保护性自噬对顺铂诱导人乳腺癌MCF-7细胞凋亡的抑制作用探讨

Protective autophagy inhibits cisplatin-induced apoptosis in human breast cancer MCF-7 cells

目的探讨顺铂对乳腺癌MCF-7细胞自噬的影响及自噬在顺铂诱导凋亡中的作用。方法顺铂处理乳腺癌MCF-7细胞,MTT检测细胞增殖的能力,Hoechst 33342染分析细胞的凋亡,吖啶橙染色分析细胞的自噬,Western blot分析自噬蛋白LC3Ⅰ/Ⅱ和p62的表达和凋亡蛋白多聚ADP-核糖聚合酶PARP表达。结果顺铂呈时间和剂量依赖性抑制乳腺癌MCF-7细胞的增殖,并且凋亡细胞数量随顺铂浓度的递增而增加;同时顺铂能诱导微管相关蛋白轻链3-Ⅱ(LC3Ⅱ)蛋白的增加,p62蛋白的减少以及酸性自噬溶酶体的增加,顺铂联合氯喹明显增加了LC3Ⅱ和p62的蛋白的表达;与单药顺铂相比,自噬抑制剂氯喹明显降低细胞存活率(89.17%±2.56%)vs(74.63%±1.51%),(P<0.05),而且PARP蛋白发生了明显的裂解(P<0.05)。结论顺铂诱导乳腺癌MCF-7细胞保护性自噬,抑制自噬可以增加顺铂诱导乳腺癌MCF-7细胞凋亡,自噬抑制剂联合顺铂为乳腺癌提供了新的治疗策略。

Objective To investigate whether cispaltin induces autophagy in human breast cancer cell line MCF-7 and to identify the role of autophagy in cisplatin-induced cell apoptosis.Methods After MCF-7 cells were treated with cispatin,cell proliferation was measured using MTT assay;cell apoptosis was determined by Hoechst 33342 staining assay;quantitative analysis of autophagy after Ac-ridine orange （AO）staining was performed using fluorescence microscopy;and LC3Ⅰ/Ⅱ,p62 and poly ADP-ribose polymerase PRAP proteins expression were detected by Western blot.Results Cell proliferation was inhibited by cisplatin in a dose-dependent and time-dependent manner,and cisplatin induced condensed bright blue apoptotic nuclei in breast cancer cells in a dose-dependent manner. Simultaneously,cisplatin led to the increase of microtubule-associated protein light chain 3-Ⅱ（LC3-Ⅱ）protein,the formation of acidic vesicular organelles （AVOs）and the increase of p62 protein.The levels of LC3Ⅱand p62 were obviously increased by combination of autophagy inhibitor chlorochine and cisplatin.Compared with cisplatin alone,the combination of autophagy inhibitor chlorochine and cis-platin significantly decreased cell viability[（89.17% ±2.56%）vs （74.63% ±1.51%）,P 〈0.05],moreover,PARP protein was sig-nificantly clearaged （P 〈0.05）.Conclusions Cisplatin-induced protective autophagy in human breast cancer MCF-7 cells.Inhibition of autophagy can promote apoptosis,and combination therapy with cisplatin and autophagy inhibitors may be a promising therapeutic strategy for breast cancer.