小分子抑制剂Torin-2抑制小细胞肺癌NCI-H446细胞增殖和促进细胞凋亡的作用和分子机制

Small molecule inhibitor Torin-2 induces growth inhibition and apoptosis of small cell lung cancer NCI-H446 cell lines

目的:研究哺乳动物雷帕霉素(mammalian target of rapamycin,mTOR)新型小分子抑制剂Torin-2对人小细胞肺癌NCI-H446细胞增殖及凋亡的影响并探讨其机制。方法:体外培养并取对数生长期人小细胞肺癌NCI-H446细胞,分别给予不同浓度Torin-2或1μmol/L处理不同时间,然后于镜下观察细胞离巢凋亡,通过克隆形成实验检验细胞生长抑制,采用MTS比色法测定吸光度值并计算相对细胞活性,流式细胞术检测细胞周期,免疫印迹法检测mTOR、p-mTOR(Ser2448)、p-Akt(Ser473)、Cyclin-B1、Cyclin-D1、Cyclin-E1、Cleaved Caspase-3等相关蛋白表达。结果:与对照组相比,Torin-2可有效抑制小细胞肺癌NCI-H446细胞增殖及相对细胞活性(P<0.05);诱导细胞周期阻滞(P<0.05);抑制p-mTOR、p-Akt磷酸化激活,降低Cyclin-D1、Cyclin-E1等细胞周期蛋白表达并诱导Cleaved Caspase-3蛋白激酶切割激活。结论:Torin-2可能通过抑制Akt-mTOR相关信号通路和激活凋亡相关蛋白诱导人小细胞肺癌NCI-H446细胞增殖抑制并促进其凋亡。

Objective:To investigate the effects of small molecule inhibitor Torin-2 on the growth and apoptosis of human small cell lung carcinoma NCI-H446 cells and explore the underlying mechanism.Methods:NCI-H446 cells in logarithmic growth phase were treated with different concentrations of Torin-2 or 1μmol/L for 0,12,24,36 and 48 h in vitro.The cell morphology was observed under the microscope to assess apoptosis reflecting by detached floating cells.Cell growth inhibition was evaluated by cloning formation assays.MTS assay was used to test cell proliferation inhibition or cell viability decrease.Cell cycle blockage was measured by flow cytometry(FCM).Primary antibodies including mTOR,p-mTOR(Ser2448),p-Akt(Ser473),Cyclin-B1,Cyclin-D1,Cyclin-E1,and Cleaved Caspase-3 were used to detect protein levels through Western blot.Results:Compared to the control group,Torin-2 inhibited the growth and proliferation of NCI-H446 cells in a concentration-or time-dependent manner.We found that Torin-2 incubation triggered apoptosis and growth inhibition by blocking cell cycle in the G 0/G 1 phase and the G 2/M phase(P<0.05).Moreover,mechanism analysis revealed that Torin-2 not only reduced phosphorylation activation of mTOR and Akt,but decreased Cyclin-D1,Cyclin-E1,whereas there was no effects on Cyclin-B1.Cleaved Caspase-3 activation further showed that Torin-2 was able to induce apoptosis in lung cancer cells.Conclusion:Torin-2 single treatment can potentially exert anti-cancer effects by triggering growth and proliferation inhibition and apoptosis in human small lung cancer cells.