冬凌草甲素通过PINK1/Parkin通路诱导三阴性乳腺癌发生线粒体自噬的机制研究

Mechanism of Oridonin Inducing Mitophagy in Triple Negative Breast Cancer Through PINK1/Parkin Pathway

    摘要
  • 摘要:
    目的 探讨冬凌草甲素（Oridonin，Ori）对三阴性乳腺癌（Triple negative breast cancer， TNBC）细胞增殖、凋亡和线粒体自噬的影响及具体的作用机制。
    方法 采用CCK8法探究Ori对TNBC细胞MDA-MB-231活力的影响并确定有效抑制肿瘤细胞生长的药物浓度；流式细胞术检测Ori对细胞凋亡和活性氧（Reactive oxygen species，ROS）的影响；Western blot测检测细胞凋亡相关蛋白Caspase-9、Bax和调控线粒体自噬相关蛋白LC3B、p62、PINK1、Parkin的表达；MDC染色法观察药物干预后MDA-MB-231细胞是否发生自噬；免疫荧光检测细胞内LC3B、PINK1和Parkin蛋白的表达及其与线粒体共定位；并通过皮下移植瘤动物实验验证Ori对TNBC增殖、自噬的影响。
    结果 Ori能显著抑制TNBC细胞活力，IC50为20.57 μmol·L-1。同时，20 μmol·L-1 Ori能显著促进细胞凋亡（P<0.001），提高细胞中ROS含量（P<0.05）；上调细胞凋亡蛋白Bax的表达（P<0.01），上调活化Caspase-9蛋白表达（P<0.01）。此外，Ori能促进TNBC细胞发生自噬，上调了自噬相关蛋白LC3BⅡ/Ⅰ、p62的表达（P<0.05，P<0.001），并上调线粒体自噬相关蛋白PINK1、Parkin的表达（P<0.01）及其与线粒体共定位。皮下移植瘤动物实验证实，与对照组相比，Ori高剂量组能显著抑制肿瘤生长，促进肿瘤细胞发生线粒体自噬。
    结论 Ori通过PINK1/Parkin通路诱导TNBC发生线粒体自噬，抑制肿瘤生长。

     

    Abstract:
    OBJECTIVE To explore the effect of Oridonin (Ori) on proliferation, apoptosis and mitophagy of triple negative breast cancer (TNBC) and its specific mechanism.
    METHODS CCK8 method was used to explore the effect of Ori on the activity of triple negative breast cancer MDA-MB-231 cells and determine the concentration of drugs that can effectively inhibit the growth of tumor cells. The effects of Ori on apoptosis and Reactive Oxygen Species (ROS) were detected by flow cytometry. Western blot was used to detect the expression of cell apoptosis -related proteins Caspase-9, Bax and mitophagy-related proteins LC3B, p62, PINK1 and Parkin. MDC staining was used to observe whether autophagy occurred in MDA-MB-231 cells after drug intervention; immunofluorescence was used to detect the expression of LC3B, PINK1 and Parkin proteins in cells and their co-location with mitochondria. The effects of Ori on proliferation and autophagy of TNBC were verified by subcutaneous transplantation of tumor animals.
    RESULTS Ori could significantly inhibit the activity of TNBC cells, and the IC50 was 20.57 μmol·L-1. At the same time, Ori could significantly promote cell apoptosis (P<0.001) and increase ROS content in cells (P<0.05), up-regulate the expression of cell apoptosis-related proteins Bax (P<0.01), and up-regulated the expression of activated Caspase-9 protein (P<0.01). In addition, Ori could promote autophagy in TNBC cells, up-regulate the expression of autophagy-related proteins LC3B Ⅱ/Ⅰ and p62 (P<0.05, P<0.001), and up-regulated the expression of mitophagy-related proteins PINK1 and Parkin (P<0.01) and their co-localization with mitochondria. . The animal experiment of subcutaneous transplanted tumor confirmed that compared with the control group, the high-dose Ori group could significantly inhibit tumor growth and promote mitophagy of tumor cells.
    CONCLUSION Ori induces mitophagy in triple negative breast cancer through PINK1/Parkin pathway, which inhibits tumor growth.

     

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