Preliminary Study On The Role Of C-Flip In TRAIL-induced Apoptosis In Colon Carcinoma Cells And Its Mechanism

Objective:(1)To investigate the sensitivity of three colon adenocarcinoma cell lines SW480,HT29,HCT116 to TRAIL and the expression of c-Flip in these colon cancer cell lines.(2)To investigate the effect of PI3K/Akt signaling pathway on the expression of c-Flip induced by TRAIL and reveal the possible mechanism that TRAIL sensitize colon carcinoma cell.Methods:Colon carcinoma cell line SW480;HT29,HCT116 was used:(1)The cells which were treated by different concentration(0,25, 50,100ng/ml)TRAIL for different lengths of time(6,12,24,48h)were measured by flow cytometry and MTT assay for its apoptosis rates(FCM:48h,100ng/ml).(2)Western blot was used to detecte the expression of c-Flip protein in this three colon cancer cells.(3) According to the different sensitivity to TRAIL and the expression of c-Flip protein,we choose HT29 for further study.The expression of c-Flip in this cell line was determined by western blot in three different treated-groups including control group,TRAIL(100ng/ml)group, ly294002+TRAIL(100ng/ml)Group.Meanwhile,the cell apoptosis rates were observed by flow cytometry.(4)The phosphorylation of Akt(p-Akt) in HT29 cells was determined by western blot after HT29 cells were stimulated by 100ng/ml TRAIL for different lengths of time(0,15,30, 60,120min).Resluts:(1)Different colon carcinoma cells have different sensitivity to TRAIL.In this study,HT29 was found insensitive to TRAIL-induced apoptosis while SW480 and HCT116 is relatively sensitive to it.SW480 and HCT116 had high antitumor activity in a time and concentration dependent manner.(2)C-Flip were expressed differently in three colon carcinoma cell lines,which might correlated with their different sensitivity to TRAIL.(3)In HT 29 cells,the expression of c-Flip protein was detected by western blot in three different treated-groups including control group, TRAIL(100ng/ml)group,ly294002+TRAIL(100ng/ml)group which showed that c-Flip increased obviously in ly294002+TRAIL(100ng/ml) group along with the increased cell apoptosis rates.(4)The expression of p-Akt proteins in HT29 decreased after cultured with TRAIL for 15,30,60,120min.Conclusion:(1)Different colon carcinoma cells have different sensitivity to TRAIL,in our study,HT29 is insensitive to TRAIL-induced apoptosis while SW480 and HCT116 is relatively sensitive to it.(2)The PI3K/Akt pathway could decrease the expression of c-Flip in colon cancer line HT29 and sensitize it to TRAIL.