JUDUL PENELITIAN THE ROLE OF HEAT SHOCK PROTEIN IN CELL ACTIVITY
PHENOMENA Proteomic is a comprehensive study about protein, especially about structure and function. Heat Shock Proteins (Hsps) are a chaperones and act as a central integrity for protein homeostasis in the inner cells.
PROBLEM Until now the role of heat shock protein 40 (hsp40) and hsp70 in pathogenesis of occurred oral squamous cell carcinoma (OSSC) patient was unclear
THE AIM This research were to explain the role of heat shock protein 40 (hsp40) and hsp70 in pathogenesis of occurred oral squamous cell carcinoma (OSSC) patient.
THE CONCEPT
HPV
Inti sel
p53
hsp
pRb
P53 (Unfold)
e2f
c-myc
PRb (Unfold) Hsp40 Hsp70
MISFOLDED P53
APOPTOSOME (Cytocrome C)
MISFOLDED PRB
E2F
Apaf 1
mitokondria
Caspase 9 Caspase 3
C-MYC sitoplasma
APOPTOSIS
CELL DIVISION
KARSINOMA SEL SKUAMOUS RONGGA MULUT
Tujuan Khusus 1. Mengkonfirmasi perbedaan ekspresi Hsp40 dan Hsp70 antara pasien LJSSRM dan KSSRM. 2. Menemukan pola diskriminator untuk menjelaskan kejadikan KSSRM.
Rumusan Masalah Sejauh mana perbedaan ekspresi Hsp40 dan Hsp70 antara penderita LJSSRM dan KSSRM ?
Hipotesis Terdapat perbedaan ekspresi Hsp40 dan Hsp70 antara penderita LJSSRM dan KSSRM.
Manfaat Teoritis Menemukan patogenesis KSSRM baru
Manfaat Praktis Hasil penelitian ini diharapkan dapat digunakan untuk memperbaiki penanganan KSSRM, setelah dilakukan penelitian di manusia dan penelitian multi senter
METODE PENELITIAN
Jenis Penelitian Observasional (Crossectional) – Analytic Desain dengan rancangan post test only control group
Sampel pada penelitian ini adalah semua penderita lesi jinak sel skuamous rongga mulut dan penderita KSSRM yang datang ke RSUD dr Muwardi dari 1 Agustus 2007 hingga 31 Juli 2008. Atau dihitung menggunakan rumus besar sampel Steel &Torrie (1980)
Kriteria Inklusi 1. tidak ada mutasi pada gena p53 dan c-myc 2. tidak ada keganasan lain dan bukan penderita kanker kambuhan.
Variabel Penelitian
Hsp40 dan Hsp70
Besar sampel
{(Z1-+Z) σ)}2 n = ------------------d2 Karena σ2 sulit ditaksir dari literatur, studi yang sama sebelumnya atau studi pendahuluan oleh peneliti, maka diasumsikan --- σ2=d2 untuk =0,05, nilai Z1- = 1,654 dan =0,1, nilai Z = 1,234 maka n yang didapatkan adalah 9. Karena 2 variabel maka menjadi 18 (Steel dan Torrie, 1980)
Jaringan Biopsi Frozen SectionSel Skuamous Rongga Mulut (Jenis Kelamin, Umur, Asal Biopsi) Sel Skuamous Jinak
HE
Histopatologi
Isolasi DNA
PCR : gena L1-HPV, p53 dan c-myc SSCP : p53, c-myc
Mikroskop Cahaya
Elektroforesis
+
+
Karsinoma Sel Skuamous
Isolasi Protein
HE
Elektroforesis SDS PAGE Histopatologi Blooting Hsp40 dan Hsp70 Densitometry
Isolasi DNA
PCR : gena L1-HPV, p53 dan c-myc SSCP : p53, c-myc
Mikroskop Cahaya
Elektroforesis
+
+
Membedakan dan Mendapatkan Descriminator
Isolasi Protein
Elektroforesis SDS PAGE Blooting Hsp40 dan Hsp70 Densitometry
Analisis data Analisis Data Menggunakan 1. Uji F (Manova) 2. Analisis diskriminan
HASIL PENELITIAN
ELEKTROFORESIS TOTAL PROTEIN
LJSSRM
70 kDa
40 kDa
KSSRM
HASIL DOT BLOTT J (JINAK) DAN G (GANAS)
Tabel 3. Data hasil pembacaan densitometri hasil dot blott No.
Hsp70
Hsp40
SIFAT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
429,52 520,48 100,01 356,70 352,30 100,02 1230,39 1017,54 661,42 293,56 1771,15 2287,43 879,63 1622,84 619,05 1968,23 1062,77 1612,16
520,55 575,92 100,04 316,59 100,05 1653,15 326,37 1728,55 873,57 605,21 975,83 323,20 2132,96 965,02 1249,96 2994,47 1173,16 1771,46
jinak jinak jinak jinak jinak jinak jinak jinak jinak Ganas Ganas Ganas Ganas Ganas Ganas Ganas Ganas Ganas
PERBEDAAN Hsp70 Jinak Ganas
Hsp40
5.298.190
6.883.092
13.463.129
13.545.854
KADAR Hsp40 DAN Hsp70 antara LJSSRM DAN KSSRM
14.000.000 12.000.000 10.000.000 8.000.000
HSP70 HSP40
6.000.000 4.000.000 2.000.000 0
Jinak
Ganas
Tests of Equality of Group Means
HSP70 HSP40
Wilks' Lambda ,61210 ,8093
F ,164 ,768
df1 1 1
df2 16 16
Sig. ,006 ,070
Classification Function Coefficients
Group
Hsp70 (Constant)
Jinak
Ganas
,002 -1,169
,005 -3,764
Fisher's linear discriminant functions
Hsp70 Jinak Ganas
10.596 67.316
POLA KEKUATAN PERAN Hsp70 PADA LJSSRM DAN KSSRM
HSP70
80.000 60.000 40.000 20.000 0
Jinak
Ganas
PEMBAHASAN
Stress Perceotion and Stress Respons in Oral Squamous Cell Stress HPV
Native Polypeptides Misfolded Unfolded Proteins
Stress Gene
Nascent Polypeptides
mRNA
Stress Protein Heat Shock Protein
During distress (hypoxia), the cells appear to adapt, as described in hepatocytes, by downregulating consumption of ATP in response to a decreasing oxygen availability.24,38
P
K = DnaK = Hsp40 ; J = DnaJ = Hsp70 ; dan U = protein unfolded
HPV
CELL DISTRESS
STRESS PROTEINS EXPRESSION
HOMEOSTASIS PROTEINS EXPRESSION
Hsp40 dan -70
ATP PROTEINS MISFOLDING
PCDs (i.e. cancer)
THE CONCLUSION
Conclusion This experiment proved that OSCC patients indicated an up regulated of Hsp70 concentration, so that there was occurs misfolding of the proteins cell. The misfolding was ensue obstacle of apoptosis and to raise cell proliferation which to storm carcinogenesis. Hsp40 was role as a co-chaperone.
THANK YOU
Adi Prayitno. “Cervical Cancer with Human Papilloma Virus and Epstein Barr Virus Positif”. Journal of Carcinogenesis (BioMed Central; PubMed; MedLine), 5:13 (10 May 2006) 2006. Adi Prayitno, Mandojo Rukmo, Ambar Mudigdo, Widya Asmara, Suhartono, TP. "The expression of Hsp10 in oral lesion with positive HPV”. 4th International Eijkman Symposium (Indonesia – Belanda Meeting), Oktober, 2007. Adi Prayitno, Mandojo Rukmo, Ambar Mudigdo, Widya Asmara, Suhartono, TP. “Ekspresi Hsp 10 Dan 40 Pada Neoplasma Jinak Sel Skuamous Rongga Mulut dengan Positif HPV”,. Konker IAPI dan PIT, Menado 15-18 Juli 2008. Adi Prayitno, Mandojo Rukmo, Ambar Mudigdo, Widya Asmara, Suhartono, TP. "The expression of Hsp10 in oral lesion with positive HPV”. Folia Medica Indonesiana, edisi 1 2009.
24 Chandel NS, Budinger GR, Choe SH, Schumacker PT. Cellular respiration during hypoxia. Role of cytochrome oxidase as the oxygen sensor in hepatocytes. J Biol Chem. 1997; 272: 18808–18816.[Abstract/Free Full Text] 38 Hicke L, Dunn R. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu Rev Cell Dev Biol. 2003; 19: 141– 172.[CrossRef][Medline] [Order article via Infotrieve]
KARSINOMA SEL SKUAMOUS RONGGA MULUT
Terkumpul 40 sampel LJSSRM dan 15 sampel KSSRM
Gambaran histopatologi KSSRM dengan pengecatan HE
AMPLIFIKASI GENA L-1 HPV (450bp)
9/40 (23%) LJSSRM positif HPV 450 bp
11/15 (73%) KSSRM positif HPV
AMPLIFIKASI GENA p53 (216bp)
216 bp 9/9 (100%) LJSSRM tanpa mutasi gena p53 9/11 (81%) KSSRM tanpa mutasi gena p53
Terlihat 2 band yang sama
AMPLIFIKASI GENA c-myc (314bp)
314 bp 9/9 (100%) LJSSRM tanpa mutasi gena c-myc 10/11 (91%) KSSRM tanpa mutasi gena c-myc
Terlihat 2 band yang sama
ANOVA of Hsp70
Sum of Squares df Between Groups Within Groups Total
144,768 93,136 237,905
1
144,768 16 17
Mean Square
F
24,870 ,000 5,821
Sig.
p≤0,070
Classification Function Coefficients group jinak Ganas HSP70 ,002 ,004 HSP40 ,001 ,002 (Constant) -1,518 -5,010 Fisher's linear discriminant functions
ANOVA of Hsp40
Sum of Squares
Mean Square
df
Between Groups
18,382
1
18,382
Within Groups
24,811
16
1,551
43,193
17
Total
F 11,854
Sig. ,003
