[Effect of inhibiting survivin expression with antisense oligodeoxynucleotides on sensitivity of hepatocellular carcinoma cell lines HepG2 and HepG2/ADM to adriamycin].
OBJECTIVE
Survivin deserves attention as a selective target for cancer therapy because it is silenced in differentiated adult tissues, but is expressed in a variety of human tumors, and is involved in tumorigenesis and chemoresistance. Antisense oligodeoxynucleotides (ASODN) can be used to inhibit the expression of survivin to induce apoptosis or enhance chemosensitivity of tumor cells. This study was to investigate the effect of inhibiting survivin expression with ASODN on sensitivity of hepatocellular carcinoma cell lines HepG2 and HepG2/ADM to Adriamycin (ADM).


METHODS
The expression of survivin in HepG2 and HepG2/ADM cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Sensitivities of HepG2 and HepG2/ADM cells to ASODN and ADM were detected by MTT assay. ASODN was transfected into HepG2 and HepG2/ADM cells; expression of survivin was detected by RT-PCR. Synergetic effects of low concentrations of ASODN and subtoxic concentrations of ADM on HepG2 and HepG2/ADM cells were detected by isobolography. The expression of active Caspase-3 and cell apoptosis were evaluated by flow cytometry.


RESULTS
mRNA level of survivin in HepG2/ADM cells was 15 folds as that in HepG2 cells; protein level of Survivin in HepG2/ADM cells was 18 folds as that in HepG2 cells. Both HepG2 and HepG2/ADM cells were sensitive to ASODN-mediated cytotoxicity in a concentration-dependent manner. The 50% inhibitory concentration (IC(50)) of ASODN was 317.90 nmol/L for HepG2 cells, and 480.74 nmol/L for HepG2/ADM cells. The maximal cytotoxicity was observed at 500 nmol/L of ASODN; the inhibitory rate was 71.10% in HepG2 cells, and 53.67% in HepG2/ADM cells. The IC(50) of ADM was 0.36 microg/ml for HepG2 cells, and 2.12 microg/ml for HepG2/ADM cells; the resistance index was 6. ASODN efficiently down-regulated mRNA level of survivin in both cell lines in a concentration-dependent manner. For HepG2 cells, with the IC(50) of 271.93 nmol/L, the maximal effect of ASODN was achieved at a concentration of 400 nmol/L, at which mRNA level of survivin was down-regulated by 69.12%; for HepG2/ADM cells, with the IC(50) of 365.72 nmol/L, its maximal effect was achieved at a concentration of 400 nmol/L, at which mRNA level of survivin was down-regulated by 60.01%. ASODN in combination with ADM synergetically enhanced sensitivity of HepG2 cells to ADM by 6 folds, and HepG2/ADM cells by 4 folds. Combination treatment of ASODN and ADM gradually enhanced activity of Caspase-3 and cell apoptosis in a concentration-dependent manner, and resulted in caspase-dependent cell death in HepG2/ADM cells.


CONCLUSIONS
Inhibiting survivin expression with ASODN could sensitize hepatocellular carcinoma cells to ADM, and the combination of ASODN and ADM may be a reasonable approach for clinical treatment of ADM-resistant hepatocellular carcinoma.
Dai
De-Jian
DJ
Department of General Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, P. R. China.
Lu
Cai-De
CD
Guo
Jun-Ming
JM
Zhang
Jun
J
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D020128:Inhibitory Concentration 50; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000077022:Survivin; D014162:Transfection
D000903:Antibiotics, Antineoplastic; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D000077022:Survivin; D004317:Doxorubicin; D053148:Caspase 3