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隐丹参酮通过抑制帽子依赖的翻译促进人肺癌细胞A549的凋亡

Cryptotanshinone Promotes Apoptosis of A549 Cells via Inhibiting Cap-dependent mRNA Translation

  • 摘要:
    目的 探索隐丹参酮对人肺癌细胞A549中帽子依赖翻译的影响及帽子依赖翻译在隐丹参酮促A549细胞凋亡中的作用。
    方法 免疫印迹实验检测隐丹参酮对A549细胞中mTOR及4EBP1磷酸化的影响,随后通过m7GTP pull down实验检测隐丹参酮对A549细胞中eIF4F蛋白复合物形成的影响,最终通过免疫印迹实验、Annexin V和CCK8法检测敲低4EBP1表达前后隐丹参酮对A549细胞中抗凋亡蛋白Bcl-2表达、细胞凋亡和存活的影响。
    结果 隐丹参酮以剂量依赖的方式抑制A549细胞中mTOR及4EBP1的磷酸化、eIF4F蛋白复合物的形成和Bcl-2蛋白的表达。细胞凋亡分析发现,对照组中,隐丹参酮显著地诱导了A549细胞的凋亡(4.68±0.77)% vs.(31.1±4.1)%, P < 0.05,但敲低4EBP1的表达显著抑制了隐丹参酮的促凋亡作用(31.1±4.1)% vs.(8.85±1.2)%, P < 0.05。CCK8法检测结果发现,隐丹参酮显著抑制了A549细胞的存活(1±0.09)% vs.(0.43±0.05)%, P < 0.05,但敲低4EBP1的表达拮抗了EMD638683的抑存活作用(0.43±0.05)vs.(0.87±0.06), P < 0.05。
    结论 隐丹参酮通过抑制mTOR激酶的活性抑制4EBP1磷酸化,从而抑制A549细胞中帽子依赖的翻译,进而抑制抗凋亡蛋白Bcl-2的表达诱发人肺癌细胞A549的凋亡。

     

    Abstract:
    Objective To investigate the effect of Cryptotanshinone on the cap-dependent translation in A549 cells and the role of cap-dependent translation in promoting Cryptotanshinone induced A549 cells apoptosis.
    Methods A549 cells were treated with various concentrations of Cryptotanshinone. The phosphorylation of mTOR and 4EBP1 were determined using Western blot assay. The formation of eIF4F complex was detected using m7GTP pull down assay. The expression of Bcl-2 was examined by Western blot assay. Cell apoptosis was detected using Annexin V Apoptosis Assay and cell survival was determined using CCK8 assay.
    Results Cryptotanshinone inhibited the phosphorylation of mTOR and 4EBP1, the formation of eIF4F complex and Bcl-2 expression of A549 cells in a concentration-dependent manner. Cryptotanshinone profoundly promoted the apoptosis of A549 cells in the control group (4.68±0.77)% vs. (31.1±4.1)%, P < 0.05, while silencing the expression of 4EBP1 suppressed the promoting effect of Cryptotanshinone on cell apoptosis (31.1±4.1)% vs. (8.85±1.2)%, P < 0.05. Cryptotanshinone significantly inhibited A549 cells survival (1±0.09) vs. (0.43±0.05), P < 0.05, while 4EBP1 depletion suppressed the inhibitory effect of Cryptotanshinone on cell survival (0.43±0.05) vs. (0.87±0.06), P < 0.05.
    Conclusion Cryptotanshinone suppresses the phosphorylation of 4EBP1 in A549 cells by inhibiting mTOR activity, thus inhibits cap-dependent mRNA translation and the expression of Bcl2 and promotes the apoptosis of A549 cells.

     

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