylated promoter region. Neither promoter methylation nor underexpression of MT-3 correlated with patient survival or with any known prognostic factor (age, gender, smoking and drinking history, clinical stage, lymph node involvement and tumor volume) [213]. MT expression detected immunohistochemically is significantly increased along with the histological progression of Barrett's esophageal cells towards adenocarcinoma [214]. MT expression at the protein level correlates with survival allowing the identification of an unfavourable group of patients with gastric cancer [215,216]. Moreover, many cases of dysplasia and metaplasia also exhibited intense MT expression as detected by immunohistochemistry [215]. MT protein expression was higher in H. pylori-negative patients than in positive patients and the MT labelling index (percentage of MTs immunohistochemically positive cells) was higher in H. pylori-positive patients without cancer compared to those with cancer. A real time RT PCR analysis of MT-2A revealed a similar result with the immunohisto-
4.4. Excretory System Renal cells. The inverse correlation between MTs immunoexpression and tumor nuclear grade in renal cell carcinoma suggests their role in tumor differentiation. MTs examination may be used as a clinical predictive parameter. The majority of the renal carcinoma cases expressed MTs in both cytoplasm and nucleus, the expression being cytoplasmic only in one third of the cases [175,210]. Urinary bladder. MTs protein expression in the urinary bladder primary tumor of transitional cell carcinoma specimens is significantly associated with overall and disease-free survival, with grade and with stage. In this case MTs expression was correlated with a more aggressive behaviour [211,212]. MT-3 mRNA is up-regulated in human bladder cancer and this expression increases with tumor grade. The
4.5. Digestive System
Metallothioneins and Cancer
chemical data. Those authors suggested that the decrease of MT expression did not prevent gastric mucosa damage caused by H. pylori , which led to more severe gastritis. Eradication of H. pylori increases MT expression, which may reduce the risk of early gastric cancer. A positive correlation between the MT labelling index and the apoptosis/ proliferation ratio (ratio of percentage of apoptotic cells divided by percentage of Ki-67 positive cells) suggested that high MT expression might suppress gastric cancer development [217]. Increased expression of MTs in colorectal cancer is associated with a high degree of lymph node involvement and poor overall survival. MT expression demonstrated by immunohistochemistry and radioimmunoassay in colorectal cancer may be a predictor of a worse prognosis, particularly in patients with synchronous liver metastases [218,219]. On the other hand, another immunohistochemical study did not find a significant correlation between MT expression and colorectal cancer prognosis when a multivariate analysis was carried out [186]. This difference may be correlation of MT expression with other prognostic factors. 4.6. Other Types of Cancer Melanoma. High expression of MTs in melanoma is associated with an increased risk for progression and reduced survival. Multivariate analysis with other prognostic markers (tumor thickness – Breslow scale, Clark level, tumor ulceration, age, gender, and location) confirms MT overexpression detected by immunohistochemistry as a significant and independent prognostic factor in melanoma [220]. Expression of MT immunopositivity in more than 20% of lymphoma cells was associated with a significantly poorer survival rate that was independent of other prognostic factor in diffuse large B-cell lymphoma [221]. MT expression detected by immunohistochemistry in malignant fibrous histiocytoma, liposarcoma and synovial sarcoma correlates with intensity of proliferation evaluated as a percentage of Ki-67 positive cells and grade and could be useful in defining the extent of malignancy and in a prognostic appraisal of the tumors. MT expression was demonstrated in epithelioid cells in all cases of biphasic type and in spindle cells in all cases of monophasic type synovial sarcoma. Patients with higher MT expression in the tumors exhibited a shorter survival time [222,223]. 4.7. MTs in Pertipheral Blood Our preliminary results shows that MT levels detected by electroanalytical methods in serum from patients with melanoma, chronic lymphocytic leukemia, breast, colorectal, and kidney cancer were elevated in a majority of samples [184]. Thus, it will be possible to elucidate the potential role of MT levels in serum of patients suffering from malignancies, as well as its clinical usefulness as a tumor marker and as a tool for selecting patients for chemotherapy. 4.8. Conclusion We may conclude that MT expression, usually detected by immunohistochemistry that cannot identify different isoforms, appears to be a sign of worse prognosis, higher tumor
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grade and/or worse response to chemotherapy in many malignant tumors particularly in breast, prostatic, ovarial, head and neck, non-small cell lung cancer, melanoma, and soft tissue sarcoma. On the other hand, detection of MTs has not be utilized as a prognostic factor in clinical practice until now. For implementation of MT immunostaining as a routine prognostic factor, other studies with multivariate analysis, which include all clinical and laboratory prognostic factors are necessary because only an independent prognostic factor can have utility for clinical practice. The significance of serum MT levels as a tumor marker requires a larger study, which is currently under way. 5. METALLOTHIONEINS AND RESISTANCE TO CYTOSTATIC DRUGS Mechanisms involved in resistance of cancer cells to cytostatic drugs include reduced drug uptake, increased drug efflux, increased DNA repair, increased tolerance of DNA damage, changes in target structure, or increased levels of intracellular thiols such as glutathione and metallothionein [21]. Chemoresistance is a multifactorial phenomenon with no single mechanism predominating even within the histological type of tumor [224]. Chemoresistance to cisplatin and carboplatin is mediated through two broad mechanisms, first, a failure of a sufficient amount of platinum to reach the target DNA and, second, a failure to achieve cell death after binding of platinum to DNA [225]. Transfer of platinum from cisplatin and carboplatin to MTs result in inactivation of those drugs. [226]. A newly developed platinum-derived cytostatic compound, heptaplatin, is more effective against the MT-overexpressing gastric cancer cell line resistant to cisplatin than either cisplatin or carboplatin. In addition, pretreatment with zinc can induce MT’s reduced cytotoxicity of cisplatin and carboplatin but not of heptaplatin [227]. The influence of the zinc pre-treatment on the increase in resistance to cancer treatment can be viewed in two perspectives: a) as inducers of MTs as mentioned above and b) as a protector itself according to the scheme shown in Fig. (4). Zinc probably obstructs ionic channels, which are used to facilitate heavy metal ion entry into a cell. This mechanism is demonstrated, for example, with zinc and cadmium ions (Fig. 4). Metallothionein plays a crucial role in the obstruction of the cadmium entering due to transporting of essential zinc to membrane and binding of toxic cadmium. Similar mechanisms can be expected in the case of platinum-based cytostatics, which may be pumped out of the cells by different transport systems [228]. We showed that addition of cisplatin or carboplatin to culture media for a neuroblastoma-derived cell line resistant to cisplatin significantly increased MT levels measured by adsorptive transfer technique. Meanwhile, in a sensitive cell line, neither cisplatin nor carboplatin induced significant MT increase. Cisplatin-resistant neuroblastoma cell lines were established by exposing parental neuroblastoma lines to increasing concentrations of cisplatin [224]. The 50% inhibitory concentration (IC50) for both cisplatin and carboplatin of resistant lines was approximately 4-fold higher than the IC50 of sensitive lines. IC50 was determined using an MTT assay. Because normal MT levels both in chemosensitive and chemoresistant cancer cells are low, their levels examined in bioptic samples taken from tumors at the time of diagnosis
10 Current Protein and Peptide Science, 2009, Vol. 10, No. 4
do not appear to be a useful sign of platinum cytostatics resistance. Other experimental studies demonstrated that a cisplatin-resistant ovarian cancer-derived cell line exposed to cisplatin manifested a nuclear MT expression, which was detected using immunocytochemistry, while this line cultivated in platinum-free medium and a cisplatin-sensitive line expressed MTs in cytoplasm but not in nuclei [229]. Recent evidence suggests that MTs may cause also resistance to several “non-metal cytostatic drugs“. They reduce apoptosis induced by etoposide in lung and liver cancerderived cell lines, and the effect is increased with higher MT levels induced by pre-treatment with zinc or cadmium. Cellular MT concentrations were estimated by the cadmiumhemoglobin radioassay method in this study [230]. Because etoposide induces apoptosis by uncorrected DNA damage, MTs may play a generalized role in prevention of apoptosis. The exact mechanism by which MTs defer cell death from etoposide exposure was not established until now. Microarray analyses on paired gastric cancer effusion samples obtained before and after irinotecan treatment identified five MT isoforms to have significantly higher signal in non-responders when compared with responders. When compared with control cells, a human gastric adenocarcinoma-derived cell line transfected by MT-1 X showed 1.4 times higher irinotecan IC50 [231]. Microarray gene expression analysis suggests the roles of MTs and proapoptotic genes down-regulation in medulloblastoma and rhabdomyosarcoma cell lines resistant to alkylating drug BCNU [232]. Esophageal squamous cell carcinomas, which did not express MTs, respond well to chemoradiotherapy (5fluorouracil and cisplatin) [48]. Increased MT concentration in the heart prior to exposure to doxorubicin prevents cardiotoxicity. Levels of MTs in myocardial may be pharmaceutically inducible by bismuth subnitrate, isoproterenol, and tumor necrosis factor- [233]. Studies using transgenic mice with high levels of MTs showed that MT protection against anthracycline cardiotoxicity is related to its anti-apoptotic effect by inhibiting both p38-MAPK-mediated and mitochondrial cytochrome crelease-mediated apoptotic signal pathways. MTs interfere with oxidative stress caused by anthracycline metabolism in the heart [234]. 6. CONCLUSIONS AND FUTURE PROSPECTS MT overexpression, their different isoforms and/or their cellular location could be used as a predictive marker of worse prognosis, sign of higher grade in selected tumors or marker of malignant transformation of benign lesions. Prediction of chemoresistance to platinum cytostatics or to other cytostatic drugs (etoposide, irrinotecan) based on MT expression needs to be confirmed in a larger data set. Examination of MT levels in serum, which may be used as a tumor marker, seems to be promising for clinical practice. One may speculate that pharmacological inhibition of MTs may reverse chemoresistance to platinum drugs or other cytostatic drugs and induction of MTs in heart may prevent doxorubicin-induced cardiomyopathy. Hyphenated analytical techniques have the potential to be interesting alternatives to the classical methods such as metal
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saturation assays or immunoassays for the characterization and quantification of metallothionein isoforms. On the other hand, electrochemical techniques cannot distinguish single isoforms but are very sensitive, easy-to-use, low cost and easy to be miniaturized. Moreover, electrochemical methods have the potential to be miniaturized and then employed in clinical practice. ACKNOWLEDGMENT The financial support from the Grant Agency of the Czech Academy of Sciences (grant no. GA AV IAAA401990701) and from the Ministry of Education, Youth and Sports (grant no. MSM 0021620813) is greatly acknowledged. REFERENCES [1]
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cance of metallothionein, p53 protein and Ki-67 antigen expression in laryngeal cancer. Anticancer Res., 2007, 27, 335-342. Theocharis, S.; Karkantaris, C.; Philipides, T.; Agapitos, E.; Gika, A.; Margeli, A.; Kittas, C.; Koutselinis, A. Expression of metallothionein in lung carcinoma: correlation with histological type and. Histopathology, 2002, 40, 143-151. Joseph, M. G.; Banerjee, D.; Kocha, W.; Feld, R.; Stitt, L. W.; Cherian, M. G. Metallothionein expression in patients with small cell carcinoma of the lung - Correlation with other molecular markers and clinical outcome. Cancer, 2001, 92, 836-842. Isik, R.; Metintas, M.; Gibbs, A. R.; Metintas, S.; Jasani, B.; Oner, U.; Harmanci, E.; Demircan, S.; Isiksoy, S. p53, p21 and metallothionein immunoreactivities in patients with malignant pleural mesothelioma: correlations with the epidemiological features and prognosis of mesotheliomas with environmental asbestos exposure. Respir. Med., 2001, 95, 588-593. Zhang, X. H.; Takenaka, I. Incidence of apoptosis and metallothionein expression in renal cell carcinoma. Br. J. Urol., 1998, 81, 9-13. Ioachim, E. E.; Charchanti, A. V.; Stavropoulos, N. E.; Athanassiou, E. D.; Michael, M. C.; Agnantis, N. J. Localization of metallothionein in urothelial carcinoma of the human urinary bladder: An immunohistochemical study including correlation with HLADR antigen, p53, and proliferation indices. Anticancer Res., 2001, 21, 1757-1761. Yamasaki, Y.; Smith, C.; Weisz, D.; van Huizen, I.; Xuan, J.; Moussa, M.; Stitt, L.; Hideki, S.; Cherian, M. G.; Izawa, J. I. Metallothionein expression as prognostic factor for transitional cell carcinoma of bladder. Urology, 2006, 67, 530-535. Smith, E.; Drew, P. A.; Tian, Z. Q.; De Young, N. J.; Liu, J. F.; Mayne, G. C.; Ruszkiewicz, A. R.; Watson, D. I.; Jamieson, G. G. Metallothionien 3 expression is frequently down-regulated in oesophageal squamous cell carcinoma by DNA methylation. Mol. Cancer, 2005, 4, 9. Li, Y.; Wo, J. M.; Cai, L.; Zhou, Z. X.; Rosenbaum, D.; Mendez, C.; Ray, M. B.; Jones, W. F.; Kang, Y. J. Association of metallothionein expression and lack of apoptosis with progression of carcinogenesis in Barrett's esophagus. Exp. Biol. Med., 2003, 228, 286-292. Ebert, M. P. A.; Gunther, T.; Hoffmann, J.; Yu, J.; Miehlke, S.; Schulz, H. U.; Roessner, A.; Korc, M.; Malfertheiner, P. Expression of metallothionein II in intestinal metaplasia, dysplasia, and gastric cancer. Cancer Res., 2000, 60, 1995-2001. Galizia, G.; Ferraraccio, F.; Lieto, E.; Orditura, M.; Castellano, P.; Imperatore, V.; La Manna, G.; Pinto, M.; Ciardiello, F.; La Mura, A.; De Vita, F. p27 downregulation and metallothionein overexpression in gastric cancer patients are associated with a poor survival rate. J. Surg. Oncol., 2006, 93, 241-252. Mitani, T.; Shirasaka, D.; Aoyama, N.; Miki, I.; Morita, Y.; Ikehara, N.; Matsumoto, Y.; Okuno, T.; Toyoda, M.; Miyachi, H.; Yoshida, S.; Chayahara, N.; Hori, J.; Tamura, T.; Azuma, T.; Kasuga, M. Role of metallothionein in Helicobacter pylori-positive gastric mucosa with or without early gastric cancer and the effect on its expression after eradication therapy. J. Gastroenterol. Hepatol., 2007, doi: 10.1111/j.1440-1746.2007.05124.x. Hishikawa, Y.; Kohno, H.; Ueda, S.; Kimoto, T.; Dhar, D. K.; Kubota, H.; Tachibana, M.; Koji, T.; Nagasue, N. Expression of metallothionein in colorectal cancers and synchronous liver metastases. Oncology, 2001, 61, 162-167. Janssen, A. M. L.; van Duijn, W.; Kubben, F.; Griffioen, G.; Lamers, C.; van Krieken, J.; van de Velde, C. J. H.; Verspaget, H. W. Prognostic significance of metallothionein in human gastrointestinal cancer. Clin. Cancer Res., 2002, 8, 1889-1896. Weinlich, G.; Eisendle, K.; Hassler, E.; Baltaci, M.; Fritsch, P. O.; Zelger, B. Metallothionein - overexpression as a highly significant prognostic factor in melanoma: a prospective study on 1270 patients. Br. J. Cancer, 2006, 94, 835-841. Poulsen, C. B.; Borup, R.; Borregaard, N.; Nielsen, F. C.; Moller, M. B.; Ralfkiaer, E. Prognostic significance of metallothionein in B-cell lymphomas. Blood, 2006, 108, 3514-3519. Dziegiel, P.; Salwa-Zurawska, W.; Zurawski, J.; Wojnar, A.; Zabel, M. Prognostic significance of augmented metallothionein (MT) expression correlated with Ki-67 antigen expression in selected soft tissue sarcomas. Histol. Histopath., 2005, 20, 83-89.
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Received: October 21, 2008
Revised: April 11, 2008
Accepted: July 01, 2008
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Shimoda, R.; Achanzar, W. E.; Qu, W.; Nagamine, T.; Takagi, H.; Mori, M.; Waalkes, M. P. Metallothionein is a potential negative regulator of apoptosis. Toxicol. Sci., 2003, 73, 294-300. Chun, J. H.; Kim, H. K.; Kim, E.; Kim, I. H.; Kim, J. H.; Chang, H. J.; Choi, I. J.; Lim, H. S.; Kim, I. J.; Kang, H. C.; Park, J. H.; Bae, J. M.; Park, J. G. Increased expression of metallothionein is associated with irinotecan resistance in gastric cancer. Cancer Res., 2004, 64, 4703-4706. Bacolod, M. D.; Johnson, S. P.; Ali-Osman, F.; Modrich, P.; Bullock, N. S.; Colvin, O. M.; Bigner, D. D.; Friedman, H. S. Mechanisms of resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea in human medulloblastoma and rhabdomyosarcoma. Mol. Cancer Ther., 2002, 1, 727-736. Sun, X. H.; Kang, Y. J. Prior increase in metallothionein levels is required to prevent doxorubicin cardiotoxicity. Exp. Biol. Med., 2002, 227, 652-657. Kang, Y. J. Antioxidant defense against anthracycline cardiotoxicity by metallothionein. Cardiovasc. Toxicol., 2007, 7, 95-100. Schafer, F. Q.; Buettner, G. R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med., 2001, 30, 11911212.
B. Literární rešerše o možnostech detekce metalothioneinu
69
TRAC - Trends in Analytical Chemistry
Metallothioneins as Target Molecules for Analytical Techniques
Vojtech Adam1,2, Ivo Fabrik1, Tomas Eckschlager3, Marie Stiborova4, Libuse Trnkova5, Rene Kizek1
1
Department of Chemistry and Biochemistry, and 2Department of Animal Nutrition and
Forage Production, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic 3
Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles
University, V Uvalu 84, CZ-150 06 Prague 5, Czech Republic 4
Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-128
40 Prague 2, Czech Republic 5
Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37
Brno, Czech Republic
Corresponding author:
Rene Kizek, Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Czech Republic; E-mail:
[email protected]; phone: +420-5-4513-3350; fax: +420-5-4521-2044
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Abstract Metallothioneins (MT) area a family of ubiquitous, biologically interesting proteins which have been isolated and studied in a wide variety of organisms, including prokaryotes, plants, invertebrates and vertebrates. Due to the property of MT being metal-inducible and, also, due to their high affinity to metal ions, homeostasis of heavy metal levels is probably their most important biological function. In addition, MT are involved in other important biochemical pathways including scavenging of reactive oxygen species, activating of transcription factors or participating in carcinogenesis. Analytical measurement of MT is not simple due to high content of cysteine in metallothionein structure and relatively low molecular mass. Detection is based on a) analysis of bonded metal ion, b) analysis of free –SH groups, c) protein mobility in electrical field and d) interaction with different types of sorbent. This review highlights techniques used for analytical detection and determination of these proteins in last decades with attention on discussion of the advantages and disadvantages of particular approaches.
Keywords: metallothionein; electrochemistry; voltammetry; high performance liquid chromatography; capillary electrophoresis; gel electrophoresis; spectrometry; structure; heavy metals
Abbreviations: MT – metallothionein; EDTA – ethylenediaminetetraacetic acid; SPE – solid phase extraction; CV – cyclic voltammetry; DPV – differential pulse voltammetry; DPASV – differential pulse anodic stripping voltammetry; SWCSV – square wave cathodic stripping voltammetry; CPSA – chronopotentiometric stripping analysis; ELISA – enzyme-linked immunosorbent assay; RIA – radioimmunoassay; PVDF – polyvinylidene fluoride; PAGE – polyacrylamide gel electrophoresis; SDS – sodium dodecyl sulfate; CE – capillary
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electrophoresis; CZE – capillary zone electrophoresis; MS – mass spectrometry; QMS – quadrupole mass spectrometry; ID – isotope dilution method; SFMS – sector field mass spectrometry; ESI – electrospray ionization; MALDI – matrix-assisted laser desorptionionization; TOF – time of flight; ICP – inductively coupled plasma; ICP-OES – ICP atomic emission spectroscopy; GF-AAS – graphite-furnace atomic absorption spectrometry; SPETDI-AAS – SPE terylenedi-imide AAS; HPLC – high performance liquid chromatography; RP-HPLC – reverse phase-HPLC; AE-HPLC – anion exchange-HPLC; SEC – size-exclusion chromatography; UV – UV spectrometer; FD – flame detector; SBD-F – 4-aminosulfonyl-7fluoro-2,1,3-benzoxadiazole.
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1.
Introduction Metallothioneins (MT) belonging to the group of intracellular and low molecular mass
proteins (from 2 to 16 kDa) [1] were discovered in 1957, when Margoshes and Valee isolated them from a horse renal cortex tissue [2]. These proteins have been isolated and studied in a wide variety of organisms, including prokaryotes, plants, invertebrates and vertebrates [3-7]. Concerning their primary structure, they are rich in cysteine and have no aromatic amino acids. The metal binding domain of MTs consists of 20 cysteine residues juxtaposed with basic amino acids (lysine and arginine) arranged in two thiol-rich sites called α and β (Fig. 1A). The cysteine sulphydryl groups bind 7 moles of divalent metal ions per mol of MT, while the molar ratio for monovalent metal ions (Cu and Ag) is twelve [1,8,9]. Although the naturally occurring protein has Zn2+ in both binding sites, this ion may be substituted for another metal ion that has a higher affinity for thiolate such as Pb, Cu, Cd, Hg, Ag, Fe, Pt and/or Pd [10-13]. Besides the metalthiolate clusters and the absence of aromatic amino acids, MT do not have characteristic structural features. The primary structure is extremely variable; it is only conserved within closely related species, which makes the classification of MT problematic [6]. A classification system containing three groups of MT has been proposed and revised several times [14]. Class I comprises all proteinaceous MT with locations of cysteine closely related to those in mammals. Some molluscs and crustacean MT belong to this class, such as those characterized in mussels, oysters, crabs and lobsters [14]. Class II includes proteinaceous MT which lacks this close similarity to mammalian MTs, while Class III consists of non-proteinaceous MTs, to which some authors include plant heavy-metal-binding peptides called phytochelatins [15]. Often several paralogues (mostly called isoforms) exist in the genome of a species; four major isoforms (MT-1 through MT-4) have been identified in
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mammals [16]. In addition, at least thirteen closely related MT proteins have been described in humans[5]. Based on their metal-inducible properties and their high affinity for metal ions, homeostasis of heavy metal levels is probably MT's most important biological function (Fig. 1B). MT can also serve as “maintainers” of the redox pool [17]. In mammals, these proteins may serve as a reservoir of metals (mainly zinc and copper) for synthesis of apoenzymes and zinc-finger transcription regulators [18]. Moreover, new roles of these proteins have been discovering including those needed in the carcinogenic process [18,19].
According to Web of Sciences, since 2000 approximately five hundreds papers including term metallothionein* in title, abstract and/or keywords have been published per a year (Fig. 1C). Considerable attention is paid mainly to involving of these proteins in various biochemical pathways and their probable usage as markers of stress or diseases. To fulfil the requirements of multifariously aimed studies, a wide range of bio-analytical instruments is needed. It is not surprising that techniques using for detection and determination of MT have been reviewed several times [14,16,20-28] and compared to each other [29-31]. Isolation, separation, detection and/or quantification of MT are not easy tasks for modern bioanalytical chemistry. Thanks to MTs low molecular mass and unique primary structure, commonly used methods for detection of proteins suffer from many _deficiencies including insufficient specificity and sensitivity. The most frequent methods used for detection of these proteins are indirect and are based on quantification of heavy metal ions occurring in their structure or on high content of sulfhydryl groups (Fig. 1D).
2.
Isolation procedures
2.1
Blood, blood serum and cells
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Isolation and consequent detection of MT in blood and/or blood serum samples is not so frequently compared to tissues analysis. Heat treatment of a sample (app. 100 °C for more than five minutes) to denature and remove high molecular mass proteins from samples proposed by Erk et al. [32] is nowadays successfully applied to blood and blood serum samples [33,34]. Moreover, Petrlova et al. showed that using of tris(2-carboxyethyl)phosphine as a reducing agent could be beneficial for quantification of MT. This method has been utilized for preparation of blood and blood serum samples of patients with various tumour diseases [35-37] or patients treated with heavy-metal based drugs [38], or even fish sperm [39]. Caulfield et al. used heat treatment for preparation of human red blood cells [40]. Cells were disrupted by repeated freeze-thawing cycles. The lysates obtained were treated and analysed. The authors had drawn blood from patients by venipuncture into tubes containing heparin. The presence of heparin or others compounds such as ethylenediaminetetraacetic acid (EDTA) can seriously influence quantification of MT in blood, blood serum or blood fractions, when electrochemical methods are used. Adam et al. showed that the presence of EDTA influenced voltammetric signals markedly [41].
2.2
Plant tissues To isolate MT from tissues, a preparation of crude extract from a tissue and
purification of such extract by using of gel filtration is one of the most commonly used protocols [42]. Tissue extract is prepared in the presence of Tris HCl with added sucrose [43], glucose and antioxidant specie (mercaptoethanol, dithiothreitol and/or TCEP [33]) [28]. This extract is centrifuged or heat treated with subsequent centrifugation. Erk et al. reported on comparison of different procedures to purify MT from the digestive glands of mussels (Mytilus galloprovincialis) exposed to cadmium: heat treatment (at 70 and 85 °C), solvent precipitation, and gel-filtration [32]. They found that the most convenient was using heat
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treatment for preparation of both heavy metal stressed and non-stressed tissues. Moreover, Beattie et al. successfully utilized solid phase extractors for MT isolation [44].
2.3
Plant tissues Preparation of plant tissues, cells and parts to isolate phytochelatins (included into MT
Class III) have been shown in many papers and reviewed several times [15]. MT Class I and II cannot be found in plant tissues without genetic modification of a plant genome. Macek et al. inserted MT genes from yeast and human into tobacco to enhance their ability to accumulate metal ions [45]. To detect MT, Diopan et al. prepared crude extracts from these plants and heat treated the extracts. Results on content of MT were similar to those detecting expression of mRNA [46].
3.
Direct detection of metallothioneins Although MT are low molecular mass proteins with very unusual primary structure,
several methods have been proposed to detect it directly without using robust electrophoretic and chromatographic separation techniques. Besides standard immunochemistry and mass spectrometry, electrochemistry can be also used for the direct detection of these proteins (Fig. 1).
3.1
Electrochemical methods Determination of MT by electrochemical methods is based on electroactivity of –SH
moieties, which tend to be oxidized or catalyze evolution of hydrogen from supporting electrolyte (Fig. 1). To prevent interferences and lower detection limits, an adsorptive transfer stripping technique is often used. The main improvment is based on removing the electrode from a solution after accumulation of a target molecule on its surface, rinsing of the electrode
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and transferring to a pure supporting electrolyte, where no interferences are present [41]. To detect MT, linear sweep, cyclic, differential pulse and square wave voltammetry have been used. The Use of of these techniques was reviewed by Sestakova and Navratil [47]. After the previously mentioned voltammetric methods, differential pulse voltammetry with a modification named after its founder “Brdicka reaction” is the most commonly used electrochemical method for detection of MT in various types of samples since Olafson optimized it on fish tissues [48,49]. Over several decades, the method has been optimized with detection limit under fM [33]. Temperature of the supporting electrolyte (app. 5 °C) and concentration of cobalt(III) ions (app. 1 mM) play the key role in the reaching the lowest detection limit. Raspor attempted to elucidate the exact mechanisms of this reaction [50]. Based on these results, Raspor and her colleagues have done a lot of work to propose physical and chemical conditions to achieve comparable results in various laboratories [51]. Moreover, unusual sample-preparation-steps including heat treatment of a sample must precede a measurement. Measurements can be also automated and thus used for larger set of samples, as was shown by Fabrik et al. [52]. In spite of the fact that Brdicka reaction is commonly used for detection of MT, Pedersen et al. showed that differential pulse polarography was found to be unsuitable for crustacean tissues due to unidentified interfering compounds which led to 5to 20-fold overestimation of metallothionein levels [31]. The interfering compounds such as other low molecular mass thiols, ionic strength or surfactants contained in a sample can be considered [53]. Besides voltammetric methods, chronopotentiometric stripping analysis (CPSA) can be also utilized for detection of MT. This method is the most sensitive analytical tool to detect and determine MT with detection limits estimated as units of aM [34]. Reaction and therefore sensitivity of determination depends on many parameters such as pH and ionic strength of supporting electrolyte, isoelectric point of measured protein. Temperature is not a concern
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compared to Brdicka reaction. Another study discovered that addition of [Co(NH3)6]Cl3 to a supporting electrolyte can increase sensitivity up to 30 % [54]. The signal amplification is probably caused by complex salt-protein formation. AdTS coupled with the CPSA method was used for the study of MT expressed in yeast Yarrowia lipolytica exposed to Zn, Ni, Co and Cd [55]. However, Petrlova et al. found that the CPSA signal of MT is dependent on content of metals in the sample, because MT-metal complex gives lower CPSA signal compared to metal free MT [56]. The results can re-calculated on the content of metals. Detection limits of electrochemical methods are summarized in Table 1. It is obvious that electrochemical methods are the most sensitive, however, they can suffer from faulty or inadequate preparation of a sample.
3.2
Immunochemical methods The second group of methods for MT detection includes use of antibodies (Fig. 1D). It
comprises techniques based on immunological detection of MT in whole tissues [57], Enzyme-linked immunosorbent assay (ELISA) with enzymatically labelled antibodies, radioimmunoassay (RIA) using isotopically labelled antibodies and Western blotting. In pathology, the technique of immunohistochemistry is widely used to visualise MT distribution in tissues. Frequent targets of analysis are tumour tissues, in which MT expression is changed between perineural and nonperineural prostate cancer [58]. More sensitive techniques than RIA with fluorescence detection using lanthanide-labelled antibodies have been proposed [59]. ELISA was optimized for detection of MT-1 in plasma and urine [60]. This method can be widely modified and optimized for direct MT detection in many types of samples without difficult sample preparation procedures. Potential oxidation of MT which can cause ten-fold overestimation of results can be avoided by addition of reducing or chaotropic agents, e.g. mercaptoethanol [61] or Tween-20 [62], respectively. To enhance sensitivity, gel separation
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can be used before immunoanalysis [63]. A protocol was also developed for a competitive ELISA method [64], which uses IgG rabbit antibodies and can be applied to the detection of MT in tissues and serum. MT was analyzed by the ELISA technique in oyster Ostrea edulis [65], fish Lithognathus mormyrus [66] and after separation by liquid chromatography (size exclusion and anion exchange) in fish Carassius carassius [67]. ELISA is often utilized for analysis of human tissues [18]. ELISA was also compared with Brdicka reaction and the results were in good agreement [68]. Detection limits of the ELISA method are in the region of ng per ml (Tab. 1). Another group of immunological methods for MT detection, blotting techniques, uses immobilization of protein on membrane (nitrocellulose or polyvinylidene fluoride (PVDF)). Recently, using of PVDF membranes were compared to other types of membranes and it was found that combination of PVDF membrane, chicken yolk antibodies and 3-aminoethyl-9-carbazole as chromogenic substrate was the most sensitive with detection limit estimated as 3 pg of MT per 1 µl [69]. In an in vitro study, researchers monitored MT by Western blot and dot blot during incubation with S-nitroso-glutathione, reduced glutathione and hydrogen peroxide, and reduced glutathione, and diamide considering possible nitrosilation of MT cysteines, which can occur in a real sample [70]. The main obstacles in using ELISA and other immune methods are the need to avoid degradation of the target molecule, cross reactivity of polyclonal antibodies and possible interferences of higher metal content. These methods are more suitable for qualitative detection of MT than quantification.
3.3
Mass spectrometry of metallothioneins For analysis of proteins by mass spectrometry two ionization techniques are mainly
used: electrospray ionization (ESI) and matrix-assisted laser desorption-ionization (MALDI). These techniques are characterised by a soft type of ionization which lowers the probability of protein destruction. ESI is used in detection systems for on-line protein measurements within
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masses from 500 to 3,000 (m/z), in which the separation step is preceded by capillary electrophoresis or liquid chromatography. The presence of multiple charged ions is not unique to this ionization technique. Thanks to this phenomenon, ESI can be used even for masses exceeding 3,000 Da [23,71,72]. Soft ionization due to using of ESI is capable of preventing the formation of metal-protein complexes. Therefore, coupling of this ionization with mass spectrometry (MS) can help in identification of protein isoforms and the stoichiometry of metal complexes [73]. Recently, capillary electrophoresis coupled with ESI-MS was used for validation of results obtained from electrophoretic mobility equations of MT isoforms depending on pH [74]. ESI-time of flight analyser (TOF) was used for MT detection in Mytilus edulis, whose gene was inserted into Escherichia coli culture [75]. Due to its low molecular mass, ESI became topical in MT research [76]. MALDI is soft ionization technique suitable for protein analysis and it is more tolerant than ESI to higher sample mass and to content of salts, buffers and other substances. MALDI ionization coupled with TOF/TOF analyser was used for rapid identification and characterization MT isoforms from a prostate cell line [77]. MALDI-MS was used for studying of cadmium bonds in MT of the fungus Heliscus lugdunensis after cadmium exposure [78]. Binding characteristics of human MT-2 for As3+ were investigated among other methods by MALDI-TOF [79] and results were shown to be strongly pH dependent. Moreover, MALDI TOF seems to be suitable technique for in vitro interaction studies of MT with other molecules, mainly metals and metal-based drugs [80]. It can be concluded that ESI-MS and MALDI-TOF and their analogues are very convenient for detecting MT and analysis of in vivo prepared mixtures. Using of these techniques for quantification or for analysis of crude or impure samples without good purification steps should be a concern and needs to be taken into account.
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4.
Separation methods Previously mentioned detection methods can be used separately, but coupling with
appropriate separation techniques can bring many advantages including sensitivity, selectivity, reduced interferences etc. Among separation techniques used for MT analysis various types of electrophoreses and chromatographic instruments can be used.
4.1
Gel electrophoresis Polyacrylamide gel electrophoresis (PAGE) belongs to the so-called gold standard in
proteomics. Native or denaturating protocols can be utilized for MT detection. Due to low MT mass and its easy reoxidation during electrophoretic run it is necessary to use gels with acrylamide concentration app. 15 – 17.5 % or gradient gel electrophoresis. Native electrophoresis in Tris-glycine buffer with Coomassie-blue and silver staining visualization was used for densitometric quantification of MT-1 and MT-2 isoforms in animal tissues. Due to this staining protocol the authors were able to specifically amplify MT band intensity, however, the detection limit (2,000 ng of MT) was relatively high in comparison to other proteins [81]. If a real sample is analysed with good resolution, the proteins can be denaturated by sodium dodecyl sulfate (SDS) [24]. Despite all the advantages of SDS-PAGE it is not unusual that bands contain different proteins with the same mobility. Occasionally, the formation of artefacts can be seen due to reoxidation which can be observed as a smear or causing smudgy bands and migration of protein of interest at higher molecular masses than expected. This can be prevented by carboxymethylation of a protein by iodoacetic acid prior to the electrophoresis [82,83]. Nevertheless, detection limits of unspecific Coomassie-blue staining, which is difficult due to the absence of aromatic amino acids in MT structure, or silver staining, which is time-consuming and may be problematic in cases of some types of metal ions in MT, mainly Cu [81], vary by microgram amounts. Lower detection limits can be
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reached by autoradiography, storage phosphorimaging or fluorescent staining [84]. From the fore-mentioned types of detection, the last one, fluorescent staining, is very convenient for detection and quantification of MT in biological samples, mostly of invertebrates [85-87]. Detection of MT by amido black can be a concern [88]. Orthogonal separation techniques are needed to separate MT in the presence of large amounts of other low molecular mass proteins or analyse of a sample containing numerous MT isoforms, e.g. coupling of isoelectric focusing with PAGE [89], or immunoelectrophoresis based on protein interactions with antibodies incorporated in a carrier [90]. Other interesting coupling was done by Otsuka et al., whose used metal-chelating column chromatography for protein separation according to affinity for Zn and Cd metals and then employed SDS-PAGE [91]. Krizkova et al. used a chip-based capillary electrophoresis for quantitative study of MT oxidation. After oxidation of MT by H2O2 they observed marked decrease in peaks heights and shift of peaks positions to higher molecular mass, which corresponded with the time of the oxidation [92]. In general, MT detection and quantification by gel electrophoresis is a relatively difficult task thanks to small protein mass, nevertheless the method is popular for its indisputable advantages including low cost and ease-of-use.
4.2
Capillary electrophoresis Capillary electrophoresis (CE) is hot topic in proteomics, mainly for its excellent
resolution, rapidity, low sample volume demand or ability to separate differently charged and neutral molecules per a run [93]. The use of his technique for detecting of MT has been reviewed several times [22,61,94]. However, this technique has limitations, most of all, protein adsorption, which can be prevented by the use of lower pH of background electrolytes. Due to this, many proteins then become unstable. To suppress protein adsorption capillaries coated by derivatives of cellulose, polysterene nanoparticles, polyamides,
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polyacrylamides and other polymers utilized also for electroosmotic flow modulation are used [71]. A recently published study aimed at MT isoform separation and detection by capillary electrophoresis coupled with ESI-TOF, demonstrated the ability of these techniques to distinguish even subtypes of MT-1 and MT-2 isoforms [95]. Other authors compared the separation properties of capillary liquid chromatography and capillary electrophoresis using MT and superoxide dismutase as models. Both separation instruments were coupled with inductively coupled plasma (ICP)-MS detectors [96]. Results, although very similar, support using capillary liquid chromatography instead of capillary electrophoresis, nevertheless opinions differs [93]. Due to several optimization steps Chamoun and Hagege enhanced the sensitivity of capillary electrophoresis-ICP-MS for the detection of metalloproteins by more than six fold [97]. The great advantages of CE in detection and quantification of MT are its ability to distinguish MT isoforms in crude samples and ultralow detection limits (Tab. 1). Commercially available standards of MT isoforms are not adequate to be used CE due to impurities and poorly defined mixtures of various MT isoforms in a standard. In addition, heat treatment, molecular filtration or solid-phase extraction must be performed before the analysis. Therefore, adding of internal standards such as carbonic anhydrase to a real crude sample is needed.
4.3
Chromatographic methods Previously mentioned electrophoretic methods are convenient for detection of MT, but
they have also several limitations. Due to the relatively small molecular mass of MT we can also use chromatographic methods. Gel chromatography fractionating molecules according their size is obviously the first separation step. For MT separation silicates and organic polymers with pores (10 – 100 nm) as a column packing are employed. Water or cytosol-like
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buffers are used as the mobile phase for preservation of MT nativity, MT-metal complexes and for minimization of ligand competition. However, under these conditions MT binds tightly to the stationary phase. Higher ionic strength is required to disrupt the interaction between stationary phase and MT, but can also damage higher structures of MT and their complexes [98]. Another solution to the problem of the interaction between MT and stationary phase is to use different column packing material (e.g. co-polymeric styrenedivinylbenzene). For samples containing larger amounts of heavy metals, it is appropriate to use orthogonal separation techniques such as capillary electrophoresis or other chromatographic methods.. Another commonly used technique is high performance liquid chromatography (HPLC), particularly ionex chromatography or reverse phase-HPLC (RPHPLC). Column packings in ionex chromatography for MT analysis are mostly anionexchange molecules e.g. weakly basic diethylaminoethyl cellulose [23]. MT isoforms from the bivalve Laternula elliptica after cadmium exposure were separated using anion-exchange chromatography [99]. Packing of columns for RP-HPLC as alternative for anion-exchange chromatography consists from aliphatic hydrocarbons (C8 and C18). Decreasing of mobile phase polarity by the addition of methanol or acetonitrile is often used to elute MT from hydrophobic chains. Based on the results published it can be concluded that RP-HPLC is more convenient for metallocomplexes including MT separation than anion-exchange chromatography. Due to the absence of potent ligands for MT in stationary phase RP-HPLC measurement is however time-consuming [23,100]. Size-exclusion chromatography (SEC) followed by RP-HPLC were used for separation of MT isoforms of snails (Helix aspersa), which were exposed to Cd2+ ions [101]. In a study focusing on MT in fish (Dicentrarchus labrax) RP-HPLC served as a separation technique [102] following exposure to doses of Cu, Cd and Zn. Mass spectrometry is most often used to identify proteins from fractions obtained after
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chromatographic separation. If there are no library spectra available, enzymatic digestion of analyte or tandem mass spectrometry can be used to determine the amino acid sequence. Of concern is the connection of a hyphenated separation technique with a mass detector. Unfortunately each separation technique has its own characteristic flow rate which does not match the optimum for ionization sources for mass detector and, moreover, salt and organic solvents in the mobile phase needed for separation are the main cause of interference. Particularly, a flow rate suitable for ICP ionization may be very similar to that of HPLC, however, the high content of organic substances results in carbon sedimentation on the ICP torch and the plasma, thus, become unstable. This can be prevented by addition of oxygen to the plasma gas and also by the removal of the solvents. Another solution of this negative phenomenon is using capillary HPLC or nanoHPLC due to lower content of interfering substances, but flow rates (capillary HPLC 4 µl/min and nanoHPLC 200 nl/min) are insufficient for ICP [23]. Trace element analysis of cerebrospinal fluid was performed by high resolution-ICP-MS, whereas MT isoforms in this liquid were separated by SEC-HPLC. Due to non-denaturating conditions in mobile phase, MT-metal complex formation was prevented [103]. SEC-HPLC coupled with ICP-MS was also used to discover Cu-MT complexes in whey from bovine milk [104]. Chromatographic methods under wisely chosen conditions represent very suitable tools for detection of MT. Nevertheless, detection limits are higher compared to CE and others (Tab. 1). The advantage of chromatography is the possibility to use an extract from a crude sample without numerous laborious and time-consuming purification steps.
5.
Indirect detection of metallothioneins Besides direct separation and detection of MT we can measure these proteins
indirectly via the content of heavy metal ions occurring in their molecules. This way of
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determination has several advantages especially
sensitivity and precision. Saturation
protocols and spectroscopic methods belong to the most commonly used ones for this purpose.
5.1
Saturation assays Saturation assays, whose principle depends on the different affinity of MT to heavy
metal ions (affinity decreases in order Hg(II) > Ag(I) ~ Cu(I) > Cd(II) > Zn(II) [105]), can be used for indirect MT determination (Fig. 1). Unsurprisingly, the displacement of metals by Hg(II), for which MT has strong affinity, is the most commonly used [106,107] approach. Displacement of the unstable isotope
203
Hg is most commonly utilized for this purpose.
Recently a new method with stable Hg isotope in low concentrations was proposed [108]. Removing of non-specifically bounded metals can be achieved by addition of egg-white solution [109]. In addition to mercury, titration by Cd(II) or Ag(I) are employed for detection of MT [105,110]. When using Ag(I), it is necessary to take into account that higher chloride levels cause precipitation with silver(I) ions. Van Campenhout et al. quantified MT level by cadmium thiomolybdate titration in carp tissues (Cyprinus carpio), but total content of individual metal ions was determined by ICP-TOF [111]. Saturation assays belong to the first methods used for quantification with sufficient detection limit (Tab. 1), however, there are several limitations of using this method mainly in the case of high copper content.
5.2
Spectroscopic methods The main advantage of metal detection in proteomics is the fact that we easily quantify
target protein, whereas direct protein detection can be difficult [23,73]. ICP-MS, which is even capable of distinguishing other heteroatoms than metals, is the most commonly used tool in indirect detection of proteins. It is possible to detect and distinguish single elements and
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their isotopes with simple sample preparation [112]. In spite of the fact that ICP-MS is highly sensitive, specificity related to detection of individual proteins is doubtful, because it is very difficult to distinguish the origin of detected elements when a mixture of different analytes is measured. Therefore, ICP-MS is connected with appropriate separation techniques, mainly with capillary electrophoresis and/or liquid chromatography [93]. As a detector, quadrupole, sector field and/or time-of-flight mass spectrometers are employed. Sector field mass spectrometry overtakes the most frequently used quadrupole mass spectrometry (QMS) particularly in resolution [113]. ICP-TOF-MS has higher detection limits than QMS, however new technological devices to overcome this lack are still being proposed. Moreover, there is no troubleshoots with time pause between analysis of different masses (spectral skew error) [114]. This distortion negatively influences the signal height of different masses from fraction with the same retention time. Due to the specific sulphur and heavy metal content of MT, the isotope dilution method (ID) using 34S isotope can be used for the detection of metalloproteins including MT [115,116]. Comparing non-isotope and isotope signal intensities it is possible to detect sulphur content and, thus, protein content. Wider application of these techniques remains limited due to un-proportional sulphur distribution in proteins (particularly amino acids cysteine and methionine) or due to the method of detection itself, because sulphur with high first ionization potential gives a low yield of ionized molecules (10 %). Moreover a high resolution analyzer is required (such as SFMS) due to the possible overlap of 16
32 +
S with ion
O2+, which common quadrupole mass spectrometers lack [117]. The overlap problem can be
also solved by the use of a collision cell. The ID method can be also used for detection of metals as shown by Van Campenhout et al., which used SEC-HPLC coupled with ICP-TOFMS with post column addition of 65Cu, 67Zn and 106Cd isotopes for detection of MT levels in eels [118].
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SEC-HPLC coupled with ICP-MS were utilized to determine MT content in gills, kidneys, livers and muscles of carps (Cyprinus carpio L.), which were exposed to Hg, Cd and Pb [119]. Results show dis-proportional distribution of saturated MT in these organs, whereas MT in gills can be considered as a biomarker of Cd and Hg pollution. The same technique enabled Esteban-Fernandez et al. to investigate the formation of aqua-complexes of cisplatin with MT in vitro and in vivo experiments after administration of to rats [120]. Narrow-bore HPLC-ICP-MS technique was used in the study of copper metabolism in mutant mice with functionless gene for ATP-ase transporters of copper Atp7a [121]. Measurements were taken of superoxide dismutase and MT as proteins with copper binding ability. Results, which were correlated with mRNA detection, showed increased occurrence of Cu-MT in intestine and in kidneys. However, higher content of Cu-MT did not correlate with mRNA expression, which can be explained by a higher transport rate of Cu-MT into that organ. In addition to the methods described above for the detection of MT in gels or on membranes, roentgen methods (X-ray fluorescence) and ICP-MS with laser ablation coupled with gel electrophoresis are also used [122]. Although the metal-content detected by these techniques can be proportional to MT, an obstacle to their use is the noise signal generated from other elements (S or P) and, moreover, the techniques make higher demands on instrumentation and operators. For these reasons, the techniques are not in general use for metallothionein detection and quantification [25]. Besides these two specific types of detectors, spectrometric techniques in general belong to the most versatile instruments for metallothionein quantification. They can be coupled almost with all separation techniques and, therefore, they can be utilized for quantification of content of all and/or specific MT isoforms and for of large scale analyses of impure samples without difficulty.
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6.
Conclusions and outlooks Metallothioneins are of interest in various fields including environmental chemistry,
biochemistry, clinical chemistry, and also analytical chemistry. Due to their unique primary structure (no aromatic aminoacids, rich in cysteine moieties) these proteins are involved in many biochemical pathways including scavenging of reactive oxygen species, detoxifying of various xenobiotics and metal ions, transporting of essential metal ions, cell proliferation etc. To cast the light on this issue, battery of precise, sensitive and selective analytical instruments is needed. Due to trends to miniaturize whole detection systems, newly designed instruments based on paramagnetic particles and small well portable devices have become to the focus of much attention. Moreover, it is reasonable to assume that hyphenated spectroscopic instruments will reveal new transport mechanism and interactions of MT with other biologically active compounds, because it seems that these proteins could also serve as a reservoir of essential heavy metals for other heavy metal binding proteins including transcription factors.
Acknowledgements Financial
support
from
GA
MSM0021622412 is highly acknowledged.
AV
IAA401990701
and
INCHEMBIOL
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Table 1: Summary detection limits of various methods used for detection of MT. Determination Sample
Method
Detection limit (µg/ml)
Reference
MTs
Human blood serum
DPV-Brdicka reaction ([Co(NH3)6]Cl3)
0.0000068*
[123]
MTs
Standard
CV
0.816*
[33]
DPV
0.00544*
DPV-Brdicka reaction ([Co(NH3)6]Cl3)
0.0000136*
MTs
Nereis (Hediste) diversicolor
SW-CSV-Brdicka reaction ([Pt(NH3)2Cl2]) 0.0003
[124]
MTs
(Cyprinus carpio L.) spleen, liver, and testes
CPSA
0.0016
[34]
MTs
Standard
DPASV (bismuth film electrode)
0.26248*
[125]
MTs
Cultured human Chang liver cells
HPLC-UV
0.031
[61,126]
MT-1, MTLP
Earthworm Eisenia andrei
HPLC-UV (SBD-F derivatization)
0.06 (5µg/g of tissue)
[127]
MTs
Mouse tissues and cells lines (L cells, HepG2 cells and HeLa)
HPLC-FD (SBD-F)
<0.9 ng/ injection
[128]
MT-1,2
Mussel hepatopancreas cytosol
CZE-UV
4 (MT-1)
[129]
MTs
Sheep foetal liver
SPE-CE-UV
3 (MT-2) 0.028 (sheep standard MT-1)
[130]
0.272 (sheep liver samples) MTs
Caretta caretta, Chelonia mydas
GFAAS
Cd - 0.2 ng/g of tissue
AAS
Zn - 0.08 µg/g of tissue
[131]
Cu - 0.12 µg/g of tissue MTs
Rat livers
SPE-TDI-AAS
0.007 (water)
[132]
< 0.009 (human fluids) MT-1,2
Rat livers
AE-HPLC-AAS
0.46 (MT-1)
[133]
0.31 (MT-2) Cd-MTs
Bakers yeast
ICP-OES
0.0012 (Cd-MT)
[134]
MT-II
Standard
HPLC-ICP-MS
0.000102 (MT-II via sulphur)*
[116]
113
Mouse Hepa 1-6 cells
2D micro HPLC-ICP-MS
22.2 ( Cd-MT-I,II)
[135]
MTs
Porcine liver
SEC-RP with ICP-MS and ESI-MS
< 0.6 (Cd7-MT)
[136]
Cd-MTs
Synechococus sp.
CE-ICP-MS
0.001 (Cd-MT-I,II, CPN nebulizer)
[137]
Cd-MT
113
0.0001 (Cd-MT-I,II MCN nebulizer)
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Determination Sample
Method
Detection limit (µg/ml) 114
MTs
Standard
CE-ICP-MS
1.52 (via
MTs
Rat tissue
CE-ICP-SFMS
80 (via sulphur)
MTs
Brain tissue
CE-ICP-SFMS
Cd)
Reference [23,138] [139]
0.011 (MT-1 via
114
0.023 (MT-2 via
114
Cd)
[140]
Cd)
MTs
Rat liver cytosol
ICP-MS (isotope dilution)
0.03
[141]
MTs
Artemia, Procambarus clarkii
Saturation method (Ag)
Ag 0.03 ppm
[142]
MTs
Urine, hepatic cytosol
ELISA
0.008
[63]
MT-1
Rabit liver and kidney cytosols
ELISA
0.004
[143]
MT-3
Cerebrum (rats)
Western blot
10 µg of protein
[144]
* MT (Mr) = 6800 Da
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Captions for Figures
Figure 1 (A) Metallothionein structure. Model of two binding sites of metallothionein according to [33]. (B) Detoxification of metal ions by metallothionein. Nucleus. A heavy metal ion enters through a cytoplasmic membrane of a cell via ionic channels or special transporters. After the entering the cytoplasm, the ion interacts with complex of metal-regulatory transcription factor-1 (MTF-1) and metal synthesis inhibitor (MTI). The ion binds to MTI. Due to this MTF-1 is released and can bind to a regulatory sequence of DNA called metal responsive element (MRE). Then, the gene responsible for synthesis of metallothioneins is transcribed. The synthesized mRNA molecule is translated into MT. MT binds to the heavy metal ion. Kidney. The metal-MT complex can be transported to the kidney, where the metal is excreted due to degradation of MT or changing of pH. (C) Number of full length articles, reviews, meeting abstracts and proceedings papers having “metallothionein*” in title, abstract and keywords per year according to Web of Science. (D) Analytical “targets” for detection of metallothioneins. Model of two binding sites of metallothionein and targets for analytical tools. The analytical instruments detect whole protein and specific sequence directly or sulphur and metal ions indirectly.
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C. Vliv kovů přirozeně obsažených ve struktuře metalothioneinu na jeho stanovení
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Determination of apo-Metallothionein Using Adsorptive Transfer Stripping Technique in Connection with Differential Pulse Voltammetry Vojtech Adam,a,b Sona Krizkova,a,b Ondrej Zitka,a,c Libuse Trnkova,d Jitka Petrlova,a Miroslava Beklova,e Rene Kizeka* a
b c d
e
Laboratory of Molecular Biochemistry and Bioelectrochemistry, Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic *e-mail:
[email protected] Department of Genetics and Molecular Biology, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic Department of Biochemistry, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic Department of Theoretical and Physical Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic Department of Veterinary Ecology and Environmental Protection, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, CZ-612 42 Brno, Czech Republic
Received: July 21, 2006 Accepted: October 5, 2006 Abstract Links between metallothionein (MT), its structure and many biologically important pathways demonstrate the necessity of taking into account of studying the behavior of MT in different well defined mediums, which could help to model conditions in an organism easily. The main aim of this work was to prepare and determine apoMT by adsorptive transfer stripping technique (AdTS) in connection with differential pulse voltammetry (DPV). Particularly, we investigated the electrochemical behavior of MT measured on the surface of hanging mercury drop electrode in the presence of sodium chloride as supporting electrolyte using AdTS DPV with respect to study the effects of MT signals by different concentrations and pH=s of the electrolyte. Then, we aimed at utilizing this technique to observe changes in MT which are dependent on using strong chelating compounds ethylendiamine-N, N, N’, N’-tetraacetic acid (EDTA). Thanks to ability of EDTA to bind heavy metals from active center of enzymes and regulation proteins, we were able to prepare apoMT; that means, MT without any metal ion bound in its structure. Detection limit of apoMT at very short time of accumulation (tA ¼ 120 s) was 3 nM (20 ng/mL and/or 15 fmol in 5 mL drop; RSD ¼ 2 – 5%) estimated by dilution of the analyzed solution until the signal disappeared. In addition, it was possible to decrease detection limit by extending of time of accumulation of apoMT on the surface of HMDE. We were able to detect 30 pM of apoMT (200 pg/mL and/or 150 amol in 5 mL drop) at tA ¼ 500 s. Keywords: Differential pulse voltammetry (DPV), Adsorptive transfer stripping (AdTS) technique, Hanging mercury drop electrode (HMDE), Metallothionein, Reaction of thiol group, Heavy metals biosensor, EDTA DOI: 10.1002/elan.200603738
1. Introduction Metallothioneins (MT) belongs to group of intracellular, high molecular and cysteine-rich proteins with molecular weight from 6 to 10 kDa [1 – 6]. The MT was discovered in 1957, when Margoshes and Valee isolated it from horse kidney [7]. MTs consist of two binding domains (a, b) that are assembled from cysteine clusters. Cysteine sulfhydryl groups participate in covalent bindings with heavy metals [8] (see Fig 1A). The N-terminal part of the protein is marked as a-domain, which has four binding places for divalent ions. bDomain (C-terminal part) has the ability to bind three divalent ions of heavy metals. In the case of univalent ions of heavy metals, MT is able to bind twelve metal ions. A crystal structure of metallothionien binding cadmium and zinc was described in 1986 [9]. Electroanalysis 19, 2007, No. 2-3, 339 – 347
The assumed structure of MT has been well described by number of different analytical techniques. Nevertheless arrangement and placement of metals binding in MT has been still discussing intensively [10, 11]. Moreover, it seems that MT could have more crucial biological properties than regulation of content of toxic metals in an organism [11 – 15]. Particularly, very interesting links between MT concentration and cell proliferation or fetus development have been described. For that reasons a question was raised: “How can be level of MT regulate in an organism, and thus, how can be MT degrade?” A few papers describing regulation of MT gene expression have been published, but not of all mechanisms are clear yet [16]. On the other hand about “fate” of metals bound to MT molecule is not clear yet. Several models and hypotheses showing their active transport to excretory system (kidney) have been suggested, but, unfortunately, what happen with M 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Fig. 1. Model of structure of rabbit liver metallothionein (A). The metallothionein with five bound atoms of cadmium and two of zinc is shown. Adsorptive transfer stripping technique in connection with differential pulse voltammetry. Typical DP voltammograms of 10 mM MT (B), 5 mM MT (C), 0.1 mM MT (D) measured in the presence of 0.5 M NaCl, pH 6.4. AdTS DPV parameters were as follows: initial potential 1.2 V, end potential 0.3 V, modulation time 0.057 s, time interval 0.2 s, step potential of 1.05 mV/s, modulation amplitude of 250 mV, time of accumulation of MT 120 s, Eads ¼ 0 V. For other details see Section 2.
MT in kidney has not been described sufficiently. There could proceed a number of interactions between heavy metals, reactive oxygen species and kidney cells [17, 18] including releasing of heavy metal ions to urine, which take place in glomerulus cells. The ions release thanks to decrease of pH. This mechanism is well known and used for preparation of MT without any metal (apoMT). Facts mentioned above demonstrate the necessity of taking into account studying of behavior of MT in different well defined medium, which could help to model conditions in an organism easily [19]. Electrochemical technique seems to be very promising tool for these purposes thanks to high sensitivity and selectivity to thiols. A number of published Electroanalysis 19, 2007, No. 2-3, 339 – 347
papers dealing with study of thiols (e.g., rabbit MT-I, MT-II , horse MT, human MT and peptide Lys-Cys-Thr-Cys-CysAla thionein fragment MT I) by electrochemical techniques in the presence of various supporting electrolytes and pHs using different working electrodes has been published [20 – 39]. Moreover, catalytic signals (peak H and Brdicka reaction) are of great importance for sensitive analysis of MT in biological samples [40]. It is supposable that a SHgroups-rich protein and ions such as Co, Ni and/or cisplatin serve as catalysts of these signals [40 – 52]. It follows from these experimentally results that a supporting electrolyte (its chemical composition and concentration of single compounds consisted from, ionic strength and pH) probably
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influence electrochemical signal of MT [23]. The changes observed relates with different MT structure including releasing of bound metal ions in the presence of the supporting electrolyte with low pH [23]. Published papers and results obtained describing electrochemical determination of MT, from 1980 to June of 2006, according to Web of Science, are summarized in Table 1. The main aim of this work was to prepare and determine apoMT by adsorptive transfer stripping technique (AdTS) in connection with differential pulse voltammetry (DPV). Particularly, we investigated the electrochemical behavior of MT measured on the surface of hanging mercury drop electrode in the presence of sodium chloride as supporting electrolyte using AdTS DPV. Then, we aimed on utilizing of this technique to observe changes of MT in dependence on different pH and concentration of supporting electrolyte, and on using of strong chelating compounds ethylendiamine-N, N, N’, N’-tetraacetic acid (EDTA) with respect to prepare apoMT.
2. Materials and Methods 2.1. Chemicals Rabbit liver MT (MW 7143), containing 5.9% Cd and 0.5% Zn, was purchased from Sigma Aldrich (St. Louis, USA). Sodium chloride, ethylendiamine-N,N,N’,N’-tetraacetic acid (EDTA) and other chemicals used were purchased from Sigma Aldrich. Stock standard solutions of MT (10 mg mL1) were prepared by ACS water (Sigma-Aldrich, USA) and stored in the dark at the temperature of 20 8C. Working standard solutions were prepared daily by dilution of the stock solutions. The pH of prepared solution was measured using WTW inoLab Level 3 (Weilheim, Germany). The pH-electrode (SenTix-H, pH 0 – 14/3 M KCl) was regularly calibrated by set of WTW buffers (Weilheim, Germany).
2.2. Spectrophotometric Measurements Spectra were recorded by means of UV-viz diode array detector (HP-Packard) in the range of 190 – 400 nm.
Table 1. Summary of electroanalytical determination of metallothionein from 1980 to June 2006. Metallothionein
Electrochemical Supporting electrolyte method
Rabbit liver ( MT-I, MT-II, MT-I þ MT-II, horse kidney DPP peptidic fragment Lys-Cys-Thr-Cys-Cys-Ala thionein human MT ( MT-I, MT-II) Human MT DPP rabbit liver MT peptide Lys-Cys-Thr-Cys-Cys-Ala thionein fragment MT I Rabbit liver MT DPP-Brdicka reaction Rabbit liver MT
Rabbit liver MT Peptide Lys-Cys-Thr-Cys-Cys-Ala thionein fragment Rabbit liver MT Rabbit liver MT Rabbit liver MT Peptidic fragment Lys-Cys-Thr-Cys-Cys-Ala MT I, rabbit liver MT apoMT Rabbit liver MT Rabbit liver MT
Rabbit liver MT Rabbit liver MT
0.01 M phosphate buffer pH 7.5 pH (2 – 12) adjusted by NaOH, HCl 0.02 and 0.01 M phosphate HEPES buffer pH (6 – 9) adjusted by NaOH, HCl
0.6 and/or 1 mM Co( NH3)6Cl3 and 1 M ammonia buffer (NH3(aq) þ NH4Cl, pH 9.6) DPV borate buffer pH 8.5 borate buffer (pH 9.1) þ 0.1 M NaClO4 þ 10% vol CH3OH AdTS DPV 0.5 M NaCl, pH 6.4 SWV 0.02 M phosphate HEPES buffer pH (6 – 9) adjusted by NaOH, HCl SVW-modified 0.5 M borate buffer pH 9.2 þ 10 mM Brdicka reaction cisplatin CV borate buffer pH 8.5 CV 0.1 and/or 0.01 M phosphate HEPES buffer pH 8 CV Tris-buffer pH 8.5 CPSA borate buffer pH 8.5 CPSA 0.4 mM Co( NH3)6Cl3 and 1 M ammonia buffer (NH3(aq) þ NH4Cl, pH 9.6) AdTS CPSA 0.1 M H3BO3 þ 0.05 M Na2B4O7, pH 8.0 EVLS borate buffer pH 8.5
References [21, 22, 58, 66, 67]
[23, 60, 61, 68]
[40 – 43, 69 – 71]
[25] [72]
[34] [60, 61, 68, 73]
[52, 74] [25] [75, 76] [72] [25] [77]
[29, 78, 79] [25]
According to ISI Web of Knowledge
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Table 2. Recovery of preparation of apoMT by means the interacting of EDTA with MT. Concentration of MT (nM )
Concentration of EDTA (nM )
Height of CdT peak (nA )
Maximal CdT peak height [a] (nA )
Recovery (%)
100 100 100 100 100 100 100 100 100 100 100 100 100
0 100 200 300 400 500 550 600 650 700 740 770 800
10.0 10.8 10.5 10.9 11.2 12.0 13.7 16.9 17.0 17.0 17.0 17.0 17.0
17.0 17.0 17.0 17.0 17.0 17.0 17.0 17.0 17.0 17.0 17.0 17.0 17.0
58.7 61.0 61.6 63.9 65.7 70.4 80.3 99.1 100 100 100 100 100
[a] Peak height of CdT measured at the highest dose of EDTA (800 nM ).
2.3. Electrochemical Measurements Electrochemical measurements were performed with the AUTOLAB Analyzer (EcoChemie, Netherlands) connected to VA-Stand 663 (Metrohm, Switzerland), using a standard cell with three electrodes. The working electrode was a hanging mercury drop electrode (HMDE) with the drop area of 0.4 mm2. The reference electrode was the Ag/ AgCl/3 M KCl electrode and the auxiliary electrode was the graphite electrode (GE). The analyzed samples were deoxygenated prior to measurements by purging with argon (99.999%), saturated with water for 120 s. All experiments were carried out at room temperature. For smoothing and baseline correction [53], the software GPES 4.4 supplied by EcoChemie was employed.
2.4. Adsorptive Transfer Stripping (AdTS) Differential Pulse Voltammetry (DPV) MT has been analyzed using AdTS DPV (principles of the transfer technique was described by Palecek [54]). The supporting electrolyte (sodium chloride: 0.5 M NaCl, pH 6.4) from Sigma Aldrich in ACS purity was purchased. DPV parameters were as follows: the initial potential of 1.2 V, the end potential 0.3 V, the modulation time 0.057 s, the interval 0.2 s, the step potential of 1.05 mV/s, the modulation amplitude of 25 mV.
3. Results and Discussion 3.1. Electrochemical Behavior of Metallothionein Primarily, we attempted to characterize metallothionein used electrochemically (rabbit liver, Mr 7065, MTCd5Zn2). We picked up the threads of our previous works and analyzed MT in the presence of 0.5 M NaCl (pH 6.4) [34, 55 – 57]. Our experiments are based on original methods Electroanalysis 19, 2007, No. 2-3, 339 – 347
using adsorptive transfer technique (MT is adsorbed on the surface of HMDE from low volume of samples 5 mL drop). Typical well reproducible AdTS DP voltammograms of rabbit liver MT (100 nM, 5 and 10 mM) measured in the presence of 0.5 M NaCl (pH 6.4) at tA of 120 s is shown in Figs. 1B, C, D, respectively. We observed all assumed electrochemical signals of MT complexes with Cd(II) and Zn(II) MT(Cd): 0.42 V; MT(Zn): 0.49 V, ZnT’: 0.87 V, CdT: 0.65 V, CdT’: 0.71 V and ZnT: 0.99 V (more detailed description of the signals are shown in following papers [23, 34, 56, 58]). In addition, we can see strong influence of MT concentration on the signals (Figs. 1B, C, D), more will be published elsewhere. The great advantage of the adsorptive technique is the possibility to study behavior of MT adsorbed on a surface of working electrode without interferences both from samples and supporting electrolyte, where the analysis last only few minutes. Thanks to this technique we are able to observe the changes of electrochemical signals caused, first of all, by experimental conditions.
3.2. Influence of a Supporting Electrolyte and its pH on Electrochemical Signal of MT As it is shown in Table 1, a supporting electrolyte and its pH influence markedly the electrochemical records of MT. A few authors shown in Table 1 have used sodium chloride (0.5 M, pH 6.4) as a supporting electrolyte that has similar pH and ionic strength as physiological environment [34, 55, 56]. We again picked up the threads of a previous work and observed influence of changes of supporting electrolyte pH (0.5 M NaCl) within the range from 6.0 to 7.6 (adjusted by additions of HCl and/or NaOH) on electrochemical signal of MT. We found out that signals called as MT(Cd) and MT(Zn) decreased with increasing pH, whereas at the highest tested pH values they were poorly detectable (Fig. 2A). When we work on an assumption that MT(Cd) and MT(Zn) signals are associated with reducing of metal
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ions bound in MT, afterwards it clearly follows from the results obtained that MT molecule is going to be “packed” with increasing pH. This cause that metal ions can not be electrochemically changed on the surface of working electrode. On the other hand, if we use a lower pH, MT molecule is slightly “unpacking” its structure, which means that sulfhydryl moieties and metal ions bound in MT are more accessible to the surface of working electrode. Moreover, marked decrease of CdT and ZnT signals with increasing pH affirm this hypothesis (Fig. 2A). Thus, the decrease probably relates with worse accessibility of SH groups to HMDE surface. Besides that behaviour of ZnT’ and CdT’ signals is very interesting, because they increase with increasing pH, which shows that they could correspond to aminoacids or metal ions, which are not contained directly in the MT clusters (Fig. 1A). That means, if the structure of MT is really going to be “packed”, other metal ions have to be bound in the outer part of the clusters. Thus, they are more accessible to the surface of HMDE. The increase in heights of ZnT= and ZnT signals have been shown during polarographic analysis of rabbit liver MT-2 [59], but the authors done their experiments directly in electrochemical cell, and then MT analysis could be influenced by metals contained in the supporting electrolyte. In spite of the differences between transfer technique and analysis in the cell, similar dependences of MT signals have been obtained [60, 61]. The exact explanations about mechanisms influencing of MT are rather difficult. Conclusions would be suitable to support by physiological experiments, when lab animals would be feed by cadmium(II) and zinc(II) ions with consequently kidney sampling, where content of MT would be analyzed. It can be expected that the whole process including binding and releasing of metal ions from MT molecule will be based not only on the changing and reorganizing of cluster structure but also on other nondescribed mechanisms.
3.3. Influence of NaCl Concentration on MT Signal As we studied the influence of pH of NaCl on MT signals, we attempted to investigate the influence of different concentrations of NaCl within the range from 0.05 to 0.5 M under constant pH 6.4 adjusting by additions of HCl and/or NaOH. We found out that MT signals were influenced by different concentrations of NaCl but not so much as by different pHs. MT(Cd) and MT(Zn) signals changed according to different ionic strength markedly, whereas MT(Zn) signal measured in the presence 0.5 M NaCl was very low in comparison with 0.4 M NaCl. Contrariwise, CdT and ZnT signals did not change with increasing ionic strength of the supporting electrolyte. In addition the ZnT= and CdT= signals changed evidently than CdT and ZnT signals (Fig. 2B). As we discussed above, the results could be associated with structural changes of MT structure and with interaction of the structures formed with the supporting electrolyte [62]. It is likely that MT structure strongly depends on the electrolyte used, thus, if it will be used other electrolyte, the results will differ.
3.4. Influence of EDTA on AdTS DPV Signals of Rabbit Liver Metallothionein Here, we aimed on investigation of structural changes of MT causing by affecting of strong chelating compounds. These investigations are strongly needed for following experiments, which enable to study of important regulation processes such interaction between transcription factors and a metal [63]. One of the most commonly used chelating compounds for biological purposes is ethylenediamine-N, N, N’, N’-tetraacetic acid (EDTA). Surprisingly, this compound is able to bind heavy metals from active center of enzymes and regulation proteins. The experiment has been suggested
Fig. 2. Dependences of heights of ZnT, ZnT’, CdT’, CdT, MT(Zn), and MT(Cd) signals on pH (6.0 – 7.8) (A) and on concentration of the supporting electrolyte (0.05 – 0.5 M) (B). pH was adjusted by additions of NaOH and/or HCl. Other experimental conditions are the same as in Figure 1. Electroanalysis 19, 2007, No. 2-3, 339 – 347
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as follows: low concentration of MT (100 nM) was added to microtest-tube (20 mL) followed by addition of EDTA. This solution has been shaken slightly for 2 min. Aliquot (5 mL) has been analyzed by AdTS DPV. The experiment was repeated with different concentrations of EDTA. MT was adsorbed (tA ¼ 120 s) on the surface of HMDE, whereas EDTA and heavy metals were washed from the surface (Fig. 3A). All of the typical DPV signals of rabbit liver MT were observed: MT(Zn), MT(Cd), CdT, ZnT, ZnT= and CdT’. We found out that the signals observed except CdT decreased with increasing EDTA concentration up to 600 nM, then did not change much (Fig. 3B). On the other hand CdT signal markedly increased with increasing concentration of EDTAup to 600 nM. It could be suggested that interaction between EDTA and MT proceeds as follows: i) MTCd5Zn2 þ EDTA ¼ cluster collapse MS-protein (M-
metal; T-thionein) ii) MS-protein ¼ Mþ S protein ¼ Mþ þ S-protein and iii) Mþ þ EDTA ¼ MEDTA [64]. Structure of MT clusters has been probably damaging markedly from 400 nM EDTA, which was confirmed by the following experiments. If we studied the change of CdT signal in the presence of 400 nM according to different times of interactions (from 30 s to 25 min.), we observed that CdT signal did not change much within the range of experimental deviation (about 10%). Thanks to ability of EDTA unbound all metal ions from the MT structure; we assumed that apoMT can be obtained by addition of 600 nM EDTA (Fig. 3B). Comparison between AdTS DP voltammograms of 100 nM rabbit liver MT without and with addition of 600 nM EDTA are shown in Figure 3C. It clearly follows from the results obtained that MT without addition of EDTA gave all typical expected signals (see in Fig 1B),
Fig. 3. Scheme of using of adsorptive transfer stripping technique for study of changes of MT in the presence of EDTA (A). Renewed surface of HMDE (1) is placed to drop containing rabbit liver MT and EDTA (2); MT binds on the surface of HMDE only (3); low molecular compounds such as heavy metals and EDTA are washed out in the following step (4); the modified HMDE electrode is placed to supporting electrolyte (5) and analyzed by DPV (6). Dependence of heights of ZnT, ZnT’, CdT’, CdT, MT(Zn), and MT(Cd) signals on different concentrations of EDTA (B); the heights are expressed as ration to highest signal, whereas heights of CdT signals are shown in the left axis, other ones in the right axis. DP voltammograms (C) and UV-vis spectra (D) of 100 nM rabbit liver MT (bold line) and 100 nM MT þ 600 nM EDTA (thin line). Other experimental conditions are the same as in Figure 1 and Section 2. Electroanalysis 19, 2007, No. 2-3, 339 – 347
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whereas MT with EDTA gave only two signals: CdT that corresponds to free SH groups in MT clusters) and peak at potential of 1 V that corresponds to Zn from the supporting electrolyte (Fig. 3C). Moreover, the same solutions were analyzed using UV-VIS spectrophotometer. The changes observed are shown in Figure 3D. It follows from the results obtained that EDTA can really unbound metal ions from MT, which means that apoMT is presented in this solution. This procedure could be an useful alternative tool for simple, rapid and undemanding preparation of apoMT to different purposes such as a suggestion of heavy metal biosensor [65].
3.5. Electroanalytical Determination of apoMT As we mentioned above, we were able to prepare apoMT which has been characterized consequently by AdTS DPV. We found out that apoMT gave CdT signal at potential of 0.70 0.01 V (n ¼ 5). Therefore, we utilized this signal to study of recovery of the preparation of apoMT by means of EDTA interacting with rabbit liver MT. We found out that this signal increased with increasing concentration of EDTA up to 600 nM and then did not change (Table 1). We assumed that we prepared apoMT, which was confirmed by spectrophotometric measurements (Fig. 3D) and by results published in [64]. Based on the obtained results we used ratio of (rabbit liver MT)/EDTA as 1/6 to prepare apoMT in the following experiments. After the optimizing of apoMT preparation step, the changes in its signal with its changing concentration have been investigated. This signals slightly shifted to more positive potential with decreasing concentration of apoMT, whereas the signal appeared at potential of 0.585 V at 2 nM apoMT. In addition, we observed dependence of CdT
signal on concentration of apoMT. Due to sustaining of constant experimental conditions, apoMT was diluted by 0.5 M NaCl (pH 6.4). We obtained the strictly linear dependence with following equation (y ¼ 0.1935x þ 0.1926; R2 ¼ 0.9986) within the whole range of concentrations used (Fig. 4A). If we analyzed concentration of apoMT from 0.15 to 12 nM, we again obtained the strictly linear dependence (y ¼ 0.2053x þ 0.1052; R2 ¼ 0.9931; Fig. 4B). The CdT signals were well developed (Fig 4B). Detection limit of apoMT at very short time of accumulation (tA ¼ 120 s) was 3 nM (20 ng/mL and/or 15 fmol in 5 mL drop; RSD ¼ 2 –5%) estimated by dilution of the analyzed solution until the signal disappeared. In addition, it was possible to decrease detection limit by extending of time of accumulation of apoMT on the surface of HMDE. We were able to detect 30 pM of apoMT (200 pg/mL and/or 150 amol in 5 mL drop) at tA 500 s (Fig. 4C). In addition the limit quantification of apoMT was 100 pM at tA 500 s. Moreover, apoMT has been successfully used for suggestion of EDTA MT biosensor for determination of heavy metals [65]. We found out that the suggested biosensor had similar behavior as MT and/or PC biosensor, whereas the biological part of EDTA MT biosensor enabled to reach lower detection limits about 10 – 30% [34, 55].
4. Conclusions Utilizing of electrochemical biosensors using heavy metal binding peptides and proteins as a biological part belongs to new tools for sensitive analysis of heavy metals. We can use EDTA for unbinding of metals naturally occurs in the structure of these peptides and proteins, whereas consequently formed compounds without any metal embody better properties than the ones with heavy metals.
Fig. 4. Electroanalytical determination of prepared apoMT. Dependence of heights of AdTS DPV signals of rabbit liver MT on its concentration within the range of 0.075 – 100 nM (A) and 0.015 – 12 nM (B); inset: typical CdT signals measured at (0.188, 0.375, 0.75, 1.5, 3, 6, and 12 nM). Dependence of CdT peak height (5 nM of MT) on time of accumulation (C). Other experimental conditions are the same as in Figure 1 and Section 2. Electroanalysis 19, 2007, No. 2-3, 339 – 347
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5. Acknowledgements The results of this work were presented on 11th International Conference on Electroanalysis, Bordeaux, France. This work was supported by grants: GACR 525/04/P132, and GA AV CR A100040602, INCHEMBIOL 0021622412 and MSMT 6215712402.
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sensors ISSN 1424-8220 © 2008 by MDPI www.mdpi.org/sensors Full Research Paper
An Electrochemical Detection of Metallothioneins at the Zeptomole Level in Nanolitre Volumes Vojtech Adam 1,2, Jiri Baloun 1, Ivo Fabrik 1,3, Libuse Trnkova 4 and Rene Kizek 1,* Department of Chemistry and Biochemistry and 2 Department of Animal Nutrition and Forage Production, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-mail:
[email protected] 3 Department of Biochemistry and 4 Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic 1
* Author to whom correspondence should be addressed; E-mail:
[email protected] Received: 15 March 2008 / Accepted: 26 March 2008 / Published: 1 April 2008
Abstract: We report on improvement of the adsorptive transfer stripping technique (AdTS) coupled with the differential pulse voltammetry Brdicka reaction to determine a thiol-protein. The current technique has been unable to generate reproducible results when analyzing very low sample volumes (nanolitres). This obstacle can be overcome technically by modifying the current transfer technique including cooling step of the adsorbed analyte. We tested the technique on determination of a promising tumour disease marker protein called metallothionein (MT). The detection limit (3 S/N) of MT was evaluated as 500 zeptomoles per 500 nL (1 pM) and the quantification limit (10 S/N) as 1,500 zeptomoles per 500 nL (3 pM). Further, the improved AdTS technique was utilized to analyze blood serum samples from patients with breast cancer. Based on the results obtained it can be concluded that the improved technique can be used to detect a thiolprotein in very low sample volumes and can also prevent interferences during the washing and transferring step. Keywords: Proteomics, metallothionein, thiols, differential pulse voltammetry, Brdicka reaction, adsorptive transfer stripping technique, human blood serum, tumour disease, zeptomole
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1. Introduction Thiols play a significant role in a number of biological activities; however, many of their functions still remain unclear. Their involvement with regulating reactive oxygen species and metal ions, as well as in transcription and translation have been and continue to be studied extensively. They could also serve as markers for many health problems [1,2]. Metallothioneins (MT) are a group of proteins rich in cysteine with molecular weights ranging from 6 to 10 kDa [3-5]. These proteins' main physiological role is to maintain heavy metal ion homeostasis. MT's biological function is possibly associated with their overexpression in patients with a tumour disease [6-9]. Several papers have discussed and investigated the detection of metallothioneins using different methods [10-18]. These approaches utilized capillary electrophoresis, liquid chromatography mass spectrometry, inductive coupled plasma mass spectrometry, immunoassays and electrochemistry. Electrochemical techniques represent an alternative to hyphenated and high cost techniques due to their sensitivity and low cost [17,18]. The aim of this paper is to improve the current adsorptive transfer stripping technique (AdTS) to analyze MT in volumes down to nanolitres. 2. Experimental 2.1 Chemicals, pH measurements and pipetting Rabbit liver MT (MW 7143), containing 5.9 % Cd and 0.5 % Zn, were purchased from Sigma Aldrich (St. Louis, USA). Tris(2-carboxyethyl)phosphine (TCEP) was prepared by Molecular Probes (Eugene, Oregon, USA). 10 μg/mL MT stock standard solutions were prepared with ACS grade water (Sigma-Aldrich, USA) and stored in the dark at –20 °C. Working standard solutions were prepared daily by dilution of the stock solutions. The pH value was measured using WTW inoLab Level 3 (Weilheim, Germany), connected to a computer and controlled by MultiLab Pilot software (Weilheim, Germany). The pH-electrode (SenTix-H, pH 0–14/3M KCl) was regularly calibrated by a set of WTW buffers (pH 4.01, 7.00 and 10.00) (Weilheim, Germany). To pipette volumes down to micro and nanolitres, pipettes used were purchased from Eppendorf Research (Eppendorf, Germany) with the highest certified deviation (±12 %). 2.2 Electrochemical measurements Electrochemical measurements were performed with an AUTOLAB Analyser (EcoChemie, Netherlands) connected to a VA-Stand 663 (Metrohm, Switzerland), using a standard cell and three electrodes. The working electrode was a hanging mercury drop electrode (HMDE). The reference electrode was a Ag/AgCl/3M KCl electrode and a glassy carbon electrode was used as the auxiliary electrode. Smoothing and baseline correction was employed by GPES 4.4 software supplied by EcoChemie.
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Adsorptive transfer stripping technique. The principle of the adsorptive transfer stripping technique is based on the strong adsorption of the target molecule on the surface of the working electrode at an open circuit (Fig. 1A). The hanging mercury drop electrode is periodically renewed (Figure 1A1). Target molecules are adsorbed on the surface of the renewed working electrode at an open circuit (Figure 1A2). The electrode is washed with a supporting electrolyte (Figure 1A3). The electrode with the adsorbed target molecules is measured in the presence of the supporting electrolyte (Figure 1A4). Brdicka reaction of MT. MT was measured by AdTS coupled with a differential pulse voltammetry (DPV) Brdicka reaction. Brdicka supporting electrolyte (1 mM Co(NH3)6Cl3 and 1 M ammonia buffer (NH3(aq) + NH4Cl, pH = 9.6) was used without surface-active agent additives. AdTS DPV Brdicka reaction parameters were as follows: an initial potential of –0.35 V, an end potential of –1.8 V, a modulation time of 0.057 s, a time interval of 0.2 s, a step potential of 1.05 mV, a modulation amplitude of 250 mV, Eads = 0 V. Temperature of supporting electrolyte was 4 °C. Figure 1. Scheme of adsorptive transfer technique (A). Typical voltammograms of 100 nM MT (solid red line), supporting electrolyte (dotted black line) (B).
A 1
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Ag/AgCl
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2.3 Clinical material Human blood serum samples from patients with breast cancer were obtained from the Department of Clinical Biochemistry and Pathobiochemistry, FN Motol, Prague, Czech Republic. The sampled sera were immediately frozen at –20 °C prior to their preparation. The sample was prepared by heat treatment followed by solvent precipitation. The samples were kept at 99 °C in a thermomixer (Eppendorf 5430, USA) for 15 min. with occasional stirring, and then cooled to 4 °C. The denatured homogenates were centrifuged at 4 °C, 15,000 g for 30 min. (Eppendorf 5402, USA). Heat treatment and solvent precipitation effectively denatured and removed high molecular weight proteins from the samples [19]. MT levels in the human blood serum samples were measured by AdTS DPV Brdicka reaction. 2.4 Descriptive statistics Microsoft Excel® (USA) was used for mathematical analyses. Results are expressed as mean ± S.D. unless noted otherwise. The detection limits (3 S/N) were calculated according to Long [20], whereas N was expressed as standard deviation of noise determined in the signal domain. 3. Results and Discussion Proteomic research demands highly sensitive analytical instruments to detect very low volumes or amounts of a biological sample. Analysis is preferably carried out on the instruments to be low cost and easy to use, and, moreover, there is great demand on miniaturization of the instruments used [2136]. The impact of these demands is well demonstrated in the field of flow microchips technology [3752]. Electrochemical devices, methods and approaches have a valuable contribution to this field. In particular, the introduction of adsorptive transfer technique by Prof. Palecek was a great advancement in the electroanalysis of low volume samples [52-58]. 3.1 Utilizing of adsorptive transfer technique for analysis of MT The AdTS technique coupled with the DPV Brdicka reaction can be used to detect metallothionein in low sample volumes and can also prevent interferences during the washing and transferring step (Figure 1A,B). The technique, however, has its limitations. This technique currently is unable to generate reproducible results when analyzing very low sample volumes. We attempted to investigate how changes in drop volume and area of the working electrode influence the repeatability and sensitivity of the measurements. Study of MT (100 µM) drop volumes of 2.5, 5.0, 10 and 15 µL by AdTS DPV Brdicka reaction at HMDE with a drop area of 400 µm2, resulted in well developed and reproducible Brdicka catalytic signals of 1.5, 3.0, 6.2 and 9.2 ng of MT, respectively (Figure 2Aa). The height of the Cat2 signal was nearly proportional to MT content with a R2 value of 0.9816. The measurements were repeated five times and relative standard deviation of Cat2 peak height did not exceed 5 %.
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MT measurements (100 nM) with drop volumes of 0.5, 1.0, 1.5, 2.5 and 5.0 µL could be carried out using HMDE with a drop area of 250 µm2. The Brdicka catalytic peaks were well apparent in the measured voltammograms, whereas Cat2 peaks were sufficiently detected even at low MT amounts of 0.3, 0.6, 0.9, 1.5 and 3.0 ng (Figure 2Ba). However, relative standard deviation (R.S.D., %) increased significantly with decreasing drop volume. The R.S.D. measurements of MT in 2.5, 1.5, 1.0 and 0.5 µL were 4 %, 8 %, 15 % and 40 %, respectively. Enhanced Cat2 peak height was observed with increasing R.S.D. The Cat2 peak measured after adsorption of MT from drop of 500 nL was five times higher compared to that measured after adsorption of MT from drop of 2,500 nL. The enhanced Cat2 peak height was almost proportional to the decrease in drop volume (Figure 2Ba). This phenomenon is possibly due to water evaporation from a drop of MT standard solution. Due to this phenomenon, MT concentration increased and a higher peak was observed. Based on the results obtained, this approach cannot be used for quantitative determination of proteins in very low sample volumes at room temperature. Figure 2. Dependence of Cat2 peak height of MT on drop volumes of 2.5, 5.0, 10 and 15 µL (A, non-cooled parafilm a and cooled parafilm b, measured at HMDE of area of 400 µm2) and of 0.5, 1, 1.5, 2.5 and 5 µL (B, non-cooled parafilm a and cooled parafilm b, measured at HMDE of area of 250 µm2). In insets: typical DP voltammograms of MT (100 nM). Peak height of 72.3 nA (Aa), 78.6 nA (Ab), 1.1 nA (Ba) and 22.3 (Bb) correspond to 100 %. HMDE area – 400 µm2 Non-cooled parafilm
b
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3.2 Improvement of the adsorptive transfer technique This obstacle can be overcome by technically modifying the current transfer technique. A small square of parafilm (10 × 10 cm, Sigma-Aldrich) is seamed on a microscope slide by a burner (Figure
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3a). The slide is washed with ethanol and distilled water (Milli Q, 18 MΩ) and transferred to a cooled space, in this case to a beaker filled with distilled water and placed in a tempered water bath (Julabo, Germany, Figure 3b) at a temperature of 2 °C. Prior to use, the slide is removed from the bath and dried using cellulose. MT low volume drops were pipetted onto the dried slide (Figure 3c) and then adsorbed on the surface of HMDE at open circuit (Figure 3d). The electrode with the adsorbed target molecule is washed and measured (Figures 3e-g). The experiment discussed in Section 3.1. was repeated and MT measurements (100 µM) with drop volumes of 2.5, 5.0, 10 and 15 µL were carried out using the improved AdTS DPV Brdicka reaction and HMDE with a drop area of 400 µm2 (Fig. 2Ab). Compared to results shown in Fig. 2Aa the signals were higher and more proportional to MT content with a R2 value of 1.000. Relative standard deviation of Cat2 (n = 5) peak height did not exceed 4 %. 3.3 Electrochemical analysis of MT in low volume samples Due to these results further studies were done on MT samples with various volumes. Investigations were done on MT containing samples (100 nM) with volumes of 0.5, 1.0, 1.5, 2.5 and 5.0 µL using HMDE with a drop area of 250 µm2 (Figure 2Bb). The improvement of the transfer technique described above enabled us to detect MT in very low volume samples in comparison to the “standard” transfer technique (Figure 2Ba,b). The height of Cat2 signal was proportional to MT content with a R2 value of 0.9928 and the relative standard deviation of Cat2 peak height was not higher than 6 %. Substantial improvement in results was due to decrease in water evaporation in low volume drops. Figure 3. Scheme of improvement of the transfer to detect MT in very low volumes of a sample. Microscopic slide, degreasing and seaming of small square from parafilm (10 × 10 cm, Sigma-Aldrich) (a), transferring it to a beaker filled with distilled water and placed in the tempered water bath (temperature of 2 °C, at least 15 min., Julabo, Germany) (b), drying it using cellulose and pipetting of a sample on it (c), adsorbing of MT on the surface of HMDE (d), transferring the electrode (e) and washing it with supporting electrolyte (f), transferring the electrode and measuring (g).
HMDE
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microscopic slide 2°C water-cooling
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washing measuring
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Additional investigations on the influence of the accumulation of MT over time (500 nL) on Cat2 peak height were studied. The peak enhanced to 120 s long accumulation, then gradually decreased (Figure 4A). Figure 4. Dependence of Cat2 peak height on accumulation time (A) and on MT concentration within the range from 25 to 5,000 pM (blue square, B) and from 25 to 500 pM (red triangles, in inset in B). Dependence of Cat2 peak potential on MT concentration (black dot, B). Volume: 500 nL, HMDE area: 250 µm2. Other experimental conditions the same as in Figure 1 and 3. A
B
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120 25
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4 y = 24.286x + 1.2732 R 2 = 0.9948
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Under 120 s long accumulation of MT on the surface of HMDE we measured the dependence of Cat2 peak height on MT concentration. The peak height increased with increasing MT concentration within the tested interval from 25 to 5,000 nM (Fig. 4B). The linear dependence was measured within the interval from 25 to 500 pM with relative standard deviation of 2.5 % (n = 5, inset in Figure 4B). Measurements were repeatable within a day and also weeks later. The relative deviation of such measurements did not exceed 5% (n = 4). The detection limit (3 S/N) of MT was evaluated as 500 zeptomoles per 500 nL (1 pM) and the quantification limit (10 S/N) as 1,500 zeptomoles per 500 nl (3 pM). 3.4 Analysis of blood serum from patients with a tumour disease Recently published studies have shown an association between metallothionein and breast cancer. MT level analysis of one hundred patients with this type of cancer was reported. The authors showed a correlation between high levels of MT and an increase in disease prognosis and vice versa [59]. Using a different approach than the latter, we attempted to utilize the improved transfer technique mentioned above for measuring MT in blood serum from patients with breast cancer. MT samples with 500 nL
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volumes were analyzed using the revised technique according to the scheme shown in Fig. 3 and MT samples with 5 µL volumes were analyzed using the “standard” technique. Results from the revised technique were compared with the “standard” transfer technique data. In both cases well developed catalytic signals were observed (Figure 5). Figure 5. DP voltammograms of human blood serum samples from four patients with breast cancer. Volume of the sample analyzed: 500 nl, HMDE area: 250 µm2. Other experimental conditions the same as in Figure 1 and 3. 50 nA Patient
scan
4
3
2
1
-0.9
-1.3
-1.6
Potential (V)
Quantification of MT levels in the samples was based on the determination of the Cat2 peak height. MT levels varied from 0.8 to 2.4 µM (1.56 ± 0.79 µM) in respect to 5 µL volumes and from 1.2 to 1.7 µM (1.46 ± 0.28 µM) in respect to 500 nL volumes. A significant difference in standard deviations was observed between the two techniques. A sample prepared according to the procedure mentioned in the “Experimental” section contained several low molecular weight thiols (e.g. glutathione) in addition to the presence of MT containing thiols. This phenomenon could be a result due to altered adsorption of these substances at a lower temperature and lower sample volume. However, the means of MT levels determined in breast cancer patients by both techniques were in good agreement with each other. 4. Conclusions Proteomic approaches to the identification of novel biomarkers for cancer diagnosis and staging have traditionally relied on the identification of differentially expressed proteins between tumour cells and their normal counterparts based on the patterns of protein expression observed by two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry. Here, we report on alternative way to detect proteins that can be carried out with very low demands on an instrument, consumable costs and of operator skill. The present paper suggested a simple but effective improvement (cooling of the
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parafilm) to the adsorptive transfer technique, which consequently resulted in several advantages when conducting measurements. Diminished water evaporation in low volume drops and greater MT adsorption under improved controlled conditions were the major changes observed which allowed measurements down to several hundred nanolitres with relatively low standard deviations and low detection and quantification limit. Acknowledgements The authors wish to express their thanks to Dr. Jiri Kukacka for providing clinical samples and to Dr. Grace J. Chavis for language corrections and discussions. Financial support from the Grant Agency of the Czech Academy of Sciences (Grant No. GAAV IAAA40199071) is greatly acknowledged. References 1.
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E. Vliv cisplatiny na hladinu metalothioneinu u buněčných linií, laboratorních krys a pacientů s nádorem v oblasti hlavu a krku
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Employment of Electrochemical Techniques for Metallothionein Determination in Tumor Cell Lines and Patients with a Tumor Disease Ivo Fabrik,a, b Sona Krizkova,a Dalibor Huska,a Vojtech Adam,a Jaromir Hubalek,c Libuse Trnkova,d Tomas Eckschlager,e Jiri Kukacka,f Richard Prusa,f Rene Kizeka* a
Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic *e-mail:
[email protected] b Department of Biochemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic c Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno University of Technology, Udolni 53, CZ-602 00 Brno, Czech Republic d Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic e Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University, V Uvalu 84, CZ-150 06 Prague 5, Czech Republic f Department of Clinical Biochemistry and Pathobiochemistry, 2nd Faculty of Medicine, Charles University, V Uvalu 84, CZ-150 06 Prague 5, Czech Republic Received: December 17, 2007 Accepted: April 4, 2008 Abstract In the present paper we employed adsorptive transfer stripping technique coupled with chronopotentiometric stripping analysis for determination of metallothionein (MT) in tumor cell lines and differential pulse voltammetry Brdicka reaction for determination of MT in blood serum of patients with head and neck cancer or retinoblastoma, and of rats treated with cisplatin with respect to discuss the role of MT in formation of resistance on treatment with heavy metal based cytostatics. The cisplatin or carboplatin sensitive and resistant neuroblastoma cell lines were derived from the maternal cell line isolated from the bone metastasis of patients with neuroblastoma. Based on the results obtained it can be concluded that level of MT increases with higher dose of platinum based cytostatics at cells. Further we focused on determination of MT in blood serum of rats treated with cisplatin (two doses 1.05 mg and/or 2.1 mg of cisplatin per kg). The highest level of MT at rats treated with 1.05 mg cisplatin was determined after four hours as 4.9 mmol/L. In the case of the second experimental group the maximum was reached even after two hours of the treatment as 4.8 mmol/L. In addition we were interested in the effect of cisplatin or carboplatin treatment of patients with a tumor disease. At patients with tumor in head and neck area treated with cisplatin we observed that the level of MT was going higher due to administration of the drug. This phenomenon was observed at all patients. However at patients with retinoblastoma treated with carboplatin we observed various phenomena including decreasing, increasing or no changes in MT level. Progression of MT levels was therefore individual and probably depended on tumor resistance to carboplatin. Keywords: Metallothionein, Carboplatin, Cisplatin, Neuroblastoma cell, Retinoblastoma, Head and neck cancer, Resistance, Voltammetry, Brdicka reaction DOI: 10.1002/elan.200704215
1. Introduction Cancer is a leading cause of death in the world, particularly in developing countries according to World Health Association and a third of cancers could be cured if detected early and treated adequately. This fact leads to the faster development of new diagnostic, prognostic and therapeutic methods [1, 2]. Based on many clinical studies it is well known that a resistance on a treatment by cytostatic agents is a crucial complication of anticancer therapy [3 – 5]. The resistance of tumor cell on the cytostatics originates by various mechanisms and the process itself is thought to be multifactorial. The anticancer therapy is complicated espeElectroanalysis 20, 2008, No. 14, 1521 – 1532
cially in the case of forming of multidrug resistance (MDR). There are several mechanisms of the resistance. The decreasing of concentration of the cytostatic in the site of its action is one of the causes of the tumor cells resistance on the therapy [6]. Some membrane proteins like membrane phosphoglycoprotein with ATPase activity called Pgp, MRP (membrane transport protein) and cytosol protein called LRP are involved in such decreasing and thus in formation of MDR [7 – 11]. A broad range of genes involved in the process of apoptosis or expression of tumor suppressors is changed in the case of MDR. The changed expression of tumor suppressor genes of protein family Bcl-2 is frequently found E 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Fig. 1. Influence of anticancer drug based on heavy metal, a) anticancer drug effect (stopping DNA replication); b) mutated genes (reparation of damaged DNA); c) cisplatin binds on metal responsive element (MRE); d) mRNA-MT; e) MT interacts with a drug; f) treatment strategy to suppress MT level.
in chemo resistant tumor cells. It is a common knowledge that the function of tumor suppressors, e.g., p53, is blocked in certain resistant tumor cells [12]. Moreover the multidrug resistance can be associated with increased activity of DNA reparation mechanisms [13]. The direct detoxification of cytostatics belongs to the factors of MDR too. The activity of oxidation and detoxification enzymes which oxidize or conjugate the xenobiotics with endogenous conjugative agents has been increased in most cases of MDR. The microsomal monooxygenases belongs to the most abundant group of enzymes oxidizing xenobiotics. Besides those glutathione-S-transferases are conjugative enzymes inactivating cytostatics. The glutathione-transferase system has been studied intensively in context of cytostatics resistance in leukemia cells [14 – 16]. Except above-mentioned mechanisms the MDR resistance can be connected with increased expression of low-molecular proteins called metallothioneins (MT) [5, 17 – 20]. Metallothioneins are a group of proteins rich in cysteine with molecular weights ranging from 6 to 10 kDa [21, 22]. Due to high affinity of metallothioneins to heavy metals, e.g., zinc, copper and/or cadmium, homeostatic control and detoxification of the metals are their main physiological function at evolutionary different animal organisms. It is less known about molecular mechanisms of the MT expression. In the case of MT expression caused by heavy metals, metal ions probably have a key role in this process because they are able to bind on specific transcriptional factor called as metal transcription factor 1 (MTF-1). Then, the formed metal-MTF-1 complex interacts with metal-responsive element (MRE) of MT promoter to activate its transcription Electroanalysis 20, 2008, No. 14, 1521 – 1532
[17]. Among others, the concentration of MT increases in the moment of administration of heavy metal (e.g., the anticancer drug) [23, 24]. The expressed MT binds the administered anticancer drug rapidly, which results in the decrease of drug concentration below the effective level [24]. The simplified scheme of the forming of a resistance to cytostatic treatment is shown in Figure 1. To detect level of MTs at various biological samples as human body liquids, cells and tissues effective, sensitive and easy to use analytical instruments are needed. The techniques and methods using for these purposes can be divided to several groups on spectrometric [25], immunoassays [26], hyphenated [27, 28], mRNA detection based [29] and electrochemical [30 – 40]. In the present paper we employ two most sensitive electroanalytical methods, adsorptive transfer stripping technique (AdTS) coupled with chronopotentiometric stripping analysis (CPSA) for determination of MT in tumor cell lines and differential pulse voltammetry Brdicka reaction (DPV Brdicka reaction) for determination of MT in blood serum of patients with head and neck cancer or retinoblastoma, and of rats treated with cisplatin with respect to discuss the role of MT in formation of resistance on treatment by heavy metal based cytostatics.
2. Experimental 2.1. Chemicals and pH Measurements Rabbit liver MT (MW 7143), containing 5.9% Cd and 0.5% Zn, was purchased from Sigma Aldrich (St. Louis, USA). Tris(2-carboxyethyl)phosphine (TCEP) was produced by
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Molecular Probes (Evgen, Oregon, USA). Other chemicals used were purchased from Sigma Aldrich. The stock standard solutions of MT at 10 mg/mL with 1 mM TCEP were prepared with ACS water (Sigma-Aldrich, USA) and stored in the dark at 20 8C. Working standard solutions were prepared daily by dilution of the stock solutions. The pH was measured using pH meter WTW inoLab (Weilheim, Germany). The pH-electrode (SenTix-H, pH 0 – 14/3 M KCl) was regularly calibrated by set of WTW buffers (Weilheim, Germany).
2.2. Tumor Cell Lines The following cell lines obtained from Department of Paediatric Haematology and Oncology, Charles University, Prague, Czech Republic were used: UKF-NB4 – cisplatin or carboplatin sensitive (prepared from recurrence of neuroblastoma into bone marrow, with MYCN amplification, del 1p34.2ter, del 13, ISO 17q) and UKF-NB4 – cisplatin or carboplatin resistant (the line derived from the previous line - UKF-NB4 with in vitro induced resistance to cisplatin). The cell lines were prepared by cultivation with increasing concentration of cisplatin. The cells were cultivated in IMDM medium with 10% of fetal calf serum at 37 8C, the chemo resistant cell lines were cultivated in medium with cisplatin addition [41, 42].
2.5. Preparation of the Samples for Electroanalytical Determination of Metallothionein 2.5.1. Blood Serum Samples The sample of human and/or rat blood serum was prepared by heat treatment and solvent precipitation. Briefly, the sample was kept at 99 8C in a thermomixer (Eppendorf 5430, USA) for 15 min. with occasional stirring, and then cooled to 4 8C. The denatured homogenates were centrifuged at 4 8C, 15000 g for 30 min. (Eppendorf 5402, USA). The supernatant (5 mL) was analyzed by differential pulse voltammetry Brdicka reaction [23, 38, 43, 44]. 2.5.2. Tumor Cell Lines Samples The harvested cells were transferred to a test tube and then deep frozen by liquid nitrogen to disrupt cells. The frozen cells were mixed with extraction buffer (100 mM potassium phosphate, pH 8.7) to a final volume of 1 mL and homogenized using hand-operated homogenizer ULTRA-TURRAX T8 (IKA, Germany) placed in an ice bath for 3 min at 25 000 rpm [45]. The homogenate was centrifuged at 10 000 g for 15 min and at 4 8C (Eppendorf 5402, USA). The supernatant were processed in the same way as blood serum samples mentioned in Section 2.5.1. The processed samples were measured by adsorptive transfer stripping technique coupled with chronopotentiomeric stripping analysis [33].
2.3. Human Blood Serum The samples of intravenous blood was obtained using vein tapping to the closed tapping units without another reagents during therapy of patients suffering from head and neck cancer at Department of Otolaryngology and Maxillofacial Surgery, University Hospital in Brno, Czech Republic, Generally forty five samples of blood obtained from the patients were analyzed. Blood serum samples of patients with retinoblastoma were obtained from the Department of Clinical Biochemistry and Pathobiochemistry, 2nd Faculty of Medicine Charles University, Czech Republic. All patients subscribed informed consent with utilization of their blood samples for the research.
2.4. Rat Blood Serum Male Wistar rats (Faculty of Medicine, Masaryk University, Brno, Czech Republic), 8 weeks old (270 – 280 g), were divided into two experimental groups per six specimens. The first experimental group was exposed to one dose of 1.05 mg of cisplatin per kg, the second group to one dose of 2.1 mg of cisplatin per kg. Cisplatin was administered intraperitoneally. Each hour after administration one of the experimental animals were put to death. Blood from the heart of the animal and liver were sampled.
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2.6. Electrochemical Measurements 2.6.1. Electroanalytical Determination of Metallothionein by Adsorptive Transfer Stripping Technique Coupled with DPV Brdicka Reaction An adsorptive transfer stripping technique (AdTS) coupled with DPV Brdicka reaction was employed for the determination of metallothionein in cell lines extract. The electrochemical measurements were performed using an AUTOLAB analyzer (EcoChemie, The Netherlands) connected to VA-Stand 663 (Metrohm, Switzerland), using a standard cell with three electrodes. The three-electrode system consisted of hanging mercury drop electrode as working electrode, an Ag/AgCl/3 M KCl reference electrode and a glassy carbon auxiliary electrode. For smoothing and baseline correction the software GPES 4.9 supplied by EcoChemie was employed [46]. The Brdicka supporting electrolyte containing 1 mM Co(NH3)6Cl3 and 1 M ammonia buffer (NH3(aq) þ NH4Cl, pH 9.6) was used; surface-active agent was not added. AdTS DPV Brdicka reaction parameters were as follows: initial potential of 0.6 V, end potential 1.6 V, modulation time 0.057 s, time interval 0.2 s, step potential of 1.05 mV, modulation amplitude of 250 mV, Eads ¼ 0 V. Temperature of the supporting electrolyte was 4 8C. For other experimental conditions see [36].
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2.6.2. Automated Electroanalytical Determination of Metallothionein by DPV Brdicka Reaction DPV Brdicka reaction was employed for the determination of metallothionein in human blood serum samples. The electrochemical measurements were performed with 747 VA Stand instrument connected to 746 VA Trace Analyzer and 695 Autosampler (Metrohm, Switzerland), using a standard cell with three electrodes and cooled sample holder (4 8C). A hanging mercury drop electrode (HMDE) with a drop area of 0.4 mm2 was the working electrode. An Ag/AgCl/3 M KCl electrode was the reference and glassy carbon electrode was auxiliary electrode. The supporting electrolyte (1 mM [Co(NH3)6]Cl3 and 1 M ammonium buffer; NH3(aq) and NH4Cl, pH 9.6) was changed after five measurements [23, 47]. The DPV parameters were as follows: initial potential of 0.7 V, end potential of 1.75 V, modulation time 0.030 s, time interval 0.8 s, step potential 2 mV, modulation amplitude 25 mV, Eads ¼ 0 V. All experiments were carried out at temperature 4 8C (Julabo F25, Germany). The data obtained were collected by using of VA-Database 2.2 (Metrohm G.B, Switzerland). Further the data were transferred and processed by GPES 4.9 supplied by EcoChemie. For smoothing and baseline correction the software GPES 4.9 supplied by EcoChemie was employed [46]. Other details on automated analysis of MT will be published elsewhere. 2.6.3. Electroanalytical Determination of Metallothionein by CPSA An AdTS coupled with chronopotentiometric stripping analysis (CPSA) was employed for the determination of metallothionein in cell lines extract through recording the inverted time derivation of potential (dE/dt)1 as a function of potential E [33]. These electrochemical measurements were performed with AUTOLAB Analyzer (EcoChemie, Netherlands) connected to VA-Stand 663 (Metrohm, Switzerland), using a standard electrochemical cell with three electrodes. The working electrode was a HMDE with a drop area of 0.4 mm2. The reference electrode was an Ag/AgCl/ 3 M KCl electrode and the auxiliary electrode was a graphite stick electrode. For smoothing and baseline correction the software GPES 4.9 supplied by EcoChemie was employed [46]. CPSA parameters were as follows: Istr of 1 mA, temperature of supporting electrolyte 20 8C, supporting electrolyte 0.1 M H3BO3 þ 0.05 M Na2B4O7 (pH 9.2). Other experimental details on CPSA analysis of MT were published in [33, 40, 48, 49].
2.7. Determination of Esterase Activity The culture was treated by trypsin for 2 min and then shaken (60 rpm) to release the sessile cells. The cells were separated by centrifugation (50 g, 5 min) and washed one times with PBS (pH 7.4). The cell lysis was carried out on ice with Triton X100 in result concentration 0.1% (v/v) for 20 min. Electroanalysis 20, 2008, No. 14, 1521 – 1532
The mechanical impurities were removed from the lysate by the centrifugation (10 000 g, 15 min, 4 8C). The supernatant was then processed immediately. The intracellular esterase activity determination was carried out by using fluoresceindiacetate test with modifications: for incubation of the reaction mixture was used 37 8C [50 – 53]. The whole proteins were evaluated according Bradford [54]. The cell density (cell per 1 mL of suspension) was determined by using the Fuchs – Rosenthal counting cell.
2.8. Polyacrylamide Electrophoresis The cell samples were also analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis was performed according to Laemmli [55] using a Biometra maxigel apparatus (Biometra, Germany). First 15% (w/v) running, then 5% (w/v) stacking gel was poured, the gels were prepared from 30% (w/v) acrylamide stock solution with 1% (w/v) bisacrylamide concentration; the polymerization of the running gel was carried out at room temperature for 1 h and 30 min for the stacking gel. Prior to analysis the samples were mixed with sample buffer containing 5% (v/v) 2-mercaptoethanol in a 1 : 1 ratio. The samples were boiled for 2 min, and then loaded onto a gel in 60 mL aliquots. For determination of MW, the protein ladder NPrecision plus protein standardsO from Biorad was used. The electrophoresis was run at 150 V for 3 hours with cooling until the front dye reached the bottom of the gel. The silver staining of the gels was performed according Blum [56]. After silver staining the gels were scanned and analyzed by Biolight software (Vilber-Lourmat, USA).
2.9. Descriptive Statistics MICROSOFT EXCEL (USA) was used for statistical analyses. Results are expressed as mean SD unless noted otherwise. Accuracy, precision and recovery of metallothioneins were evaluated with homogenates (human blood serum) spiked with standard. Before extraction, 100 mL metallothioneins standards and 100 mL water were added to human blood serum samples. Homogenates were assayed blindly and metallothioneins concentrations were derived from the calibration curves. The spiking of metallothioneins was determined as a standard measured without presence of real sample. Calculation of accuracy (%Bias), precision (%CV, coefficient of variation) and recovery was carried out as indicated by Causon [57] and Bugianesi et al. [58].
3. Results and Discussion Biologically active molecules with free SH groups called thiols play numerous roles in organisms and can affect a treatment [5, 6, 19, 20, 59 – 64]. Via free sulfhydryl groups the thiols can be conjugated to various molecules, however
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their affinity to the metal ions can be considered as one the highest. One may suggest that these compounds could play role in resistance on cancer treatment by heavy metal based cytostatics, because cisplatin and others are still frequently in use. Due to the reactivity of free SH the electrochemical methods belong to the most sensitive ones employing for their determination [33, 36, 65 – 69].
3.1. Electroanalytical Methods in Metallothionein Analysis Almost all methods used for detection of metallothionein in tissues are based on immunohistochemical principles [27, 59, 61, 64]. These procedures require biopsy tissues and labor and time consuming sample handling and can not be used for exact quantification of the protein of interest. Recently we have proposed sensitive and selective procedure for sample preparation and consequent analysis to determine metallothionein in blood [23, 49, 70]. The sample preparation method is based on heat treatment and solvent precipitation, which can effectively denature and remove high molecular weight proteins out from samples [33, 40, 48, 49, 70 – 72]. The principle of the detection technique called AdTS DPV Brdicka reaction bases in adsorption of thermostable metallothionein directly with the surface of the mercury electrode. The procedure is shown in Figure 2A. The transfer technique can be considered as so-called Nsample purifying stepO separating low-molecular compounds from larger ones. Therefore metallothionein is adsorbed and low-molecular thiols, such as cysteine or glutathione, do not affect the measurements, other details will be published elsewhere. The measuring electrode system is in the connection with potentiostat/galvanostat and controlling computer (Fig. 2B). Typical DP voltammograms of the same MT (100 ng/mL) sample of are shown in Figure 2C. Two well developed catalytic signals called Cat1 and Cat2 are observable. It is shown in inset in Figure 2C that nine times measured Cat2 signal of MT (100 ng/mL) gave well repeatable response with relative standard deviation below 5%. However due to necessity of the manual control of all mentioned steps we have looked for the possibility to automate the measurements. To automate measurements 747 VA Stand instrument connected to 746 VA Trace Analyzer and 695 Autosampler with cooled sample holder was employed. A measurement is carried out automatically under the control of microprocessor within five minutes. For the quantification of MT the catalytic signal Cat2 is used. Typical voltammograms of MT (1.5, 3
and 6 mM) measured by automated electrochemical analyzer are shown in Figure 3A. The MT signals depended on MT concentration. The continuous line shows on reduction of cobalt(III) ions. The electrochemical analysis of MT resulted in appearing of three signals in voltammograms obtained. The signal of MT complex with cobalt ions called RS2Co appeared at 1.0 V. This signal shifted to more positive potentials with increasing MT concentration. Two other signals called Cat1 ( 1.2 V) and Cat2 ( 1.4 V) were catalytic. The calibration curve measured is shown in Figure 3B. The dependence of Cat2 height signal of MT on its concentration was linear within the studied range (y ¼ 2.0009x þ 1.6673, R2 ¼ 0.9965). RSD was below 5%. Recovery was tested by three additions of MT standard into human blood serum sample (Table 1). The results were within 95 – 100%.
3.2. Neuroblastoma Cell Lines Primarily we aimed our attention to determination of MT in samples of neuroblastoma cell lines resistant and sensitive to platinum based drugs. The pictures of the cell lines resistant and sensitive to cisplatin are shown in Figure 4A. The single cell lines have been characterized previously according chromosomal aberrations [41]. However the changes in metabolic activity of the cell lines are still unclear. To reveal the activity of metabolism we employed fluorimetric determination of activity of intracellular esterase [50 – 53]. The same volumes of the cell samples with the exact and same count of cells were analyzed (Fig. 4B). We found that the esterase activity of cisplatin sensitive cell lines was higher in comparison with the activity of resistant ones. This phenomenon can be associated with the toxic effects of cisplatin on the cells. In addition we attempted to utilize fluorimetric determination for evaluation of the number of living cells in the samples of neuroblastoma cell lines. Based on known cell number homogenized in defined volume the equivalents of cell number from 0 to 7000 were transferred into reaction mixture for esterase activity determination. The detected intracellular esterase activity was directly proportional to cell number and the detection limit was about 600 cells for the sensitive neuroblastoma cultures and about 1000 cells for the resistant cultures (Fig. 4B). In the following experiments the neuroblastoma cell extracts were measured by using two different techniques AdTS DPV Brdicka reaction and AdTS CPSA. The dependence of the height of Cat2 signal measured by Brdicka reaction on the count of the cells is shown in
Table 1. Recovery of MT for analysis of the human blood serum sample (n ¼ 5).
MT addition 1 MT addition 2 MT addition 3
Homogenate (mM ) [a]
Spiking (mM ) [a]
Homogenate þ spiking (mM ) [a]
Recovery (%)
6.5 0.2 (3.1) 6.5 0.2 (3.1) 6.5 0.2 (3.1)
1.10 0.03 (2.7) 2.20 0.05 (2.2) 4.40 0.15 (3.4)
7.5 0.2 (2.7) 8.4 0.3 (3.6) 10.6 0.3 (2.8)
99 97 97
[a] MT concentration; expressed as mean SD ( CV%)
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Fig. 2. A) Scheme of adsorptive transfer stripping technique used for MT detection; 1) renewing of the hanging mercury drop electrode (HMDE) surface; 2) adsorbing of MT in a drop solution onto the HMDE surface at open circuit; 3) washing electrode in ACS water; 4) measuring of MT by DPV in the presence of the Brdicka supporting electrolyte. B) Electrochemical cell with three electrodes in the connection with potentiostat/galvanostat and controlling device. C) Typical AdTS DP voltammograms of metallothionein (100 ng/mL) measured in triplicate.
Fig. 3. A) DP voltammograms of MT (1.5, 3 and 6 mM) measured by using 747 VA Stand instrument connected to 746 VA Trace Analyzer and 695 Autosampler (n ¼ 3). B) The dependence of the height of Cat2 signal on the MT concentration.
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Fig. 4. A) Images of tumor cell lines UKF-NB4 cisplatin sensitive and resistant. B) Changes of esterase activity in UKF-NB4 cell lines.
Fig. 5. Dependence of peak heights on count of tumor cells measured by A) AdTS DPV Brdicka reaction or B) peak H; in inset: the dependence for lower counts of the cells.
Figure 5A. The obtained dependence is linear with the equation y ¼ 0.0014x þ 1.733, R2 ¼ 0.9747. The lowest count of the cells, which we were able to detect, was 100. The same samples were also analyzed using chronopotentiometry. The typical concentration dependence is shown in Figure 5B. The height of the peak increased up to the number of the cells about 500 and then rose more gradually. Within the range from 0 to 100 cells the dependence was strictly linear Electroanalysis 20, 2008, No. 14, 1521 – 1532
with the equation y ¼ 61.536x þ 33.557, R2 ¼ 0.9989. Using this procedure the detection of less than 30 neuroblastoma cells was possible. In spite of the fact that CPSA is more sensitive than the Brdicka reaction we employed the Brdicka reaction due to faster and more reproducible analysis. Based on the results obtained above we always analyzed the equivalent of 1000 cells in the following experiments.
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3.3. The Changes of Metallothionein Level in the Tumor Cells The cisplatin or carboplatin sensitive and resistant neuroblastoma cell lines are derived from the maternal cell line isolated from the bone metastasis of patients with neuroblastoma. The resistant tumor cell line is constantly cultivated in the cultivation media containing anticancer drug (cisplatin or carboplatin). The typical AdTS DP voltammograms of cellular extracts obtained from maternal, sensitive and resistant cell lines are shown in Figure 6A. In the voltammograms the well distinguishable catalytic signals Cat2 are observed. These signals correspond to the presence of free SH groups in MT molecule. To confirm that we measured MT in cell extracts we carried out PAGE electrophoresis of cellular homogenates followed by silver staining. The very slight band indicating the presence of MT was detectable only in cell line resistant to cisplatin (Fig. 6A). By using PAGE we successfully confirmed presence of MT in cell extracts and also shown that the Brdicka reaction is much more sensitive to presence of this protein. The concentration of MT in the cell extracts determined by the electrochemical method is shown in
Figure 6B. The concentration of MT in the cell line resistant to cisplatin (the cells were cultivated in the presence of 1 mM cisplatin for 72 h) increased for more than 60% compared to the maternal cell line. The MT level in the cisplatin sensitive cell line (the cells were cultivated without cisplatin for 72) was also slightly higher compared to maternal line (Fig. 6B). The changes of MT level in cell lines in the exponential growth phase treated with cisplatin and carboplatin for 24 h are shown in Figure 6C,D. The cisplatin concentration 0.01 mM caused negligible rise in MT concentration compared to the sensitive cells. The applied dose of 0.1 mM of cisplatin lead to the MT concentration increase for 10% compared to the sensitive cells, however the higher cisplatin concentration induced greater increase of MT concentration. The very similar phenomenon was observed in the case of carboplatin, concentrations from 0.1 to 1 mM resulted in slightly enhanced MT biosynthesis compared to the sensitive cells. The concentration of carboplatin higher than 1 mM led to very rapid MT biosynthesis. Based on the results obtained it can be concluded that level of MT increases with higher dose of platinum based cytostatics at cells. Thus we were interested in the effect of various treatment strategies on MT level at patient with malignant tumors.
Fig. 6. A) AdTS DP voltammograms of the cell lines UKF-NB4 a) maternal line, b) resistant line (cultivated in the presence of 1 mM cisplatin), c) sensitive line (cultivated without cisplatin). PAGE analysis of the samples, on the left part of the panel it is a signal of 300 ng of MT standard, on the right part of the panel the signal of the sample is shown. B) The changes of MT signal measured in UKF-NB4 cell lines, a) maternal line, b) resistant line, c) sensitive line. The MT peak height measured in the cells cultivated in the presence of various concentrations of C) cisplatin and/or D) carboplatin. Electroanalysis 20, 2008, No. 14, 1521 – 1532
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Fig. 7. Changes in MT level in A) blood serum or B) liver of rats treated with 1.05 mg of cisplatin per kg or 2.1 mg of cisplatin per kg. In bottom insets: typical DP voltammograms. In upper inset: photography of sampling of blood serum and liver.
3.4. Changes of Metallothionein Levels at Rats after Intraperitoneal Application of Cisplatin In addition we tested the automated electrochemical instrument on analysis of MT in blood serum of rats treated with cisplatin. We found out that synthesis of MT enhanced quickly. Even one hour after administration of cisplatin the level of MT increased at both experimental groups. Particularly, the level of MT in blood serum of untreated rats was app. 2.9 mmol/L. At rats treated with 1.05 mg and/or 2.1 mg of cisplatin for one hour the MT level was 4.2 and/or 4.3 mmol/L, respectively. The highest level of MT at rats treated with 1.05 mg cisplatin was determined after four hours as 4.9 mmol/L. In the case of the second experimental group the maximum was reached even after two hours of the treatment as 4.8 mmol/L. Moreover we quantified MT level in ratsO liver (the levels MT determined in control tissues were taken as 100%). It clearly follows from the results obtained that lower dose of cisplatin (1.05 mg/kg) induced Electroanalysis 20, 2008, No. 14, 1521 – 1532
highest MT expression in second hour after injection then MT level decreased. Higher cisplatin dose (2.1 mg/kg) resulted in enhancing of MT synthesis, which did not change it during the experiment (Fig. 7).
3.5. Changes of Metallothionein Level at Treated Patients with Malignant Tumors in Head and Neck Area A monitoring of MT level at patients with a tumor disease could be useful from various points of view. One of the points of view is the role of MT as marker of resistance to tumor disease treatment by heavy metal based cytostatics. Due to evaluation of this assumption the samples of blood of patients with a tumor in head and neck area were collecting during drug the treatment. The changes in MT level at the patients in the time manner is shown in Figure 8A. The levels of MT is several times higher compared to MT levels in blood serum of healthy volunteers [73]. Typical DP
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Fig. 8. Monitoring of MT level at three patients with a tumor in head and neck area treated with cisplatin. A) Patient 1 (0 – 148 h), in inset: typical DP voltammograms; B) patient 2, ( 168, 120, 72, 48 are hours before administration of cisplatin); C) patient 3 (0 – 80 h).
voltammograms of blood serum of patient with head and neck cancer treated with cisplatin are shown in inset in Figure 8A. The effect of cisplatin on MT level before and after administration of this drug is clearly shown in Figure 8B. The level of MT was going higher due to administration of the drug. This phenomenon was observed at all patients (Fig. 8A,B,C). Moreover the MT level rapidly decreased even after tens of hours after administration. Then the changes in MT level were negligible.
change. On the contrary level of MT determined at patients Nos. 1, 3, 5 and 9 decreased up to 4 hours after administration. This can be related with binding of MT to cytostatic. Treatment of patients Nos. 7 and 8 resulted in almost twofold MT level enhancing as same as at patients and at rats treated with cisplatin (Figs. 7, 8 and 9). Progression of MT levels was individual and probably depended on tumor resistance to carboplatin.
4. Conclusions 3.6. Changes of Metallothionein Level at Patients with Retinoblastoma Treated by Carboplatin We were interested in the issue if the administration of second generation of the platinum based drugs called carboplatin to patients with a tumor disease could influence MT level. Patients with rare tumor disease – retinoblastoma (n ¼ 9) were treated by carboplatin. We determined MT level during 24 hours after the administration (Fig. 9). Average concentration of MT in plasma of patients with retinoblastoma before treatment with carboplatin was 3.4 mM. During analysis of MT level we can observe various phenomena. At patients Nos. 2, 4 and 6 MT level did not Electroanalysis 20, 2008, No. 14, 1521 – 1532
Electrochemical instrument belongs to the one of the most sensitive instrument for thiols determination [31, 37]. The whole electroanalytical process is very simple and fast. In the present paper we employed the electrochemical methods for evaluation of role of metallothionein in resistance on tumor disease treatment. Particularly we aimed our attention on tumor cell lines, patients with tumor in head and neck area and with retinoblastoma, and rats treated with cisplatin. In conclusion MT could participate in resistance to platinum based cytostatics and therefore should be investigated more deeply.
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Fig. 9. Changes in MT level at patients with retinoblastoma treated with carboplatin.
5. Acknowledgements We gratefully acknowledge the Grant Agency of the Academy of Sciences of the Czech Republic (grant No. GA AV IAA401990701) and the Ministry of Education, Youth, and Sports (Grant No. MSMT 0021620813) for the financial support to this work. The authors wish to also express their thanks to Dr. Hana Binkova for providing of clinical samples.
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Chronopotentiometric Stripping Analysis of Gelatinase B, Collagen and Their Interaction Dalibor Huska,a Vojtech Adam,a, b Ondrej Zitka,a, c Jiri Kukacka,d Richard Prusa,d Rene Kizeka* a
Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic *e-mail:
[email protected] b Department of Animal Nutrition and Forage Production, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic c Department of Biochemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic d Department of Clinical Biochemistry and Pathobiochemistry, 2nd Faculty of Medicine, Charles University, V Uvalu 84, CZ-150 06 Prague 5, Czech Republic Received: July 23, 2008 Accepted: October 12, 2008 Abstract Matrix metalloproteinases (MMP) belong to a group of zinc-dependent proteins that play a central role in the breakdown of extracellular matrices. Collagen, elastin, gelatin and casein are the main components of extracellular matrix cleaved by MMP. This paper aims to analyze the interaction between gelatinase B (MMP-9) and collagen using chronopotentiometric stripping analysis with adsorptive transfer stripping technique (AdTS CPSA). Under optimized experimental conditions (time accumulation of 90 s, supporting electrolyte 0.2 M acetate buffer pH 5, stripping current 1 mA), the detection limit (3 signal/noise) for MMP-9 was estimated as being 100 pM. The interaction between MMP-9 and collagen was studied according to the following scheme: i) HMDE surface was renewed. ii) Renewed surface of HMDE collagen (1 mg/mL) was accumulated for 90 s under open circuit. iii) The electrode was rinsed in ACS grade water and immersed in 5 mL drop of MMP-9. iv) The interaction between MMP-9 with collagen took place at open circuit. v) The electrode was then rinsed in ACS grade water. vi) The rinsed electrode was transferred into an electrochemical cell and measured in acetate buffer (pH 5). The CPSA signal of collagen after its interaction with MMP-9 increased more than 30% compared to that of only collagen. This increase in signal is likely due to the cleavage of collagen by MMP-9, hence its easy access to the electrodes surface. Keywords: Matrix metalloproteinases, Chronopotentiometric stripping analysis, Collagen, Protein – protein interaction, Cancer DOI: 10.1002/elan.200804440
Dedicated to Professor Joseph Wang, on the Occasion of His 60th Birthday
1. Introduction The matrix metalloproteinases (MMP), also known as matrixins, belong to a group of zinc-dependent proteins, which are thought to play a central role in the breakdown of extracellular matrix. Collagen, elastin, gelatin, and casein are the main components cleaved by MMP. The breakdown of these components is essential for many physiological processes such as embryonic development, morphogenesis, reproduction, and tissue resorption and remodeling [1]. MMP also participate in pathological processes such as arthritis, cancer, cardiovascular and neurological diseases [2 – 6]. The primary structure of MMP, for twenty different vertebrates, is comprised of several domain motifs, as illustrated in Figure 1. The domains have been divided according to their structure and function: collagenases, stromelysins, matrilysins, gelatinases, membrane type MMP and others MMP [7, 8].
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Chronopotentiometric stripping analysis (CPSA) measures the evolution of hydrogen from the supporting electrolyte catalyzed by the presence of a protein. This method is a highly sensitive technique commonly used for the analysis of proteins with detection limits at subnanomolar and lower levels. Disadvantages include high standard deviations and time of analysis at high stripping currents [9]. CPSA has been used for the detection of several biologically important peptides [10, 11] and proteins such as metallothionein [12 – 18], a-synuclein protein [19], MutS protein [20], glutathione-S-transferase [21], thrombin [22]. Moreover, Ostatna et al. showed this electrochemical method can be employed to study structural changes of bovine serum albumin [23, 24]. Serrano et al. studied metal – protein interactions using CPSA [25, 26]. Redox states of peptides and proteins can also be determined using CPSA [27]. However, CPSA has not been utilized for the detection of MMP, yet. The main aim of this paper is to characterize MMP-9, collagen and
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Fig. 1. Characterization of single MMPs according to their structural differences.
their interaction by using chronopotentiometric stripping analysis with adsorptive transfer stripping technique.
2. Experimental 2.1. Chemicals and pH Measurements Human MMP-9 was purchased from Chemicon International (Temecula, USA). Collagen was supplied from Vyzkumny ustav pletarsky (Brno, Czech Republic). ACS grade Co(NH3)6Cl3 and other chemicals (chemicals meet the specifications of the American Chemical Society) used were purchased from Sigma Aldrich (Sigma-Aldrich, USA) unless noted otherwise. The stock standard solutions (10 mg/mL) were prepared with ACS water (Sigma-Aldrich, USA) and stored in the dark at 20 8C. Working standard solutions were prepared daily by the dilution of the stock solutions with ACS certified water. The pH and conductivity were measured using inoLab Level 3 (WissenschaftlichTechnische Werksttten GmbH; Weilheim, Germany).
2.2. Electrochemical Measurements Electrochemical measurements were performed with AUTOLAB Analyzer (EcoChemie, Netherlands) connected to VA-Stand 663 (Metrohm, Switzerland), using a standard cell with three electrodes. A hanging mercury drop elec 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
trode (HMDE) with a drop area of 0.4 mm2 was employed as the working electrode. An Ag/AgCl/3 M KCl electrode served as the reference electrode. Glassy carbon electrode was used as the auxiliary electrode. For smoothing and baseline corrections, the software GPES 4.9 supplied by EcoChemie was employed. The analyzed samples were deoxygenated prior to measurements by purging with argon (99.999%) and saturated with water for 120 s. All experiments were carried out at room temperature. The temperature of supporting electrolyte was maintained by the flow electrochemical cell coupled with thermostat JULABO F12/ED (Labortechnik GmbH, Germany). Adsorptive transfer stripping technique (AdTS) with chronopotentiometric stripping analysis (CPSA) was used to determine the presence of MMP-9 and/or collagen by recording the inverted time derivation of potential (dE/dt)1 as a function of potential E [12]. Peptides and proteins produce a well-developed peak at highly negative potentials [10]. The behavior of this peak suggests the presence of catalytic evolution of hydrogen [16]. CPSA parameters were optimized as seen in Section 3.
2.4. Descriptive Statistics and Estimation of Detection Limit Data were analyzed using MICROSOFT EXCEL (USA). Results are expressed as mean SD unless noted otherwise. The detection limits (3 signal/noise, S/N) were calculated
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according to Long and Winefordner [28], whereas N was expressed as standard deviation of noise determined in the signal domain unless stated otherwise.
3. Results and Discussion The analysis of MMP raises methodical questions and concerns such as the type of sample (serum or plasma or blood), target molecule (total content of MMP or specific MPP) or form of MMP detected (MMP molecules or proMMP). Most commonly used methods for MMP analysis are immunochemistry and enzymatic-based ones [29].
3.1. Chronopotentiometric Stripping Analysis of MMP-9 Coupling adsorptive transfer stripping technique with chronopotentiometric stripping analysis has several advantages which include low detection limits for target molecules [9, 12, 13, 17, 23]. This coupled technique was employed to detect MMP-9 (Fig. 2A). Even though the amino acid cysteine forms only 3% (total count 19) of the total amino acid content in MMP-9 (total count 707), they are known to be responsible for most of the CPSA measured electroactivity of MMP-9. Experimental conditions were optimized to detect MMP-9 by AdTS CPSA. Time accumulation of MMP-9 onto HMDE was the first parameter optimized. Working MMP-9 concentrations used (1 ng/ mL) were low, which showed high CPSA sensitivity to
proteins. Measurements were carried out in borate buffer (pH 7.6) according to previously published results [12, 15, 16, 30]. MMP-9 was adsorbed onto HMDE at various times of accumulation: 30, 60, 90, 120, 150 and 180 s. Dependence of peak height on accumulation time is shown in Figure 2B. Peak height enhanced up to 90 s, and then decreased more than 50% possibly due to increase formation of complex structures on the working electrodes surface. Similar behavior of protein adsorption on HMDE surface was observed by Petrlova et al. [13, 31] and Adam et al. [32]. The pH of the supporting electrolyte plays an essential role in CPSA analysis. Hence, the electrochemical behavior of MMP-9 was studied in the following buffers: acetate buffer (pH 4.6), Britton – Robinson buffer (pH 6.5), phosphate buffer (pH 6.95) and borate buffer (7.6). MMP-9 signals measured in the buffers are shown in Figure 2C. MMP-9 gave signals at different potentials according to the pH and type of buffer: acetate buffer at 1.47 V; Britton – Robinson buffer at 1.62 V, phosphate buffer at 1.71 V and borate buffer at 1.74 V. The highest MMP-9 signal response was detected in acetate buffer (Fig. 2D). The lower pH values were determined to be more suitable for metalloproteinase electrochemical analysis than higher pH values. The value of MMP-9 isoelectric point is roughly 5.7 (http://www.signaling-gateway.org/molecule and is overall positively charged making MMP-9 detection advantageous. Fasciglione et al. showed experiments were unsuitable below pH 6.0 for accurate evaluation of MMP-9 enzymatic activity. Nevertheless, based on their results, MMP-9 is a relatively stable protein at pH values above 4.
Fig. 2. A) Scheme of adsorptive transfer stripping technique used for the detection of collagen and/or MMP-9 or for the study of interactions between these molecules; 1) renewing of the hanging mercury drop electrode (HMDE) surface; 2) adsorption of MMP-9 or collagen in a drop solution onto the HMDE surface at open circuit; 3) rinsing electrode in water of ACS purity; 4) measuring by chronopotentiometric stripping analysis. B) Dependences of MMP-9 (1 ng/mL) peak height on accumulation time and C), D) type of supporting electrolyte. Electroanalysis 2009, 21, No. 3-5, 536 – 541
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Fig. 3. Dependences of MMP-9 (1 ng/mL) peak height on A) pH of acetate buffer, B) stripping current and C) and inset, MMP-9 concentration.
The dependence of MMP-9 (1 ng/mL) peak height on various pH acetate buffers were investigated. This dependence is shown in Figure 3A. The highest CPSA response was observed at pH 5 0.2 M acetate buffer. Additionally, its potential shifted to more negative values with increasing pH. Lower pH values possibly facilitate hydrogen evolution from the supporting electrolyte during the catalytic reaction. Stripping current (1, 2, 4, 6, 8, 10 and 12 mA) was another experimental condition that influenced MMP-9 peaks (Fig. 3B). The lower stripping current resulted in a higher signal response. However, lower stripping currents (below 1 mA) produced lower reoccurring signals and increased relative standard deviations up to 10%. Based on results obtained a stripping current of 1 mA was selected for the following experiments. The shape of the dependence of the CPSA peak height on MMP-9 concentration was obtained (Fig. 3C). Concentration ranging from 1 to 10 nM MMP-9 assigned a linear dependence (y ¼ 2299.6x þ 220.36, R2 ¼ 0.9975). The detection limit (3 S/N) was estimated to be 100 pM.
The effect of hydrochloric acid on collagen solubility was studied in greater detail. HCl solutions with concentrations ranging from 0.1 to 20% (m/m) were used to dissolve 100 mg of collagen. This solution (1 mL) was placed onto a shaker and agitated for 30 min. at 400 rpm. Collagen disintegration increased with increasing hydrochloric acid concentration. However acidic conditions (pH 0.5 – 1.5), can negatively influence the native structure of a protein. Usha and Ramasami found charge repulsion disrupts the stability of rat tail tendon collagen fiber at low pH values [33]. At pH lower then 6, there is a significant decrease in shrinkage temperature. This may partly be due to osmotic forces that lead to acid swelling. Extensive hydration could lead to significant volume changes and the rupture of the matrix structure. Furthermore, protonation of the ionizable group may dominate at pH values lower than the isoelectric point which could decrease intermolecular ion pair formation. Lower pH does not digest collagen fibers although MMP does. Collagen was dissolved with 9% HCl (m/m) and used in the following experiments.
3.2. Collagen Modified HMDE
3.3. Interaction of MMP-9 with Collagen
Preparation of standard collagen solutions is a difficult task. Dissolving collagen in water is limited due to its low solubility and its compact structure. With agitation and stirring the solubility of collagen can be enhanced, but the natural folding structure could be lost. Estimating collagen concentration by using spectrometry is also difficult. Electrochemical methods including chronopotentiometry are convenient alternative methods for estimating collagen concentration. The effect of two solvents, deionized water and HCl (9% m/m), tested solubility properties of collagen. Collagen suspensions were agitated using Vortex 2 (Eppendorf, Germany) at 400 rpm for 15 min. Collagen decomposition improved in HCl compared to water (Fig. 4A).
This work studied the interaction of MMP-9 with collagen using AdTS CPSA. The dependence of collagen peak height (1 mg/mL) on time accumulation is shown in Figure 4B. The highest response was measured at 90 s. In order to maintain the optimum conditions for the enzymatic collagen cleavage by MMP-9, MMP-9 dissolving solution, which contained 0.05 M Tris-HCl pH 7.6 þ 0.2 M NaCl þ 0.01 M CaCl2, was used. At this pH, MMP-9 is activated and cleaves collagen [34]. MMP-9 concentration of 1 ng/mL gave a signal at 1.65 V (Fig. 4C). The signal of collagen appeared at slightly more positive potentials ( 1.64 V). The interaction itself was studied according to the following scheme: i) HMDE surface was renewed. ii) Collagen (1 mg/mL) accumulated (90 s) on renewed HMDE surface at open
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Fig. 4. A) Height of peaks of collagen dissolved in ACS water or 9% HCl. B) Dependence of collagen peak height on accumulation time. C) Signals of collagen, MMP-9 and collagen after interaction with MMP-9 measured by AdTS CPSA (interaction time: 30 s). D) Height of CPSA peaks of collagen (0.5 or 1 mg/mL) after interaction with MMP-9 (0.5 or 1 ng/mL).
circuit. iii) The electrode was rinsed in ACS grade water and immersed in 5 mL MMP-9 solution (1 ng/mL). iv) The interaction between MMP-9 and collagen was studied from 30 to 300 s under open circuit. v) The electrode was then rinsed in ACS water. vi) The electrode was transferred into an electrochemical cell and measured in acetate buffer (pH 5). The change in CPSA peak is shown in Figure 4C. CPSA signal of collagen after interaction with MMP-9 increased more than 30% compared to CPSA signal of collagen only. The potential of the signal was shifted 20 mV toward positive values. With increased MMP-9 interaction, the signal of collagen adsorption onto HMDE enhanced. The experiment was repeated with lower collagen and MMP-9 concentrations. The concentration of both components were halved: 0.5 mg/mL collagen itself (Fig. 4Dcolumn 3) and collagen after interaction with MMP-9 (0.5 ng/mL) (Fig. 4D-column 4). The signals measured were cut in half compared to previous results. Based on the obtained results, it is possible collagen is cleaved into smaller fragments by MMP-9. These fragments are conveniently accessible to the HMDEs surface, resulting in a higher signal (Fig. 4D). Similar phenomenon were observed Electroanalysis 2009, 21, No. 3-5, 536 – 541
during the analysis of denatured protein p53 [35, 36], urease [37] and lactoferrin [38 – 40].
4. Conclusions Study of protein-protein interactions in the past have required expensive, time consuming and labor intensive methods, techniques and approaches. Adsorptive transfer stripping technique coupled with chronopotentiometric stripping analysis is an easy and low cost approach to detect MMP-9 interaction with collagen. This technique determines the cleavage of collagen catalyzed by MMP-9 using enhanced CPSA signals. The well observed signal is probably due to the collagen moieties open access to the electrodes surface.
5. Acknowledgements Financial support from the Grants IGA MZLU MP 12/AF and 2A-1591/122-MPO is highly acknowledged. The au-
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thors wish to express their thanks to Dr. Grace Chavis for English correction and discussion.
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www.electroanalysis.wiley-vch.de
Electroanalysis 2009, 21, No. 3-5, 536 – 541
V.
KOMENTÁŘ K PUBLIKACÍM Hledání nových onkologických markerů je v dnešní době úkol, který zaměstnává
řadu odborníků od lékařů, přes molekulární biology až po analytické chemiky. Toto multioborové propojení přináší zajímavé výsledky a především otevírá nové možnosti pro navržení nových diagnostických postupů, metodik a přístrojů. Dalším přínosem je usnadnění při hledání a ověřování vhodnosti použití nových a slibných markerů pro jednotlivá nádorová onemocnění. Do této velké skupiny látek, které jsou dnes intenzivně studovány, patří i protein metalothionein.
4.
Literární
rešerše
o
vztahu
metalothioneinu
a
nádorových
onemocnění V tomto příspěvku jsme se pokusili sumarizovat výsledky dosažené v oblasti studia vztahu MT a vzniku, progrese a léčby nádorových onemocnění. V posledním desetiletí se objevila řada publikací ukazující MT jako užitečný diagnostický faktor nádorové progrese a rezistence u různých malignit např. leukémie, tumory kůže, prsu, vaječníků, ledvin, plic, slinivky,
žlučníku,
jícnu
a
bazálních
buněk
karcinomů
[335,336,347-358].
Z publikovaných výsledků vyplývá, že se MT může aktivně zapojovat do ochrany nádorových buněk proti apoptóze a zároveň podporovat metastazující chování nádoru a buněčnou proliferaci. Na druhé straně je nezbytné zmínit, že ve studiích věnovaných nádorovým onemocněním v oblasti tlustého střeva a močového měchýře a dalších, nebyl pozorován žádný významný vztah mezi hladinou MT a prognózou daného onemocnění [359,360]. Při podrobnějším pohledu na jednotlivá nádorová onemocnění dle jejich lokalizace bylo zjištěno, že zvýšená MT exprese u pacientů trpících karcinomem prsu se ukázala být známkou horší prognózy [361,362]. V případě karcinomu prostaty byl objeven velmi zajímavý fenomén lokalizace MT detekované pomocí imunohistochemie. Benigní léze vykazovaly vyšší obsah MT v jádru epitelových buněk, zatímco buňky karcinomu produkovaly MT především v cytoplazmě [305]. Pozitivní korelace mezi MT, vyzráváním nádoru („gradingem“) a expresí Ki-67 byla nalezena v nedávno publikované studii [363]. MT exprese u ovariálních epiteliálních nádorů je známkou malignity [364]. Podobný jev lze pozorovat u nádorů v oblasti děložní sliznice, kde exprese MT opět pozitivně koreluje nejen s gradingem a expresí Ki-67, ale také s obsahem proteinu p53 a negativně s receptory
142
progesteronu. Tyto údaje naznačují, že exprese MT detekovaná imunohistochemicky je pod hormonální kontrolou ve zdravém endometriu a může být použita jako marker progrese endometriálních karcinomů [365]. Dalším typem nádorového onemocnění, kde můžeme pozorovat pozitivní korelaci mezi fadingem, expresí Ki-67 a MT je karcinom plic [347]. Navíc bylo pozorováno, že zvýšená exprese MT je nezávislým prediktivním faktorem kratšího přežití pacientů s karcinomem plic podstupujících chemoterapii ve srovnání s pacienty s nádory vykazujícími nízkou expresi MT [366]. Co se týče dalších nádorů v oblasti dýchacího řetězce, doposud publikované výsledky naznačují možnou korelaci mezi spinocelulárním karcinomem v dutině ústní a MT [367,368]. V případě vylučovacího systému je možné použít MT jako potencionální klinický prediktivní marker u renálních buněk [350,369]. Studie zaměřené na karcinom močového měchýře ukazují na korelaci exprese MT s agresivním chováním nádoru [370,371]. Vztahem karcinomu trávicího traktu a MT se zabývala řada autorů [372-378]. Z jejich výsledků vyplývá pozitivní korelace exprese MT a Ki-67. Melanomy se řadí mezi další typy nádorových onemocnění, u kterých lze pozorovat pozitivní expresi MT a Ki-67 [379]. Zvýšená exprese různých isoforem MT anebo jejich buněčná lokalizace může sloužit jako prediktivní marker horší prognózy, vyššího vyzrávání nádoru nebo markeru maligní transformace benigních lézí. Predikce chemorezistence na cytostatika platiny nebo jiných cytostatik (etoposid, irrinotecan) na základě hladiny MT musí být potvrzeno větším souborem dat. Navíc, stanovení hladiny MT v séru se zdá být perspektivní pro klinickou praxi [335,336,352].
5.
Literární rešerše o možnostech detekce metalothioneinu Detekce MT, ať již z pohledu environmentálního nebo klinického prognostického
markeru, je objektem zájmu řady vědeckých týmů. Souhrnný článek diskutuje řadu přístrojů a metod, které se běžně používají pro detekci MT vzhledem k jejich sensitivitě, selektivitě, nákladech a možnostech použití. Díky nízké molekulární hmotnosti a unikátní primární struktuře (MT neobsahuje žádné aromatické aminokyseliny nebo disulfidické vazby) běžně používané metody detekce proteinů trpí nedostatečnou specifičností a citlivostí. Nejčastější metody používané pro detekci těchto proteinů jsou nepřímé a jsou založené na kvantifikaci iontů těžkých kovů běžně obsažených ve struktuře MT a na vysoký obsah –SH skupin.
143
Separace MT je nejčastěji prováděna pomocí gelové elektroforézy v různých provedeních a modifikacích [380-384]. Speciální místo v separačních metodách má kapilární elektroforéza, která umožňuje snadné a rychlé rozlišení jednotlivých isoforem MT [385]. Vysoce účinná kapalinová chromatografie má oproti výše zmíněným technikám minoritní použití [386,387]. Pro samotnou detekci MT lze použít nejen řadu analytických nástrojů včetně elektrochemie a hmotnostní spektrometrie, ale také metody molekulární biologie v čele s imunologickými metodami [383,388]. Každá z technik má své výhody a nevýhody, které ji upřednostňují pro jednotlivé typy materiálů, potřeby získat specifické informace či cenové dostupnosti. Budoucnost technik lze spatřit v miniaturizaci celého detekčního zařízení a tím usnadnění použití přímo na místě.
6.
Vliv kovů přirozeně obsažených ve struktuře metalothioneinu na
jeho stanovení MT ve své struktuře obsahuje esenciální ale i toxické těžké kovy. Tento fakt na jednu stranu může usnadnit jeho stanovení pomocí spektrometrických technik, na straně druhé se může stát překážkou pro jeho správnou a přesnou kvantifikaci pomocí dalších metod včetně elektrochemických. Proto je velmi žádoucí studovat chování MT v různém dobře definovaném prostředí a tak zjednodušeně modelovat podmínky v živém organismu. 6.1
Elektrochemické chování metalothioneinu Nejdříve
jsme
přistoupili
k
základní
elektrochemické
charakterizaci
metalothioneinu (MTCd5Zn2). V této práci jsme navázali na experimentální znalosti z našich předešlých prací a analýzu MT jsme uskutečnili v základním elektrolytu 0,5 M NaCl (pH 6,4) [389,390]. MT jsme analyzovali pomocí adsorptivní přenosové rozpouštěcí techniky (AdTS) ve spojení s diferenční pulsní voltametrií (DPV). Značnou výhodou techniky adsorptivního přenosu je možnost sledovat chování MT adsorbovaného na povrch pracovní elektrody bez dalších interferencí. Díky tomuto originálnímu postupu můžeme sledovat změny elektrochemických signálů způsobené vlivem přesně definovaných experimentálních podmínek (Obr. 4). Prvním studovaným parametrem byl vliv pH (od 6,0 do 7,6) na strukturu MT. Ze získaných experimentálních dat je zřejmé, že molekula MT se vzrůstajícím pH je více sbalená a díky tomu se atomy iontů stávají nepřístupné elektrodové reakci. V případě 144
sníženého pH dochází k postupnému rozprostření cysteinových klasterů a tím pravděpodobně dochází ke snadnějšímu uvolnění vázaného iontu kovu ze struktury MT a také k lepší dostupnosti sulfhydrylových skupin a kovů k povrchu pracovní elektrody. Lze očekávat, že celý pozorovaný mechanismus (zachytávání a uvolňování iontů kovů z molekuly MT) nebude založený pouze na výměně a reorganizaci struktury klasterů MT, ale i na dalších nepopsaných mechanismech. Dalším sledovaným parametrem byla iontová síla v podobě koncentrace NaCl od 0,05 M do 0,5 M. Zjistili jsme, že signály MT jsou koncentrací základního elektrolytu ovlivněny, ale ne tak významně. Ze získaných výsledků vyplývá, že iontová síla neovlivňuje strukturu tak, jako je to v případě změn pH.
Obrázek 4. Schéma využití adsorptivní techniky pro studium změny ve struktuře metalothioneinu v přítomnosti EDTA. (1) Obnovený povrch pracovní HMDE je umístěn do malé 5 µl kapky, (2) ve které je přítomen metalothionein a EDTA. (3) Na povrch HMDE se přednostně váže pouze metalothionein, (4) slabě adsorbované nízkomolekulární látky jako těžké kovy a EDTA jsou v následujícím kroku odstraněny z povrchu omytím elektrody ve vodě ACS čistoty. (5) Následně je modifikovaná HMDE umístěna do základního elektrolytu, (6) kde probíhá analýza pomocí diferenční pulzní voltametrie.
6.2
Vliv EDTA na strukturu metalothioneinu V další části práce jsme se zaměřili na studium změny struktury MT vyvolané
působením silného chelatačního činidla. Takové experimenty jsou potřebné pro další studie, které umožní studovat regulačně významné procesy jako je například interakce mezi transkripčními faktory a kovem [391].
Mezi látky velmi hojně využívané
i v biologických experimentech patří ethylendiamin-N, N, N`, N`-tetraoctová kyselina (EDTA). EDTA je dokonce schopna intenzivně vázat těžké kovy i z aktivních center enzymů a regulačních proteinů [392]. Námi navržený experiment měl následující uspořádání – MT v nízké koncentraci (100 nM) byl umístěn do mikrozkumavky (20 µl) a následně byla přidána EDTA. Vzniklý roztok byl mírně protřepán po dobu 2 minut, bylo odebráno 5 µl a toto množství bylo analyzováno pomocí AdTS DPV. Celý postup byl opakován pro rozdílné výsledné koncentrace EDTA. Na povrch HMDE byl akumulován pouze MT (tA 120 s), molekuly EDTA a uvolněné těžké kovy byly z povrchu HMDE
145
omyty. Zjistili jsme, že všechny signály MT, kromě CdT, který je přímo úměrný volným –SH skupinám, pozvolna klesají až do výsledně přidané koncentrace EDTA (600 nM). Poté jsou již změny signálů velmi nízké. Naopak signál CdT se vzrůstající koncentrací výrazným způsobem vzrůstal také do výsledné koncentrace EDTA (600 nM). Předpokládá se,
že
interakce
mezi
EDTA
a
MT
probíhá
podle
následujících
kroků
i) MTCd5Zn2 + EDTA = dochází ke kolapsu klusteru M-S-protein (M- kov; T – thionein) ii) M-S-protein = M + S- protein = M+ + S-protein a iii) M+ + EDTA- = M-EDTA [393]. Struktura MT klasterů se pravděpodobně začíná rozpadat při koncentraci EDTA 400 nM a vyšší. Tento fakt jsme ověřili studiem závislosti změny signálu CdT v přítomnosti 400 nM EDTA na době interakce (30 s až 25 min). Pozorovaný signál CdT se měnil velmi málo. EDTA postupně z molekuly MT intenzivně vyvazuje molekuly těžkých kovů. Lze předpokládat, že od aplikované koncentrace 600 nM EDTA je molekula MT na povrchu HMDE již jako apoMT (Obr. 5A).
Obrázek 5. (A) Změny výšky voltametrických signálů matlothioneinu ZnT, ZnT`, CdT`, CdT, MT(Zn) a MT(Cd) v závislosti na změně koncentrace EDTA; výšky signálů jsou ukázány jako poměr k maximální hodnotě, na levé ose je výška CdT, výška ostatních signálů je ukázána na pravé ose. (B) Voltamogramy a (C) UV-Vis spektra MT (100 nM, černá křivka) a 100 nM MT + 600 nM EDTA (červená křivka).
146
Na Obr. 5B jsou porovnány AdTS DPV voltamogramy 100 nM MT, kde jsou pozorovatelné všechny charakteristické signály, avšak v případě přídavku 600 nM EDTA dochází k vymizení všech signálů a je zřetelný pouze signál CdT (odpovídající -SH skupinám v klasterech MT). Navíc jsme stejný roztok analyzovali pomocí UV-VIS spektrometrie. Pozorované změny ve spektrech jsou ukázány na Obr. 5C. Získané výsledky naznačují,
že
EDTA
skutečně
z
molekuly
MT
vyvazuje
těžké
kovy
a v roztoku zůstává MT bez navázaných iontů kovů tzv. apoMT. Tento uvedený postup by mohl představovat velmi vhodnou alternativní metodu pro jednoduchou, rychlou a nenáročnou přípravu apoMT.
7.
Elektrochemické stanovení metalothioneinu v krevních vzorcích
pacientů s nádorem prsu Proteomický výzkum vyžaduje vysoce senzitivní analytické techniky umožňující detekci velmi malého množství biologického vzorku. Navíc pro vlastní analýzu je vyžadováno, aby nebyla příliš nákladná a bylo možné uskutečnit její další miniaturizaci. Tento trend je velmi dobře viditelný v oblasti průtokových mikročipů [394-398]. Elektrochemické metody mohou být v této oblasti velmi přínosné. V této práci jsme testovali možnosti analýzy vzorku o objemu několika set nanolitrů. Vypařování vzorku, jako největší překážky, jsme zabránili pomocí modifikace přenosové techniky. Využili jsme běžně dostupných komponent a navrhli následující postup analýzy MT. Na běžné mikroskopické sklíčko je pomocí kahanu nataven malý čtvereček parafilmu (Obr. 6a). Upravené sklíčko je omyto v etanolu a destilované vodě. Takto upravené sklíčko se umístí do chlazeného prostoru v našem případě kádinky s destilovanou vodou umístěnou v temperované vodní lázni na teplotu 2°C (Obr. 6b). Těsně před použitím se sklíčko s parafilmem vytáhne z vodní lázně, jemně otře buničinou a pomocí mikropipety je nanesen vzorek (Obr. 6c). Vzorek je následně analyzován pomocí pracovní elektrody umístěné do tohoto vzorku (Obr. 6d). Po akumulaci analytu na povrch pracovní elektrody je elektroda omyta v destilované vodě a pufru s následnou elektrochemickou detekcí (Obr. 6e,f,g). Jednoduchý technický prostředek (podchlazení povrchu) přináší pro analýzu vzorku několik výhod. Mezi nejvýznamnější náleží snížení procesu vypařování z povrchu kapky vzorku. Takto modifikovaná metoda adsorptivního přenosu ve spojení s Brdičkovou reakcí byla využita pro stanovení MT v reálných vzorcích krevních sér pacientek trpících 147
zhoubným nádorem prsu. Před patnácti lety byla ve světové databázi Web of Science zveřejněna první informace o vztahu metalothioneinu a zhoubného nádoru prsu [399,400]. Později byla publikována práce na stovce pacientek, kde se autorům podařilo prokázat, že zvýšená hladina MT je asociována se špatnou prognózou onemocnění a naopak nízká hladina MT v nádorové tkáni indikovala dobrý prognostický typ [401]. Vzorek upraveného krevního séra o objemu 500 nl byl nanesen na vychlazené sklíčko a analyzován. Získali jsme velmi dobře vyvinuté katalytické signály (Obr. 7). Signál Cat 2 byl vyhodnocen a z jeho výšky určena koncentrace MT ve vzorcích. Množství MT u pacientů se pohybovalo v rozmezí od 0,9 do 1,9 µM, což téměř dvoj- až trojnásobně převyšuje hladinu MT v krvi zdravých dobrovolníků.
Obrázek 6. Schéma adsorptivní přenosové techniky využívané pro senzitivní stanovení obsahu MT v malém objemu analytu (stovky nl). (a) Příprava mikroskopického sklíčka, odmaštění, přitavení parafilmu, (b) umístění do vychlazené vodní lázně minimálně po dobu 10-15 min, (c) osušení a nanesení malého množství analyzovaného vzorku, (d) umístění pracovní elektrody do vzorku, (e) omytí elektrody ve vodě ACS čistoty a pufrem, (f) přenesení elektrody do základního elektrolytu a analýza.
Obrázek 7. Voltamogramy krevního séra získaného od čtyř pacientů trpících zhoubným karcinomem prsu. Analyzovaný objem vzorku byl 500 nl.
148
8.
Vliv cisplatiny na hladinu metalothioneinu u buněčných linií,
laboratorních krys a pacientů s nádorem v oblasti hlavu a krku V předchozí práci vyvinutou techniku jsme aplikovali pro analýzu na cisplatinu senzitivní a rezistentní buněčné linie neuroblastomu vycházejí z mateřské linie izolované z kostních metastáz pacientů s neuroblastomem. Na Obr. 8A jsou ukázány typické AdTS DP voltamogramy buněčných extraktů získaných z mateřské, senzitivní a rezistentní buněčné linie. Rezistentní buněčná linie je trvale kultivována v kultivačním médiu obsahujícím protinádorové léčivo. Na voltamogramech jsou dobře rozlišitelné katalytické signály Cat2 ukazující na přítomnost volných –SH skupin v molekulách o vyšší molekulové hmotnosti. U těchto buněčných homogenátů byla provedena polyakrylamidová gelová elektroforéza s následným barvením stříbrem. Pouze u linie rezistentní k cisplatině bylo možné pozorovat velmi slabý proužek indikující přítomnost MT (Obr. 8A). Je zřejmé, elektrochemická detekce je velmi výhodná pro detekci MT v buněčných vzorcích.
Obrázek 8. (A) Voltamogramy buněčných linií UKF-NB4 a to (a) mateřské buněčné linie, (b) linie rezistentní vůči cisplatině (kultivována v přítomnosti 1 µM cisplatiny), (c) linie senzitivní
149
(kultivované bez přídavku cisplatiny). V dolní části obrázků jsou ukázány výsledky z analýzy pomocí polyakrylamidové gelové elektroforézy, vlevo standard metalothioneinu (300 ng) a vpravo signál detekovaný ve vzorku. (B) Změny v hladině metalothioneinu stanovené v UKF-NB4 buněčných liniích (a, b, c odpovídá A). Hladina metalothioneinu stanovená v rezistentní linii kultivované v přítomnosti různé koncentrace (C) cisplatiny anebo (D) karboplatiny.
V případě, že byla buněčná linie vystavena působení 1 µM cisplatiny po dobu 72 h, došlo k nárůstu obsahu MT o více než 60 %. Obsah MT v rezistentní linii buněk ponechaných po dobu 72 h na médiu bez cisplatiny byl také mírně zvýšen v porovnání s mateřskou linií (Obr. 8B). Změna hladiny MT u buněčných kultur vystavených rostoucí koncentraci cisplatiny a karboplatiny po dobu inkubace 24 h je ukázán na Obr. 8C,D. Koncentrace cisplatiny 0,01 až 0,1 µM vede k nárůstu obsahu MT do 10 %, ale vyšší koncentrace způsobuje velmi prudký nárůst obsahu MT. Podobný výsledek byl pozorován i v případě aplikované karboplatiny, kdy koncentrace 0,1 až 1 µM vede k syntéze MT do 10 % v porovnání s neovlivněnou buněčnou kulturou. Vyšší koncentrace již vede k výrazné produkci MT.
8.1
Změny hladiny metalothioneinu u pacientů se zhoubnými nádory v oblasti
hlavy a krku léčenými cisplatinou Monitorování hladiny MT by mohlo být přínosné pro pacienty se zhoubnými nádory z různých hledisek. Jedním by mohlo být předcházení rezistence na protinádorovou léčbu vedenou platinovými léčivy. Pro tento účel byly sbírány vzorky krve od pacientů v průběhu jejich léčby. Změny hladiny MT u pacienta v průběhu 148 hodin od podání léčiba jsou ukázány na Obr. 9A. Hladina MT je několikanásobně vyšší než u hladiny MT v krevním séru zdravých dobrovolníků [402]. Typické DP voltamogramy krevního séra pacientů s nádory hlavy a krku léčených cisplatinou jsou uvedeny na Obr. 9A. Účinek cisplatiny na hladinu MT před a po podání tohoto léku je ukázáno na Obr. 9B. Hladina MT se zvýšila v důsledku podání léku u všech pacientů (Obr. 9A,B,C). Po několika hodinách od podání léčiva se začala hladina MT snižovat.
150
Obrázek 9. Sledování změn hladiny metalothioneinu v krevním séru tří pacientů trpících zhoubným nádorem v oblasti hlavy a krku po podání cisplatiny. (A) Pacient 1 (0 – 148 h), ve vloženém obrázku jsou zobrazeny typické DP voltamogramy; (B) pacient 2 (-168, -120, -72, -48 jsou hodiny před podáním cisplatiny); (C) pacient 3 (0 – 80 h).
8.2
Změny hladiny metalothioneinu u pacientů s retinoblastomem léčenými
karboplatinou Dále nás zajímalo, jakým způsobem ovlivní podání karboplatiny, druhé generace platinových léčiv, hladinu MT u pacientů s nádorovým onemocněním. Pacienti se vzácným nádorovým onemocněním retinoblastomem (n = 9) bylo léčeno karboplatinou. Pozorovali jsme změnu hladiny MT během 24 hodin od podání léčiva (Obr. 10). Průměrná koncentrace MT v plazmě pacientů s retinoblastomem před zahájením léčby karboplatinou
151
byla 3,4 μM. Při detekci hladiny MT můžeme pozorovat různé jevy. U pacientů 2, 4 a 6 se hladina MT neměnila. Naopak hladina MT se u pacientů 1, 3, 5 a 9 snížila již 4 hodiny po podání. To může souviset s vazbou MT na cytostatika. Léčba pacientů 7 a 8 karboplatinou měla za následek téměř dvojnásobné zvýšení hladiny MT. Průběh hladin MT je individuální a závisí pravděpodobně na nádorové rezistenci vůči karboplatině.
Obrázek 10. Změny hladiny metalothioneinu u pacientů trpících retinoblastomem po podání karboplatiny.
9.
Studium interakce želatinázy B s kolagenem MT se účastní v procesu karcinogeneze pravděpodobně více procesů, přičemž
jedním z nich by mohla být aktivace kov-dependentních enzymů, mezi které patří MMP, 152
které ke své aktivaci potřebují přítomnost dvou zinečnatých iontů ve své molekule. Proto jsme se v další práci rozhodli využít elektrochemické techniky pro studium interakce želatinázy B, jako zástupce MMP, s kolagenem. Nejprve jsme optimalizovali experimentální podmínky chronopotenciometrické rozpouštěcí analýzy, která byla využita pro detekci želatinázy B (MMP-9). V 5 µl kapce bylo tak možné analyzovat femtomolové množství MMP-9. Kolagen patří do množiny jejích hlavních substrátů v organismu. Rozhodli jsme se proto přistoupit k elektrochemické detekci interakce MMP-9 s kolagenem. Použili jsme vzorek kolagenu o koncentraci 1 µg/ml a akumulovali ho po dobu 90 s na HMDE. Po této době jsme HMDE omyli ve vodě ACS čistoty („chemicals meet the specifications of the American Chemical Society”) a pufru a následně kolagenem modifikovanou elektrodu umístili do 5 µl kapky vzorku MMP-9 o koncentraci 1 ng/ml. Interakce MMP-9 s kolagenem byla studována po dobu 30 až 300 s. Po uplynutí této doby interakce byla HMDE omyta a výsledné změny na povrchu pracovní elektrody byly zaznamenány pomocí CPSA. Signál kolagenu se po působení MMP-9 zvýšil asi o 30 % v porovnání s CPSA signálem kolagenem modifikované HMDE. S dobou interakce kolagenem modifikované HMDE docházelo k nárůstu signálu produktu štěpení kolagenu pomocí MMP-9. Experiment byl úspěšně opakován i v nižších koncentracích kolagenu a MMP-9. Lze usuzovat, že vzrůst signálu kolagenu ovlivňuje MMP-9 tím, že kolagen lyzuje na menší fragmenty, které jsou lépe přístupné na povrch HMDE a dávají tím vyšší signál. Interakci kolagenu v našich experimentálních podmínkách jsme ověřili pomocí další elektrochemické metody (diferenční pulzní voltametrie Brdičkovy reakce) a pomocí chipové gelové elektroforézy. A dále jsme se rozhodli studovat, zda-li jsme schopni zvýšit aktivitu MMP-9. Tyto proteiny ve své struktuře obsahují zinečnaté ionty, které je schopen transportovat
protein
metalothionein.
Proto
jsme
celý
experiment
zjednodušili
a analyzovali směs kolagenu, MMP-9 a zinečnatých iontů. Interakci jsme sledovali po dobu devíti hodin. Přibližně po šesti hodinách došlo ke zlomu a signál vzrostl během následujících několika desítek minut téměř o padesát procent. Struktura kolagenu je velmi komplexní a je velmi pravděpodobné, že tento zlom značí rozložení největší části hlavích struktury a následně došlo k rozkladu menších fragmentů. Nejzajímavějším výsledkem je, že aktivita MMP-9 v prostředí zinečnatých iontů je vyšší v porovnání s experimentem kolagen-MMP-9, což naznačuje možnost aktivace MMP-9 pomocí zinek-transportujících proteinů.
153
VI. ZÁVĚR Klinický výzkum se dnes opírá o řadu moderním technologií, postupů a přístupů, které jsou na straně jedné precizní a robustní, na straně druhé vyžadují školenou obsluhu, jejich pořízení i samotný provoz je finančně náročný a především nejsou vhodné pro použití na místě popř. pro plošný „screening“ neboli vyšetřování. V předkládané práci jsou ukázány možnosti elektrochemické analýzy v nádorové diagnostice. Elektrochemické techniky byly využity pro detekci metalothioneinu jako potencionálního nádorového markeru. Získané pilotní výsledky naznačují nejen možnosti využité metody pro detekci daného proteinu, ale také ukazují na možný vztah metalothioneinu se vznikem a progresí dvou typů nádorových onemocnění. Pro analýzu většího množství vzorků od pacientů by mohla sloužit elektrochemie jako velmi vhodný nástroj. Dále jsme se pokusili studovat možnou
interakci
mezi
metalothioneinem
a
jedním
zástupcem
matrixových
metaloproteináz. Zde jsme pomocí elektrochemické analýzy snadno detekovali, že přítomnost na zinek bohatého protein metalothioneinu zvyšoval aktivitu metaloproteinázy, což otevírá nové možnosti ve studiu jejich aktivace, která je esenciální při růstu a vývoji nádoru.
154
VII. SHRNUTÍ Nádorová onemocnění, „mor moderního světa“, jsou slovy, která představují hrozbu lidské populace a zároveň jsou předmětem výzkumu široké vědecké veřejnosti (podle databáze Web of Science se jen v roce 2008 objevilo více než 80 tisíc článků, které ve svém názvu, klíčových slovech či abstraktu obsahovaly výraz „cancer*“ nebo „tumour disease“, přičemž v časopisech Nature a Science jich bylo dohromady přes sto padesát). Úspěch léčby nádorových onemocnění závisí na řadě faktorů, ale především na včasné, rychlé a citlivé diagnostice. Pro diagnostické účely je možné využít řadu postupů a technik založených na různých principech, mezi něž patří detekce nádorových markerů. Jsou různé typy molekulárních nádorových markerů zahrnující DNA, mRNA, proteiny, antigeny, hormony. Výzkum markerů nádorových onemocnění je dynamicky se rozvíjející se oblast, přičemž se hledají a ověřují stále nové. Do této obrovské skupiny látek patří i proteiny s názvem metalothioneiny. Metalothioneiny (MT) jsou skupinou jednořetězcových proteinů o molekulové hmotnosti od 2 do 13 kDa (u savců okolo 6,5 kDa). Jsou to kovy vázající proteiny bohaté na cystein se zajímavou strukturní vlastností a tou je nepřítomnost aromatických aminokyselin. Předložená práce je orientována na možnosti využití elektrochemických technik v nádorové diagnostice, konkrétně je pozornost zaměřena na protein
metalothionein.
Nejdříve
jsme
přistoupili
k
základní
elektrochemické
charakterizaci metalothioneinu (MTCd5Zn2). Ze získaných výsledků vyplývá, že iontová síla neovlivňuje strukturu tak, jako je to v případě změn pH. Dále jsme se zaměřili na studium změny struktury MT vyvolané působením silného chelatačního činidla, kterým byla ethylendiamin-N, N, N`, N`-tetraoctová kyselina (EDTA). I přesto, že jsou vazebné interakce mezi kovy a MT velmi silné, při poměru EDTA:MT (4:1) došlo ke kolapsu klusteru M-S-protein (M- kov; T – thionein). Tento fakt jsme potvrdili i pomocí UV-VIS spektrometrie. Poté, co jsme úspěšně elektrochemicky charakterizovali MT, jsme se zaměřili na navržení metody pro ultra-sensitivní detekci těchto látek v krevním séru pomocí adsorptivní přenosové rozpouštěcí techniky ve spojení s diferenční pulsní voltametrií Brdičkovou reakcí (AdTS DPV Brdičkova reakce). Navrženou metodou jsme byli schopni stanovit hladinu MT v reálných vzorcích krevních sér pacientek trpících zhoubným nádorem prsu o objemu 500 nl. Množství MT u pacientů se pohybovalo v rozmezí od 0,9 do 1,9 µM, což téměř dvoj- až trojnásobně převyšuje hladinu MT v krvi zdravých
155
dobrovolníků. Navrženou metodu jsme také aplikovali pro analýzu na cisplatinu senzitivní a rezistentní buněčné linie neuroblastomu vycházejí z mateřské linie izolované z kostních metastáz pacientů s neuroblastomem. Množství MT stanovené v liniích rezistentních na platinová cytostatika bylo několikanásobně vyšší v porovnání s linií mateřskou a na cytostatika citlivou. Na základě těchto výsledků jsme se zaměřili na detekci změn hladiny MT u pacientů se zhoubnými nádory v oblasti hlavy a krku léčenými cisplatinou a u pacientů s retinoblastomem léčenými karboplatinou. Hladina MT se zvýšila v důsledku podání cisplatiny u všech sledovaných pacientů, zatímco průběh změn hladiny MT u pacientů léčených karboplatinou je individuální a závisí pravděpodobně na více mechanismech. MT se účastní v karcinogenezi pravděpodobně více procesů, přičemž jedním z nich by mohla být aktivace kov-dependentních enzymů, mezi které patří matrixové metaloproteinázy (MMP), které ke své aktivaci potřebují přítomnost dvou zinečnatých iontů ve své molekule. Proto jsme se v další práci rozhodli využít elektrochemických technik (AdTS DPV Brdičkovu reakci a chronopotenciometrickou rozpouštěcí analýzu) pro studium interakce želatinázy B (MMP-9) s kolagenem. Dále jsme se rozhodli studovat, zdali jsme schopni zvýšit aktivitu MMP-9. Interakci směsi kolagenu, MMP-9 a zinečnatých iontů jsme sledovali po dobu devíti hodin. Nejzajímavějším výsledkem je, že aktivita MMP-9 v prostředí zinečnatých iontů je vyšší v porovnání s experimentem kolagen-MMP-9, což naznačuje možnost aktivace MMP-9 pomocí zinek-transportujících proteinů.
156
VIII. SUMMARY Tumour diseases, “plague of the world”, are words, which presents a threat to humans as well as a subject of investigation for broaden scientific public (more than 80 thousands papers appearing in 2008 in Web of Science included “cancer*” nebo „tumour disease“ within article titles, keywords or abstracts, particularly, more than one hundred and fifty of them has been published in Nature or Science). The success of treatment of the disease depends on many factors such as early and sensitive diagnostics. That means the sooner a cancer is detected, the better the chances to treat it successfully. For diagnostic purposes, it is possible to use a series of procedures and techniques based on different principles including the detection of tumour markers. There are different types of molecular tumour markers, including DNA, mRNA, proteins, antigens, hormones. A research of tumour diseases markers is a dynamically developing field, while constantly seeking for new ones. This huge group of substances includes proteins called metallothioneins. Metallothioneins (MT) are a group of low molecular mass (from 2 to 13 kDa, in mammals app. 6.5 kDa) single-chain proteins. They are metalbinding cysteine-rich proteins with interesting structural feature, which is the absence of aromatic amino acids. This thesis is aimed at the possibility of using electrochemical techniques in cancer diagnostic, specifically, the attention is focused on the protein metallothionein. Primarily, we investigated the basic electrochemical behaviour of metallothionein (MTCd5Zn2). The results obtained show that the ionic strength does not affect the structure as much as the changes in pH. Furthermore, we have focused on the study of changes in the structure of MT induced by the action of strong chelating agents as ethylenediamine-N, N, N `, N`-tetraacetic acid (EDTA). Even though the binding interaction between metals and MT are very strong one, cluster MS-protein (M-metal, T-thionein) was collapsing with the EDTA: MT (4:1). This fact was confirmed by using UV-VIS spectrometry. After we have successfully characterized electrochemically MT, we focused on the development of methods for ultra-sensitive detection of these substances in the blood serum by adsorptive transfer stripping technique in connection with differential pulse voltammetry Brdička reaction (ADTS DPV Brdička reaction). Using the proposed method we were able to determine the level of MT in real samples of blood sera of patients suffering from the breast carcinoma. The volume of a sample analyzed was 500 nl. Level
157
of MT determined in blood sera of the patients ranged from 0.9 to 1.9 μM, which was nearly double to three times higher than the level of MT in the blood sera of healthy volunteers. We also applied the proposed method for analysis of sensitive- and cisplatinresistant neuroblastoma cell lines derived from the maternal line isolated from bone metastases in patients with neuroblastoma. Level of MT in cell lines resistant to platinum based antitumor drugs were several times higher than in the maternal and/or sensitive cells. Based on these results, we have focused on detecting changes in MT levels in patients with malignant tumours in head and neck treated with cisplatin and in patients with retinoblastoma treated with carboplatin. Level of MT increased as a result of administration of cisplatin with each patient, while the pattern of change MT levels in patients treated with carboplatin was individual and probably depended on multiple mechanisms. MT is involved in the more processes of carcinogenesis, whereas one of them could be activating metal-dependent enzymes, including matrix metalloproteinases (MMP), which on its activation require the presence of two zinc ions in its molecule. Therefore, we have further decided to use electrochemical techniques (ADTS DPV Brdička reaction and chronopotentiometric stripping analysis) for studying interactions gelatinase B (MMP-9) with collagen. Furthermore, we aimed at the issue whether we can enhance the activity of MMP-9. Mixture of collagen, MMP-9 and zinc ions was monitored for nine hours. The most interesting result is that the activity of MMP-9 in an environment of zinc ions is higher in comparison with the experiment of collagen-MMP-9, suggesting the possibility of activation of MMP-9 with zinc-transporting proteins.
158
IX. SEZNAM POUŽITÉ LITERATURY [1] [2] [3] [4] [5] [6] [7] [8]
[9] [10] [11]
[12] [13]
[14]
[15]
[16] [17]
[18]
[19]
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