YM155诱导乳腺癌细胞MDA-MB-231自噬并促进其凋亡

YM155 enhances apoptosis in triple negative breast cancer MDA-MB-231 cells via autophagy

目的 :研究生存素(survivin)抑制剂YM155对三阴性乳腺细胞株MDA-MB-231的凋亡及自噬的影响及其可能的作用机制。方法:采用CCK-8法检测不同浓度YM155对MDA-MB-231细胞增殖的影响,同时联合加入自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)处理细胞与单用组比较细胞增殖变化;流式细胞术检测细胞凋亡情况;Real-time PCR法检测不同加药组与对照组细胞的survivin、beclin 1和bcl-2的m RNA表达量;蛋白质印迹法检测survivin、bcl-2、caspase-3、PARP及自噬相关蛋白beclin 1和LC-3表达变化情况。结果:YM155对MDA-MB-231具有明显的生长抑制效应且呈剂量和时间依赖性。流式细胞结果显示YM155在0.5、1.0、1.5 ng/m L浓度对细胞的凋亡率分别是(11.9±2.4)%、(21.7±2.6)%、(30.8±4.5)%,与对照组(6.4±1.2)%相比具有统计学意义(P<0.01)。当使用自噬抑制剂3-MA(5 mmol/L)联合处理细胞24 h后细胞增殖率(62.5±3.3)%,与单用YM155组(54.7±2.7)%相比,细胞增殖活性增强(P<0.05)。YM155可显著下调survivn的m RNA和蛋白表达,降低bcl-2和提高caspase-3、PARP的蛋白表达,同时上调beclin 1表达及增加LC-3Ⅱ/LC-3Ⅰ的比值,促进细胞自噬的发生。结论:YM155能够有效诱导乳腺癌细胞MDA-MB-231凋亡及自噬的发生,其诱导的自噬效应进一步促进其凋亡的发生。

Objective：To investigate the effect of YM155,a survivin inhibitor,on the apoptosis and autophagy of the triple negative breast cancer MDA-MB-231 cells. Methods：MDA-MB-231 cells was treated with different concentrations of YM155,the survival rate of the cells was determined by CCK-8 assay and the IC50（half inhibitory concentration）value of YM155 was calculated. The apoptosis rate was examined by Annexin V-FITC / PI double staining. The m RNA expression of survivin,beclin 1 and bcl-2 in MDAMB-231 cells was detected by Real-time PCR. The protein expression of survivin,bcl-2,caspase 3,PARP,beclin 1 and LC-3 were detected by Western blot. Results：It was revealed that YM155 significantly inhibited the growth of MDA-MB-231 cells in a dose-and time-dependent model. The apoptosis rates of cells treated with 0.5,1.0,1.5 ng / m L YM155 were（11.9 ± 2.4）%,（21.7 ± 2.6）%,and（30.8 ± 4.5）%,respectively,which all had significant difference compared to control cells [（6.4 ± 1.2）%]. When a combination of 3-MA effect after 24 h,cell proliferation rate was significantly enhanced compared to that of the single YM155 group（P〈0.05）.With the increasing of YM155 concentration,the expression levels of m RNA and protein of survivin and BCL-2 were decreased,while the expression levels of caspase-3,PARP,beclin1 and LC-3 were increased. Compared with the YM155 group,the protein levels of LC-3and caspase-3 were lower in YM155 ＋ 3-MA group. Conclusion：YM155 could effectively inhibit MDA-MB-231 cells proliferation by inducing apoptosis and autophagy,while autophagy induction effect can enhance its apoptosis effect.