MG132联合自噬抑制剂对A2870卵巢癌细胞抗肿瘤效应的研究

Effects of MG132 and autophagy inhibitors on human ovarian cancer A2870 cells

目的:研究蛋白酶体抑制剂MG132联合自噬抑制剂对A2870卵巢癌细胞的毒性作用。方法:不同浓度蛋白酶体抑制剂MG132处理A2870细胞,MTT及Hoechst33258核染色方法检测细胞的存活率及凋亡形态学改变;吖啶橙(AO)染色和蛋白质印迹法检测酸性自噬泡的形成及自噬特异性蛋白LC3的变化。多种自噬抑制剂联合MG132处理后,MTT法检测细胞的存活率。结果:与空白对照组比较,1、2、5和10μmol/L浓度的MG132作用24 h均能明显抑制A2870细胞的生长,细胞存活率分别为(84.38±0.53)%、(65.86±2.34)%、(54.63±2.36)%和(52.16±1.27)%,P值均<0.05;同时,5μmol/L的MG132处理后,A2870细胞出现染色质浓缩和核碎片等凋亡特征性形态学改变,且细胞内酸性囊泡细胞器明显蓄积;不同浓度MG132处理细胞,蛋白质印迹法证实,1μmol/L MG132即明显增加A2870细胞中LC3-Ⅱ蛋白表达,且随MG132浓度增加,LC3-Ⅱ表达呈剂量依赖性增加。与MG132单独作用后A2870细胞存活率(58.32±1.46)%比较,早期自噬抑制剂3-MA和渥曼青霉素能显著增强MG132抑制A2870细胞的增殖作用,细胞存活率分别为(43.40±2.02)%和(45.35±2.27)%,P值均<0.05。晚期自噬抑制剂巴佛洛霉素和氯喹则无增强作用,细胞存活率分别为(58.11±2.70)%和(56.76±1.15)%,P值分别为0.558和0.667。结论:蛋白酶体抑制剂MG132抑制A2870卵巢癌细胞增殖并诱导自噬。早期自噬抑制剂能增加MG132抗A2870卵巢癌细胞的作用,而晚期自噬抑制剂无明显作用。

OBJECTIVE：To investigate the effects of proteasome inhibitor MG132 and autophagy inhibitors on hu- man ovarian cancer A2870 cells. METHODS： A2870 ceils were treated with different doses of MG132 for 24 h,cell viabili- ty was assessed by methyl thiazolyl tetrazolium（MTT） assay, and apoptosis was observed with Hoechst33258 staining. Autophagy was observed using fluorescent microscope by acridine orange（AO） staining. The autophagy-specific protein LC3 （microtubule-associated protein 1 light chain 3） as detected by Western Blot. A2870 cells were cotreated with MG132 and autophagy inhibitors,and cell viability was measured using MTT assay. RESULTS： 1,2,5,10 pmol/L MG132 resulted in suppression of cell growth,cell viabilitywere（84.38±0.53）%,（65.86±2. 34）%,（54. 63±2.36）% and （52. 16±1.27） % （P〈0.05）;5 μmol/L MG132 resulted in morphological apoptosis and accumulation of acidic vacuoles in A2870 cells. MG 1 3 2 increased the transition of LC3- Ⅰ to LC3-Ⅱ in dose-dependent manner. 3-MA or wortmannin enhancedcytotoxicity of A2870 cells mediated by MG132 ,cell viability were （43.40±2.02） %, （45.35 ±2.27）% contrasted with （58.32±1.46）% （P〈0.05）. Bafilomycin or cloroquine had no obvious effects on MG132, cell viability which were （58.11±2.70）% and （56.76±1.15）% ,and P was 0. 558 and 0. 667. CONCLUSIONS： MG132 can reduce cell growth and induce autophagy in A2870 cells. Autophagy inhibitors at early stage enhanced cytotoxicity of A2870 cells induced by MG132 ,while autophagy inhibitors at late stage demonstrated no obvious effect.