ABT199通过增加Drp-1依赖的线粒体分裂诱导人皮肤鳞癌A431细胞凋亡

ABT199 induces apoptosis in human squamous carcinoa A431 cells by increasing Drp-1-dependent mitochondrial fission

目的观察抗凋亡蛋白Bcl-2特异性抑制剂ABT199对A431细胞活力及凋亡的影响并探讨其机制。方法体外培养A431细胞,MTT法检测ABT199对A431细胞活力的影响,流式细胞术检测ABT199对A431细胞凋亡的影响,共聚焦显微镜观察ABT199联合线粒体分裂蛋白(mitochondrial dynamin-related protein-1,Drp-1)与线粒体分裂抑制剂-1 (mitochondrial division inhibitor-1,Mdivi-1)对线粒体长度变化的影响,Western blot检测ABT199联合mdivi-1对Cyto C释放、Drp-1线粒体转位及caspase-3和caspase-9活化的影响。流式细胞术检测ABT199联合mdivi-1对线粒体膜电位的影响。结果 MTT结果显示ABT199能以剂量依赖性的方式抑制A431细胞活力,24 h的IC50值为(10.33±0.93)μM。流式细胞术结果进一步表明ABT199能显著诱导A431细胞的凋亡,5μM、10μM、20μM ABT199作用A431细胞24 h,其细胞凋亡率分别为(16.10±2.65)%、(25.51±4.21)%、(54.21±4.89)%。荧光显微镜观察显示mdivi-1能减弱ABT199诱导的线粒体平均长度减短。Western blot结果显示mdivi-1能减弱ABT199诱导的Cyto C的释放、Drp-1线粒体转位及caspase-3和caspase-9的活化。流式细胞术结果表明mdivi-1减弱了ABT199诱导的线粒体膜电位下降。结论 ABT199在体外能抑制A431细胞活力并诱导其凋亡,其机制与增加Drp-1依赖的线粒体分裂有关。

Objective The objective of this study was to observe the effect of ABT199, a specific inhibitor of anti-apoptotic protein Bcl-2, on the viability and apoptosis of A431 cells, and to explore its mechanism. Methods A431 cells were cultured in vitro. MTT method was used to investigate the effects of ABT199 on viability in A431 cells. Flow cytometry was used to detect the effect of ABT199 on apoptosis. The effect of ABT199 and/or mdivi-1 on mitochondrial fission was observed by confocal laser microscopy. Western blot was used to detect release of cytochrome c （Cyto C）, mitochondrial translocation of Drp-1, caspase-3 and caspase-9 activation in A431 cells treated with ABT199 and/or mdivi-1. The effect of ABT199 and/or mdivi-1 on mitochondrial membrane potential was detected by flow cytometry. Results MTT results showed that ABT199 could inhibit A431 cell activity in a dose-dependent manner and the IC50 values of ABT199 in A431 cells for 24 h was （10.33±0.93） μM. The results of flow cytometry further showed that ABT199 could induce apoptosis of A431 cells. The apoptosis rates of A431 cells treated with 5, 10 and 20 μM ABT199 for 24 hours were （16.10±2.65） %, （25.51±4.21） %, and （54.21±4.89） %, respectively. Confocal laser microscopy studies revealed that mdivi-1 weakened ABT199-induced decrease in mitochondrial length. Western blot results showed mdivi-1 attenuated ABT199-induced release of Cyto C, mitochondrial translocation of Drp-1, caspase-3 and caspase-9 activation in A431 cells. Flow cytometry results showed that mdivi-1 decreased ABT199-induced loss of mitochondrial membrane potential. Conclusions ABT199 obviously inhibits viability and inducts apoptosis of A431 cells in vitro, and the mechanism may be related to the increase of Drp-1-dependent mitochondrial fission.