mTOR通路抑制剂依维莫司对不同胶质瘤细胞自噬作用的影响

The mTOR pathway inhibitor Everolimus in cell autophagy of different gliomas

目的探讨mTOR抑制剂依维莫司对人脑胶质瘤细胞自噬作用的影响。方法体外培养胶质瘤细胞株87-MG、U251、SHG．44，给予不同浓度的依维莫司（0．1、0．5、1、10、20、100nmol／L）作用4～6d后用MTT法检测细胞的增殖并分析各细胞株的半数抑制浓度（Ic50）值。Hoechst33342／PI荧光双染检测Ic50的依维莫司作用48h后细胞凋亡；单丹磺酰尸胺（MDC）荧光染色检测细胞自噬囊泡的形成；流式细胞术检测细胞周期的变化。Westernblotting测定IC50的依维莫司作片J24h后细胞磷酸化mTOR、p70S6K、4E．BP—l蛋白及非磷酸化mTOR、p70S6K蛋白的表达。结果MTT结果显示U87-MG、SHG-44、U251细胞的生长均受到明显抑制，IC50分别为0．1、0．5、10nmol／L；Hoechst33342／PI荧光双染结果显示细胞没有发生明显的凋亡，可能为细胞白噬：MDC染色显示IC50的依维莫司作用后，细胞中荧光颗粒增加，荧光强度增强；流式细胞术检测显示3种细胞均有特异性的G0／G1期细胞阻滞；Westernblotting检测显示随着依维莫司剂量的增加，胶质瘤细胞巾磷酸化mTOR、p70S6K、4E．BP-1的表达水平均明显减少，非磷酸化4E—BP．1的表达水平减少．而非磷酸化p70S6K无明显变化。结论mTOR抑制剂依维莫司可以促进胶质瘤细胞的自噬，mTOR通路可能为潜在的新的胶质瘤的治疗靶点．

Objective To investigate the mTOR pathway inhibitor Everolimus in cell autophagy of different human gliomas in vitro. Methods Glioma cell lines, 87-MG, U251 and SHG- 44, were cultured in vitro; different doses of Everolimus （0.1, 0.5, 1, 10, 20 and 100 nmol/L） were given to these cells for 4-6 d; MTT assay was used to observe the proliferation of these human glioma ceils and the IC50 value was observed; Hoechst 33342/PI staining was employed to determine the apoptosis after Everolimus treatment at dosage of ICso value for 48 h; monodansyl cadaverine （MDC） staining was used to detect the autophagic vesicles after Everolimus treatment at dosage of IC50 value for 48 h; flow cytometry was employed to detect the effect of Everolimus at IC50 value for 48 h on quantitative cell cycle; Western blotting was employed to further analyze the protein expressions of phosphorylated and non-phosphorylated mTOR, p70S6K and 4E-BP-1 of glioma cells after Everolimus treatment at 1C50 value for 24 h. Results The U87-MG, SHG-44 and U251 cell growth was significantly inhibited, and their ICs0 value was 0.1 nmol/L, 0.5 nmol/L and 10 nmol/L, respectively; no obvious apoptosis was noted in the cells, which indicated that cell autophagy might exist; the fluorescent panicles were gradually increased as the increment of Everolimus dosages; Everolimus could cause the cycle arrest at the G0/GI phase in these 3 kinds of cells; the expressions ofphosphorylated mTOR, p70S6K and 4E-BP-1 were significantly decreased, and the expression of non-phosphorylated 4E-BP-1 was decreased, while that ofnon-phosphorylated p70S6K showed no significant changes. Conclusion The mTOR pathway inhibitor Everolimus can induce the cell autophagy of gliomas, and mTOR pathway might be a potential new therapeutic target in treating glioma.