Preliminary Study On The Role Of TGF-β 1-induced Apoptosis In Colon Carcinoma Cells And Its Mechanism

Objective:(1) To investigate the sensitivity of five colon adenocarcinoma cell lines SW480,HT29,HCT116,SW620,LS174T to TGF-β1 and the expression of active caspase-3 in these colon cancer cell lines.(2) To investigate the effect of P38 signaling pathway and active caspase-3 on the apoptosis which induced by TGF-β1 and reveal the possible mechanism that TGF-β1 sensitize colon carcinoma cell.Methods:Colon carcinoma cell line SW480,HT29,HCT116,SW620,LS174T was used:(1) The cells which were treated by 10ng/ml TGF-β1 for the same time(48h)were measured by flow cytometry for its apoptosis rates(FCM:48h,10ng/ml).(2) According to the different sensitivity to TGF-β1,we choose SW620 for further study.The cells which were treated by different concentration(0,5,10,15ng/ml)TGF-β1 for the same time(48h)were evaluated by DAPI Staining and by the expression of active caspase-3 detected by Western blot for its apoptosis inflection.(3)The cells which were treated by the same concentration(10ng/ml)TGF-β1 for different lengths of time(6,12,24,48h)were measured by flow cytometry and the expression of active caspase-3 which were determined by Western blot for its apoptosis rates(FCM:6,12,24,48h,10ng/ml).(4)Western blot was used to examine the expression of active caspase-3 in this cell line for three different treated-groups including control group,TGF-β1(10ng/ml) group, SB203580+10ng/mlTGF-β1 group,the expression of active caspase-3 by same concentration SB203580+10ng/mlTGF-β1 for different lengths of time(6,12,24,48h) were also determined by western blot.Meanwhile,the activation of caspase-3 were observed by cytospectrophotometry.(5)The phosphorylation of p38(p-p38) in SW620 cells was determined by western blot after SW620 cells were stimulated by 10ng/ml TGF-β1 for different lengths of time(0,30,60,120min).Resluts:(1) Different colon carcinoma cells have different sensitivity to TGF-β1-induced apoptosis.In this study,SW620 was found sensitive to TGF-β1-induced apoptosis while SW480,HT29,HCT116 and LS174T is relatively insensitive to it.SW620 had high antitumor activity in a time and concentration dependent manner.(2) In SW620 cells,the expression of active caspase3 was detected by western blot in three different treated-groups including control group,TGF-β1(10ng/ml) group,SB203580+10ng/mlTGF-β1 group which showed that active caspase-3 decreased obviously in SB203580+10ng/mlTGF-β1 group along with the decreased cell apoptosis rates.(3) The expression of p-p38 proteins in SW620 increased after cultured with TGF-β1 for 30,60,120min.Conclusion:(1) Different colon carcinoma cells have different sensitivity to TGF-β1,in our study,SW620 is sensitive to TGF-β1-induced apoptosis while SW480,HT29,HCT116 and LS174T is relatively insensitive to it.(2) The P38 pathway could increase the expression of active caspase-3 in colon cancer line SW620 and sensitize it to TGF-β1.