竹节香附素A调控mTOR通路对肠癌HCT116细胞自噬及凋亡的影响

Effect of raddeanin A on autophagy and apoptosis of colon cancer HCT116 cells through regulating mTOR pathways

目的探讨竹节香附素A(RA)抑制HCT116细胞的增殖及其相关机制。方法利用四甲基偶氮唑蓝(MTT)检测RA对HCT116细胞的增殖抑制;流式细胞术(FCM)检测RA对细胞凋亡的影响;透射电镜观察RA诱导自噬;Western blot检测凋亡、自噬相关蛋白表达。结果 RA能够明显抑制HCT116细胞的增殖,并呈浓度、时间依赖性。透射电镜观察到双层膜自噬体的存在;FCM显示RA能够诱导HCT116细胞凋亡,且随着浓度的增大凋亡率增加。Western blot检测显示,自噬相关蛋白Beclin-1、微管相关蛋白1轻链3(LC3)表达增高;抑制凋亡蛋白Bcl-2表达减少;而促凋亡蛋白Bax,凋亡相关蛋白Cleaved-caspase-3、Cleaved-聚二磷酸腺苷核糖多聚酶(PARP)表达增加;RAPA靶蛋白(mTOR)信号通路中mTOR蛋白表达增加,磷酸化mTOR(p-mTOR)蛋白表达减少。加入mTOR通路抑制剂雷帕霉素(RAPA)后Beclin-1、LC3蛋白表达增加,凋亡率增加,且Cleaved-caspase-3、Cleaved-PARP表达也增加。结论 RA能够抑制肠癌细胞HCT116的增殖;并能诱导细胞自噬和凋亡,其机制可能是通过调控mTOR信号通路实现。

Objective To explore raddeanin A（RA）inhibiting the proliferation of HCT116 cells and its related mechanism.Methods The MTT method was used to observe the RA inhibition on the proliferation of gastric cancer cells HCT116.Flow cytometry was used to detect the RA effects on cell apoptosis.Transmission electron microscope（TEM）was used to observe RA induced autophagy.Western blot was used to test expression of apoptosis and autophagy related proteins.Results RA could significantly inhibit the proliferation of HCT16 cells with concentration and time dependence;double layer membrane of autophagosome was detected by TEM;FCM showed that RA induced apoptosis in HCT116 cells,moreover the apoptosis rate was increased with the concentration increase;Western blot showed that the expression of autophagy related proteins（Beclin-1 and LC3）was increased,the expression of apoptosis inhibition protein Bcl-2 was decreased.On the contrary,the expression of apoptosis promotion proteins（Bax,Cleaved-caspase-3,Cleaved-PARP）was increased,the expression of mTOR protein in the mTOR signal pathway was increased,while the p-mTOR protein expression was decreased.When the mTOR pathway inhibitor rapamycin（RAPA）was added to cells,Beclin-1,LC3,Cleaved-caspase-3,Cleaved-PARP proteins were increased,and apoptosis rate was also increased.ConclusionRA can inhibit the proliferation of HCT116 cells and can induce cellular autophagy and apoptosis,its mechanism may be realized by regulating the mTOR signal pathway.