Apoptosis Mechanisms Research In Interfering TIGAR Gene And Epirubicin Treatmnet In Hepatic Carcinoma Cells

Objective: To investigate the relative apoptosis mechanisms induced by interfering TIGAR plus epirubicin in hepatic carcinoma HepG2 cell line in vitroMethods: MTT assay was performed to determine the inhibition of proliferation and 50% inhibiting concentration (IC50) in HepG2 cells with epirubicin treatment. Western blot detected the expression of autophagic and apoptotic proteins in HepG2 cells treated with defferent concentration epirubicin. Two small interfering RNA (TIGAR siRNA1/2) targeting to interfere with TIGAR mRNA were chemosynthesized and the interfering efficacy was tested by Western blot analysis. Annexin V-FITC/PI double dyes were used to stain the cells and flow cytometry assay (FCM) was performed to observe the apoptosis. The probe 2', 7'-dichlorofluorescin diacetate (DCFH2-DA) was used to detect the levels of intracellular ROS in defferent treatments in FCM. NADPH, Z-VAD-FMK and 3-methyladenine(3-MA)were used to inhibit the increase of intracellular ROS and the activation of apoptosis and autophagy, respectively. FCM and western blot were performed to evaluate the apoptosis and the relative proteins.Results: Cell growth inhibition was apparently in a time- and dose-dependent manner in defferent concentration epirubicin and defferent incubation time. The IC50 incubated for 12 hours with defferent concentration epirubicin was 78.14±3.27μg/ml. Up-regulation of p53, PUMA and TIGAR and down-regulation of Bcl-2 and autophagy were observed in HepG2 cells treated with different concentration of epirubicin and incubated for different time. Two small interfering RNA (TIGAR siRNA1/2) were well transfected into the cells and the transfection efficiency was about 89% and 91%(*** p<0.001),respectively. Interfering TIGAR plus epirubicin notably increased not only the apoptosis but the levels of intracellular ROS, Caspase-3 activation and autophagy in HepG2 cells. The levels of intracellular ROS were facilitated and neutralization of intracellular ROS via NADPH, not completely but partially, diminished the apoptosis in interfering TIGAR plus epirubicin treatment. Activated Caspase-3 productions were high and inhibiting the activation of Caspase-3 with Z-VAD-FMK apparently attenuated the apoptosis in interfering TIGAR plus epirubicin treatment in HepG2 cells. In addition , the levels of LC3-Ⅱ/ LC3-Ⅰ, as a marker of autophagy, were increased and suppressing autophagy by 3-MA synergistically induced more extensive apoptosis in interfering TIGAR plus epirubicin treatment in HepG2 cells.Conclusion: Interfering TIGAR increased the sensitivity of chemotherapy in epirubicin treatment, which was involved in the oxidative stress, Caspase-3 activation and autophagy inhibition in hepatic carcinoma HepG2 cells.