熊果酸通过影响线粒体功能诱导TC-1肿瘤细胞自噬性死亡  

Mitochondria dysfunction and mitophagy involved in Ursolic acid induced TC-1 cancer cell death

作　　者：黄丽[1] 谢珊艳 洪乐鹏[1] 石纯[1] 冷水龙[1] 

机构地区：[1]广州医科大学基础医学院人体解剖学教研室,广东广州511436

出　　处：《解剖学研究》2015年第5期375-382,391,共9页Anatomy Research

基　　金：广东省自然科学基金(81370395;81200846)

摘　　要：目的探讨线粒体失能和线粒体自噬在熊果酸诱导TC-1癌细胞自噬相关性死亡中的作用。方法同一批TC-1癌细胞分成对照组和熊果酸(UA)处理组,通过透射电镜(TEM)观察以及PI染色标记死细胞;利用流式细胞术检测线粒体膜跨电位(Δψm)及细胞内ATP和ROS水平变化;分光光度法检测线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ和Ⅳ的活性;利用吖啶橙(AO)染色来检测自噬;荧光显微镜下观察线粒体与自噬标志蛋白LC3的位置关系;Western blot法检测线粒体自噬相关蛋白PINK1和Nix表达;以及检测对照组和UA处理组在小鼠胚胎成纤维细胞(MEFs)和自噬缺乏(Atg5-/-)的MEFs中ROS水平变化。结果 UA 30μmol/L作用TC-1细胞6 h后,可见大量细胞的线粒体出现了显著异常;UA不仅降低线粒体膜电位和ATP水平(P<0.05),而且抑制线粒体复合酶Ⅰ~Ⅳ的活性(P<0.05),且均呈现时间与剂量依赖;UA还降低TC-1肿瘤细胞和小鼠胚胎成纤维细胞(MEFs)内的ROS水平,但是在自噬缺乏(Atg5-/-)的MEFs中ROS水平未受影响(P<0.05);吖啶橙(AO)染色发现UA还可增加自噬溶酶体的数量,并呈剂量依赖;经UA处理的细胞,无论是自噬相关蛋白LC3与线粒体共定位,还是参与损伤线粒体清除的线粒体自噬相关蛋白PINK1和Nix均表达增高。结论 UA通过诱导线粒体自噬清除部分ROS并导致线粒体功能损伤;线粒体失能与线粒体自噬至少部分参与UA诱导的TC-1肿瘤细胞自噬性死亡。Objective To investigate the role of mitochondrial dysfunction and mitophagy in the autophagy-related death in- duced by UA on TC-1 cancer cells. Methods Mitochondria in TC-1 cells were observed by transmission electron microscope （TEM） after UA treated for 6 h. Propidium iodide （PI） was used to stain dead-cells. The mitochondrial membrane potential（AqJm）, intracellular ATP and ROS level were detected by FCM （flow cytometry）. Observed the activity of mitochondrial respiratory complexes I -1V （MRC I -IV） by spectrophotometry. Using acridine orange （AO） to identify autophagy. Fluorescent microscope was used to observe the localization of the mitochondria and LC3, a autophagy marker proteins. The expression of mitophagy related proteins PINK1 and Nix were detected by western blot. detected the UA-induced changes in intracellular ROS levels in autophagy deficient （AtgS-/-） MEFs and MEFs. Results Compared with the control groups, the Transmission Electron Microscope （TEM） images of UA treated TC-1 tumor cells, abnormal mitochondria were noted in many cells. And UA significantly increased the number of dead cells in a dose-/time-depen- dent manner. In this study, we found that UA not only decreased inner mitochondrial membrane potential （A^m） and ATP levels in a dose-and time-dependent manner, but inhibited the activity of mitochondrial respiratory complexes I -IV （MRC I -IV）. Unexpectedly, UA reduced cytosolic ROS levels in TC-1 tumor cells and mouse embryonic fibroblasts （MEF）, but not in autophagy deficient （Atg5-/-） MEFs. In addition, UA increased the number of autolysosomes in a dose-dependent manner. We also found that LC3, a marker of au- tophagy, was co-localized with mitochondria and that the expression of mitophagy related proteins PINK1 and Nix, which mediate dam- aged mitochondrial clearance, was increased in UA treated ceils. Conclusion UA impairs mitochondrial function, while inducing mi- tophagy, which leads to partial clearance of cytosolic ROS. UA induced

关 键 词：熊果酸 自噬 线粒体 活性氧 线粒体自噬 TC-1肿瘤细胞 

分 类 号：R73-3[医药卫生—肿瘤]