摘要
目的:观察维生素E琥珀酸酯(vitamin E succinate,VES)处理SGC-7901细胞后是否发生自噬,并探讨自噬在VES抑制细胞增殖中的作用。方法:用不同浓度(0、5、10、15、20μg/mL)VES处理SGC-7901细胞24 h后,电子显微镜观察自噬体形态;VES处理转染GFP-LC3质粒后的SGC-7901细胞,荧光显微镜观察自噬情况;Western blot检测自噬标志蛋白LC3,以及Akt/mTOR通路关键分子Akt,mTOR活性变化;采用自噬抑制剂氯喹(chloroquine,CQ)和3-甲基腺苷(3-methyladenine,3-MA)处理细胞后,MTT法检测细胞的增殖能力。结果:电子显微镜观察显示经不同浓度VES处理后,细胞中出现了典型的自噬囊泡以及自噬溶酶体等自噬各阶段的形态变化;荧光显微镜观察显示GFP-LC3质粒转染后随VES处理剂量的增加,绿色点状荧光逐渐聚集增强;Western blot结果显示,VES可使LC3-Ⅱ蛋白表达增强;并下调Akt/mTOR通路关键分子Akt,mTOR的活性;MTT结果显示VES+CQ组和VES+3-MA组的细胞增殖抑制率均明显高于VES单独处理组(P均<0.05),即自噬抑制剂CQ与3-MA预处理使细胞增殖率进一步下降,提示VES诱导的肿瘤细胞自噬对肿瘤细胞增殖具有保护作用。结论:VES可以通过抑制Akt/mTOR通路活性诱导人胃癌细胞SGC-7901细胞发生自噬,且此种自噬为保护性自噬。
OBJECTIVE： To investigate whether vitamin E succinate （VES） could induce autophagy in SGC- 7901, and to explore the role of autophagy in cell proliferation inhibited by the VES. METHODS： Human gastric cancer cells SGC-7901 were treated with VES. Electron microscopy was used to study autophagosome morphology, and fluorescence microscopy for intracellular punctuate fluorescence of green fluorescence protein-LC3 （GFP-LC3）. Western blot evaluated the level of autophagy marker protein LC3, and the activities of Akt and mTOR, the critical targets of Akt/mTOR signaling pathway. SGC-7901 cells were pre-treated with autophagy inhibitor, chloroquine （CQ） and 3- methyladenine （3-MA）, then we used MTT to detect the suppression of cell growth. RESULTS： Typical autophagic vesicles and autolysosome in VES treatment groups were identified. With increasing doses of VES, the punctuate fluorescence began to gather and intensity. LC3-II protein levels increased in cells treated with VES, suggested that VES may probably induce autophagy in SGC-7901. VES down-regulated the activities of the phosphory-Akt and phosphory- mTOR, suggested that the activity of Akt/mTOR signaling pathway was inhibited by VES. Combined action of VES＋CQ group and VES＋3-MA group significately suppressed cell growth compared with VES alone （P〈0.05）. When autophagy was inhibited , the rate of cell proliferation was greatly reduced, suggesting that VES-induced autophagy had a protective effect on cell proliferation. CONCLUSION： Vitamin E succinate induced protective autophagy in human gastric cancercells SGC-7901 via the Akt/mTOR signaling pathway.
出处
《癌变．畸变．突变》
CAS
CSCD
2013年第5期343-347,351,共6页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
国家自然科学基金资助项目(81172651)
