哇巴因诱导MCF-7自噬作用与机制探讨

Effect of ouabain on autophagy and related molecular mechanisms in MCF-7

目的探讨哇巴因诱导人乳腺癌细胞MCF-7发生自噬的作用以及分子机制。方法 MTT法和克隆形成实验测定哇巴因对MCF-7细胞增殖活力及克隆形成能力的影响,吖啶橙染色结合荧光显微镜及流式细胞术观测细胞内形态的变化及FL3/FL1荧光强度比值,蛋白质印迹法检测细胞内抗微管相关蛋白1轻链3(Light chain 3,LC3)-Ⅱ及p62蛋白表达水平的变化,流式细胞术检测细胞内活性氧自由基(reactive oxygen species,ROS)水平的变化。结果哇巴因处理MCF-7细胞24h后半数抑制浓度(IC50)为(37.1±9.0)nmol/L。25nmol/L哇巴因处理12、24、48和72h后细胞存活率组间F=12.787,P<0.001;与对照组比较差异均有统计学意义,P值均<0.01。哇巴因处理2周后给药组较对照组相对克隆形成率降至(6.03±1.51)%,差异有统计学意义,F=635.800,P<0.001。吖啶橙染色及蛋白质印迹结果显示,经25nmol/L哇巴因处理48h后,胞质中出现呈亮红色荧光的酸性囊泡,并伴随LC3-Ⅱ呈时间依赖性的蓄积及p62表达的下降;在12、24和48h时,相对FL3/FL1比值组间F=62.226,P<0.001;与对照组比较差异均有统计学意义,P值均<0.05。哇巴因能同时引起细胞内ROS水平呈时间依赖性升高;0.5、1、2、4、6、12和24h时ROS水平组间F=112.901,P<0.001;与对照组比较,除0.5h组差异无统计学意义外,其余各组均差异有统计学意义,P值均<0.05;给药同时使用抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)后,联合给药组较给药组相对ROS水平降低至(101.2±17.5)%,与给药组比较差异有统计学意义,F=185.870,P<0.001;并且LC3-Ⅱ表达水平随之下降,而IC50值增加为(81.4±4.5)nmol/L,差异有统计学意义,F=57.610,P<0.01。结论哇巴因能够诱导人乳腺癌MCF-7细胞发生自噬,其作用机制可能与促进细胞内ROS生成有关。

OBJECTIVE To investigate whether ouabain could induce autophagy in human breast cancer cells MCF-7 and the related molecular mechanisms.METHODS Cell viability and colony formation ability were assessed by MTT and clone formation assay.Acridine orange-stained acidic vesicles and FL3/FL1 fluorescence intensity were determined by fluorescent microscopy and flow cytometry,respectively.LC3-Ⅱconversion and the expression levels of p62 were evaluated by Western blot.Levels of intracellular reactive oxygen species（ROS）were detected by flow cytometry.RESULTSThe half inhibition concentration（IC50）of ouabain for 24hwas（37.1±9.0）nmol/L in MCF-7cells.The cell viability after 25nmol/L ouabain treatment was in 12,24,48,72 h,respectively（F=12.787,P〈0.001,and each group was statistically significant versus control group,P〈0.01）.The relative colony formation ability fell to（6.03±1.51）% after exposure to ouabain for 2weeks significantly（F=635.800,P〈0.001）,compared to control group.The induction of autophagy was demonstrated by acridine orange staining of intracellular acidic vesicles,the time-dependent increase in FL3/FL1 ratio,p62decrease and conversion of LC3-I to autophagosome-associated LC3-Ⅱ upon the treatment of 25nmol/L ouabain for 48 h.The FL3/FL1 ratio significantly increased in 12,24,48 hafter ouabain treatment（F=62.226,P〈0.001,and each group was statistically significant versus control group,P〈0.05）.Simultaneously,a time-dependent increase of ROS levels was found in ouabain-treated cells,and the rate was in 0.5,1,2,4,6,12 and 24h,respectively（F=112.901,P〈0.001,and each group except for 0.5hgroup was statistically significant versus control group,P〈0.05）.Antioxidant n-acetylcysteine（NAC）could significantly attenuate the increase of LC3-Ⅱ,ultimately contribute to cell survival as confirmed by increase of IC50 values to（81.4±4.5）nmol/L（F=57.610,P〈0.01）.CONCLUSION Ouabain could induce autophagy in human breast cancer cells MCF-7,and ROS generation may be involved in the induction of autophagy.