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

关键词:  榄香烯  胶质瘤  14-3-3  Raf-1  ERK

Key words:  [Key words]　elemene  glioma  14-3-3  Raf-1  extracellular signal-regulated MAP kinases

摘要:

[摘要]　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IC50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。

Abstract:

[Abstract] 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 IC50 of (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.