The Effect Of GSK-3β On TRAIL-induced Apoptosis Of Hepatoma Cells And Its Mechanism Of Action

Objects: 1) To demonstrate that down-regulation of GSK-3β protein expression may affect Hepatocellular carcinoma(HCC) cells growth. 2) To investigate the influence of GSK-3β down-regulation on TRAIL- induced apoptosis and to study its mechanism in HCC cells.Methods: Build plasmids of RNA interference targeting the GSK-3β gene(sh GSK-3β) and Adenovirus- mediated TRAIL(Ad5.TRAIL). Hep G2 cells were randomly divided into three groups: blank Hep G2 cells group, sh RNA-control group and sh GSK-3β treated group. The GSK-3β m RNA and protein level were independently confirmed by Real-time PCR and Western blot. And cell proliferation was me asured by the MTT assay. Hu H-7 cells were randomly divided into four groups: blank Hep G2 cells group, GSK-3β inhibitor treated group, Ad5.TRAIL treated group, GSK-3β inhibitor and Ad5.TRAIL treated group. Detection the apoptosis of each group by flow cytometric assay. In addition, the expressing of Bcl- x L and c IAP2, two downstream protein of NF-κB pathway, were measured by Western blot after down-regulation of GSK-3β by sh GSK-3β or GSK-3β inhibitor in HCC cells. Hu H-7 cells wre randomly divided into five groups: a) blank control; b) cells treated with Ad5.TRAIL and sh Bcl- x L; c) cells treated with Ad5.TRAIL and shc IAP2; d) cells treated with Ad5.TRAIL, sh Bcl-x L and GSK-3β inhibitor; e) cells treated with Ad5.TRAIL, shc IAP2 and GSK-3β inhibitor. Detection the apoptosis of each group by flow cytometric assay.Results: 1) Compared with the control group, the expression of GSK-3β was reduced in Hep G2 cells transfected with sh GSK-3β(P<0.05), there was a dramatically growth delay from sh GSK-3β treated cells(P<0.05). 2) There was an increase of TRAIL-induced apoptosis after adding GSK-3β inhibitor in Hu H-7 cells(P<0.05), and GSK-3β inhibitor decreased the expressing of Bcl- x L and c IAP2(P<0.05). The apoptosis of group e was significantly enhangced that the other four groups(P<0.05).Conclusion: 1) GSK-3β down-regulation can inhibite the proliferation of HCC cells. 2) GSK-3β inhibitor can sensitized HCC cells to TRAIL- induced apoptosis via NF-κB pathway.