蒲公英萜醇通过mTOR信号通路诱导乳腺癌细胞自噬

Effect of Taraxerol in Inducing Autophagy in Breast Cancer Cells via mTOR Signaling Pathway

目的:体外研究蒲公英萜醇对乳腺癌MCF-7细胞自噬的影响,并探讨相关作用机制。方法:体外培养乳腺癌MCF-7细胞,通过噻唑蓝(MTT)比色法检测蒲公英萜醇(12. 5,25,50,100,200μmol·L-1)对MCF-7细胞增殖的影响;采用吖啶橙(AO),免疫荧光染色和透射电镜观察蒲公英萜醇对乳腺癌MCF-7细胞自噬的影响;应用蛋白免疫印迹法(Western blot)检测蒲公英萜醇用药后细胞自噬相关蛋白表达以及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的变化情况。结果:MTT比色法结果显示,蒲公英萜醇组可抑制MCF-7细胞的增殖能力;吖啶橙染色结果显示,与空白组比较,蒲公英萜醇组MCF-7细胞内酸性溶酶体增加;透射电镜观察到用药组细胞内出现明显的自噬体结构;蒲公英萜醇用药后,荧光显微镜下可见MCF-7细胞中出现较多的微管相关蛋白1轻链3(LC3)绿色荧光分布;Western blot结果显示,与空白组比较,蒲公英萜醇可上调LC3-Ⅱ和酵母Atg6同系物(Beclin-1)蛋白表达水平,LC3-Ⅱ/LC3-Ⅰ升高(P <0. 05,P <0. 01);加入自噬抑制剂3-甲基腺嘌呤(3-MA)后与蒲公英萜醇组比较,3-MA+蒲公英萜组LC3-Ⅱ/LC3-Ⅰ降低(P <0. 05)。蒲公英萜醇用药后可显著下调细胞内p-mTOR和磷酸化的真核细胞翻译起始因子4E结合蛋白(p-4EBP1)蛋白表达水平(P <0. 05,P <0. 01)。结论:蒲公英萜醇能够诱导MCF-7细胞发生自噬,其机制可能与抑制mTOR信号通路有关。

Objective:To investigate the effect of taraxerol on autophagy of breast cancer MCF-7 cells in vitro,and explore the related mechanisms.Method:The effect of various doses of taraxerol(12.5,25,50,100,200μmol·L-1)on proliferation of MCF-7 cells was detected by methye thiazolye telrazlium(MTT)assay.The autophagy-inducing effect of taraxerol was observed by acridine orange staining,transmission electron microscope(TEM)and immunofluorescence.The expressions of autophagy-related proteins and the changes of mammalian target of rapamycin(mTOR)signaling pathway were determined by Western blot analysis.Result:The viability of MCF-7 cells was significantly inhibited by taraxerol.Acridine orange staining indicated that the acidic lysosomes increased significantly after treatment with taraxerol in MCF-7 cells.The autophagic structure in the treated group was observed by TEM.Immunofluorescence showed that the expression of microtubule-associated protein 1 light chain 3(LC3)in the cells of the drug group was increased.Western blot demonstrated that the protein expressions of LC3-Ⅱand Beclin-1 were increased in taraxerol-treated MCF-7 cells(P<0.05,P<0.01),respectively.Compared with 100μmol·L-1 taraxerol group,combination group(taraxerol+3-methyladenine,3-MA)showed the down-regulation of LC3-Ⅱin the MCF-7 cells(P<0.05).And expressions of phosphorylated mammal target of rapamycin(p-mTOR)and phosphorylated eukaryotic initiation factor 4 E binding protein 1(p-4 EBP1)were decreased in MCF-7 cells after treatment with taraxerol(P<0.05,P<0.01).Conclusion:Taraxerol can induce autophagy in MCF-7 cells,which may be related to the inhibition of mTOR signaling pathway.