TGF-β1/Smad3对辐射所致鼻咽癌细胞凋亡及细胞周期分布的影响

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

目的探讨TGF-β1/Smad3在辐射所致鼻咽癌细胞CNE-2凋亡中的作用及其机制。方法 CNE-2细胞分为6 Gy照射组、TGF-β1+6 Gy照射组以及对照组,免疫荧光方法检测γH2AX焦点形成试验,流式细胞仪检测凋亡细胞及细胞周期分布,MTS法检测细胞生长生长活力,绘制生长曲线并计算生长增殖率。Western blot检测P-ATM、pCHK1、pCHK2、CDC25A、p-Smad3、Smad3、P15和CDK4蛋白表达水平。结果照射组及TGF-β1+6Gy照射组凋亡细胞数明显增多,细胞核体积缩小,可见浓染致密的γH2AX表达。TGF-β1+6 Gy照射组的γH2AX表达相对于对照组与6 Gy照射组明显增加。6 Gy照射组和TGF-β1+6 Gy照射组的G2/M期细胞比例均明显高于对照组(P<0.05)。TGF-β1+6 Gy照射组与6 Gy照射组相比,S期明显增加(P<0.05)。TGF-β1+6Gy照射组和6 Gy照射组的凋亡及坏死细胞比对照组的明显增加,差异有统计学意义(P<0.01)。TGF-β1+6Gy照射组的凋亡和坏死细胞比例明显比6 Gy照射组增加,差异有统计学意义(t P<0.05)。对照组、6 Gy照射组和TGF-β1+6 Gy组的CNE-2细胞克隆形成率分别为0.60、0.03和0.01,与对照组相比,6 Gy照射组和TGF-β1+6 Gy照射组的细胞克隆形成明显减少,差异有统计学意义(P<0.01)。与6 Gy照射组相比,TGF-β1+6 Gy照射组的细胞形成率减少,差异有统计学意义(P<0.01)。Western blot结果显示TGF-β1处理组相对于单独6Gy照射组,p-ATM、p-CHK1、p-CHK2、P15、p-Smad3、CDC25A及CDK4表达下调,Smad3表达无明显变化。结论 TGF-β1可增加照射所致鼻咽癌细胞的凋亡,可能与增加S期细胞比例以及抑制Smad3磷酸化同时促进ATM磷酸化有关。

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 eytometry 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- CHKI, 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 of γH2AX 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 exposure 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 ceils 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 significantly reduced （P 〈 0. 01 ）. Western - blot results indicated that comparing with 6Gy exposure group, p - ATM, p - CHK1, p - CHK2, plS, 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 carcinoma cell apoptosis caused by radiation exposure and it may be related to the increase of S phase cell proportion, inhibition of Smad3 phosphorylation and promotion of ATM phosphorylation.