14-3-3/Raf-1复合体介导的榄香烯抗胶质瘤作用

14-3-3/Raf-1 molecular complex mediated anti-glioma effect of elemene

目的探讨莪术提取物榄香烯在体外抗胶质瘤作用及相关机制。方法采用细胞计数、免疫共沉淀、Western印迹等方法分别检测经不同浓度、不同作用时间榄香烯对U87胶质瘤细胞的增殖影响、对U87细胞中与Raf-1结合形成分子复合体的14-3-3表达水平的影响、对U87细胞14-3-3、Raf-1、ERK蛋白质表达的影响。结果榄香烯在体外具有浓度、时间依赖的抗胶质瘤效应,其IC50约为(83.2±4.3)μg/mL。分子机制研究表明:榄香烯对总14-3-3蛋白表达无明显影响(P>0.05),能明显抑制14-3-3/Raf-1复合体的形成;榄香烯明显下调ERK-1/2信号通路中关键蛋白P-Raf-1、P-ERK-1/2的表达(P<0.05),对非磷酸化的ERK-1/2和Raf-1无明显影响(P>0.05)。结论榄香烯在体外通过破坏14-3-3的分子伴侣功能,阻碍14-3-3/Raf-1复合体的形成,使Raf-1不能被活化,进一步抑制Raf/MEK/ERK通路,达到抗胶质瘤细胞作用。

Objective To study the mechanisms through which elemene inhibits the proliferation of human U87 glioblastoma cells in vitro. Methods The anti- proliferation effect of elemene on U87 glioblastoma cells was measured by cell-counting. The level of 14- 3- 3 combined to Raf- 1 and expression of 14- 3- 3,Raf- 1 and ERK protein was tested by,Co- immunoprecipitation assay and Western blotting analysis at different concentrations or at different time points,respectively. Results Elemene inhibits the proliferation of U87 glioblastoma cells in a dose- and time- dependent manner,with a IC50of（ 83. 2 ± 4. 3） μg /mL. The data of Co- immunoprecipitation assay indicates that elemene inhibits 14- 3- 3 /Raf- 1 complex formation. Downregulated phosphorylated Raf- 1 and ERK- 1 /2 expression are also observed in western blot（ P〈 0. 05）,while no significant difference of non- phosphorylated Raf- 1 and ERK- 1 /2 expression are observed in each treated group（ P 〉0. 05）. Conclusion Elemene has significant anti- glioma effects on U87 glioblastoma cells in vitro. It disrupts the molecular chaperone function of 14- 3- 3,decreases the recruitment of 14- 3- 3 in the 14- 3- 3 /Raf- 1 complex,and blocks the activation of Raf- 1,which results in downregulation of ERK pathway and consequently attained its anti- proliferation effect.