Apoptosis Of Hepatocellular Carcinoma Cells Induced By A Combination Of TRAIL, Cisplatin And IFN-α

Objective To investigate the synergistic induction of cytoxicity and apoptosis by the combination of tumor necrosis factor drelated apoptosis inducing ligand(TRAIL), cisplatin and IFN-αin hepatocellular carcinoma cells SMMC7721. And to explore the potential mechanism of apoptosis incuced by three combinative drugsMethods Human hepatocellular carcinoma line SMMC7721 cells were maintained in RPMI1640 supplemented with I0% calf serum and 1％penicillin/streptomycin. Cells were divided into the following groups:blank group,control group,TRAIL group,DDP group,IFN-αgroup,TRAIL+DDP group,TRAIL+IFN-αgroup,DDP+IFN-αgroup,TRAIL+DDP+IFN-αgroup. The growth inhibitory rates were analyzed by SRB assay and the apoptosis rates were detected by FCM. Synergetic effect was determined by Weeb coefficient. Statistical analysis was processed by SPSS12.0 software. Pvalue of less 0.05 was considered significant. The expression of TRAIL receptors DR4, DR5 in SMMC7721 was examined by RT-PCR. The changes of caspase-9, bax and bcl-2 in protein level were assayed by Western blot analysis.Result SRB assay demonstrated that the growth inhibitory rates(GIR) became lager with increasing concentrations of TRAIL, so did DDP and IFN- α.The subcytotoxic concentration of TRAIL was lug/ml; DDP was 10ng/ml;IFN-αwas 2.5×10~5U/ml. IC50 of TRAIL was above 10ug/ml, implied that SMMC 7721 was not sensitive to TRAIL. Combination of their subcytotoxic concentration, the growth inhibitory of TRAIL+DDP group was 26.00％±1.17; TRAIL+IFN-αgroup was 37.81％±5.97; DDP+IFN-αgroup was 29.09％±3.43. The TRAIL+DDP+IFN-αgroup was 65.19％±3.10. The apoptosis rate of TRAIL+DDP group was 9.1％; TRAIL+IFN-αgroup was 12.1％; DDP+IFN-αgroup was 12.6％. The TRAIL+DDP+IFN-αgroup was 40.1％.There was significant difference in the growth inhibitory and apoptosis rate when three drugs combined together.The combination of the three drugs could not effect the expression of DR4, DR5 mRNA and bcl-2 protein, but could up-regulation of caspase-9, bax protein.Conclusion Human hepatocellular carcinoma line SMMC7721.cells were not sensitive to TRAIL.The combination of any two of the three drugs showed significant inhibiting effect but no synergetic effect. The combination of the three drugs showed significant inhibiting effect and synergetic effect.The combination of the three drugs could greatly enhance SMMC7721 cells apoptosis.The up-regulation of caspase-9 and bcl-2 protein expression may related to the mechanism of the enhanced apoptosis effect.