The impacts of TGF-β1/Smad3 on radiation-induced apoptosis and cell circle distribution of nasopharyngeal cancer cells

LI Ming-yi; FENG Xiao-min; CHEN Dong-ping; LIU Jin-quan; LI Zhou-yu; YIN Wen-jing; JIANG Shao-hui; ZHOU Xiang

Abstract：Objective To investigate the effects of TGF -β1/Smad3 on nasopharyngeal carcinoma cell CNE -2 apoptosis caused by radiation and its mechanism .Methods CNE-2 cells were divided into 6Gy exposure group, TGF-β1+6Gy exposure group and control group .The immunofluorescence method was adopted for γH2AX focus formation assay;the flow cytometry was adopted to detect apoptotic cell and cell cycle distribution ;and MTS method was used to as-sess cell growth activity so as the growth curve was drawn to calculate the growth proliferation rate .Western blot was used to measure p-ATM, p-CHK1, p-CHK2, CDC25A, p-Smad3, Smad3, p15 and CDK4 protein expression levels . Results The number of apoptotic cells in the 6Gy exposure group and TGF-β1+6Gy exposure group were significantly increased, with the presentation of nucleus volume shrank and densely stained compact of γH2AX.The expression ofγH2AX in the TGF-β1+6Gy exposure group was significantly higher than control group and 6Gay exposure group.G2/M phase cell proportions of 6Gy exposure group and TGF -β1+6Gy exposure group were significantly higher than control group (P<0.05).The S phase cell count was significantly increased in TGF -β1+6Gy exposure group than 6Gy expo-sure group (P<0.05).The apoptosis and necrotic cells of TGF -β1+6Gy exposure group and 6Gy exposure group were significantly increased than control group (P<0.01).The apoptosis and necrotic cells of TGF -β1+6Gy exposure group was significantly increased than 6Gy exposure group (P<0.05).CNE-2 cell clone formation rates in control group , 6Gy exposure group and TGF -β1+6Gy exposure group were 0.60, 0.03 and 0.01, respectively.Comparing with control group, the cell clone formation rates in 6Gy exposure group and TGF -β1+6Gy exposure group were significantly reduced (P<0.01).Compared with 6Gy exposure group, the clone formation rate in TGF-β1+6Gy exposure group was signifi-cantly reduced (P<0.01).Western -blot results indicated that comparing with 6Gy exposure group, p-ATM, p-CHK1, p-CHK2, p15, p-Smad3, CDC25A, and CDK4 expression in the TGF-β1+6Gy exposure group was down -regulated, while no significant difference in Smad3 expression.Conclusion TGF-β1 can increase nasopharyngeal car-cinoma cell apoptosis caused by radiation exposure and it may be related to the increase of S phase cell proportion , inhibi-tion of Smad3 phosphorylation and promotion of ATM phosphorylation .