The Expression And Effect Of Survivin In Apoptosis Of Hepatocellular Carcinoma Induced By ATRA

Objectives:Survivin is a family member of inhibitor of apoptosis proteins (IAPs) and overexpression of survivin was observed in hepatocellular carcinoma (HCC) cell lines. However, the role of survivin in HCC is still not quite clear. In this experiment, the apoptosis was induced and proliferation was inhibited by all-trans retinoic acid (ATRA) in HCC cells. We examined the expression of survivin to discuss the function of ATRA at the treatment of liver cancer and the role of survivin in HCC and to determine the rationality that survivin will be used as the therapeutic target.Methods:To select the concentration of ATRA using MTT method. Human hepatocarcinoma cells SMMC-7721 were cultured with ATRA at different concentration (50μmol/l, 10μmol/l, and the comparing group) for 6 days, and to investigate the inhibition of ATRA on proliferation of SMMC-7721 cells by calculating the number of vital cells. To judge the morphologic change using invert microscope, micro-interference and electron microscope. To investigate apoptosis and cell cycle changes using flow cytometry. Using the assay of RT-PCR, to examine the expression of survivin mRNA. Using the assay of Western-blot and immunocytochemistry, to examine the change of survivin protein, and investigate the inhibition effect of ATRA on survivin protein in SMMC-7721 cells.Results:ATRA has inhibitive effect to the proliferation at different concentration, and the effect is dose-depedent and time-depedent. SMMC-7721 cells showed typical form change of apoptosis after 6 day's incubation with ATRA. While the concentration of ATRA is 50umol/l, 10umol/l, the inhibition rate of proliferation is 71.47%, 53.37% respectively and the apoptosis rate is 42.89%, 8.53% respectively. The apoptosis induction ability of ATRA depends on the increase of ATRA concentration. After 6 day's incubation with ATRA, the G0/G1 phase SMMC-7721 cells increased and S phase cells decreased. A sub-G1 phase cell peak was observed using flow cytometry. The results of RT-PCR showed that the survivin mRNA remained at steady state level in RA-treated SMMC-7721 cell line. The results of Western-blot and immunocytochemistry showed that the survivin protein expression was dramatically down-regulated and the effect of ATRA to survivin protein is dose-depedent.Conclusion:Survivin re-expressed in SMMC-7721 cell line. ATRA suppressed the proliferation and induced apoptosis in SMMC-7721 cells in vitro. Down-regulating the level of survivin protein may be one of the mechanisms that ATRA can induce HCC cells apoptosis.