左旋含羞草碱调控ROS/JNK/c-Jun轴诱导黑色素瘤细胞线粒体凋亡的研究

L-Mimosine Modulates the ROS/JNK/c-Jun Signaling Axis to Trigger Mitochondrial Apoptosis in Melanoma Cells

    摘要
  • 摘要:
    目的 探讨左旋含羞草碱对黑色素瘤细胞增殖、凋亡的影响及分子机制。
    方法 采用MTT法检测不同浓度左旋含羞草碱对黑色素瘤A375和B16细胞存活率的影响；平板克隆实验评估左旋含羞草碱对细胞集落形成能力的影响；流式细胞术分析左旋含羞草碱对细胞凋亡、ROS和线粒体膜电位的影响；Western blot检测细胞内Bcl-2、Bax、Cyt C、Cleaved Caspase-3、Cleaved Caspase-9、Bim-EL、Bad、p-JNK、p-c-Jun、p-MKK4及p-ASK1等蛋白表达水平的变化。荷瘤小鼠分别接受生理盐水、左旋含羞草碱及左旋含羞草碱联合NAC治疗后，进行肿瘤组织学和TUNEL染色分析。
    结果 左旋含羞草碱显著抑制了A375和B16细胞的增殖，并明显促进了细胞发生凋亡。Western blot提示左旋含羞草碱可显著提升胞质内的Apaf-1和Cyt C的含量，上调了细胞内Cleaved PARP、Cleaved Caspase-3、Cleaved Caspase-9、Bax、Bim-EL和Bad的表达，下调了抗凋亡蛋白Bcl-2的表达。进一步的实验表明左旋含羞草碱显著诱导A375细胞内活性氧(ROS)的累积，激活了JNK/c-Jun信号通路，表现为p-JNK、p-c-Jun、p-MKK4及p-ASK1蛋白水平的显著升高。此外，抗氧化剂N-乙酰半胱氨酸(NAC)能够显著拮抗左旋含羞草碱诱导的ROS积累，进而逆转左旋含羞草碱对黑色素瘤细胞凋亡的诱导作用。在体内实验中，NAC同样能够显著拮抗左旋含羞草碱对肿瘤生长的抑制作用，表现为肿瘤体积的增加和凋亡相关蛋白表达水平的逆转。
    结论 左旋含羞草碱通过增加ROS水平和激活JNK/c-Jun信号通路，诱导黑色素瘤细胞发生凋亡。

     

    Abstract:
    OBJECTIVE To investigate the effects of L-mimosine on the proliferation and apoptosis of melanoma cells and its molecular mechanisms.
    METHODS The effects of different concentrations of L-mimosine on the viability of melanoma A375 and B16 cells were examined by the MTT assay.The ability of L-Mimosine to affect colony formation was assessed by the plate colony formation assay.The effects of L-mimosine on cell apoptosis, reactive oxygen species(ROS), and mitochondrial membrane potential were analyzed by flow cytometry.The changes in the expression levels of intracellular proteins such as Bcl-2, Bax, Cyt C, Cleaved Caspase-3, Cleaved Caspase-9, Bim-EL, Bad, p-JNK, p-c-Jun, p-MKK4, and p-ASK1 were detected by Western blot. The tumor-bearing mouse model was subjected to histological and TUNEL staining analysis of tumor tissues after treatment with normal saline, L-mimosine alone, and L-mimosine in combination with N-acetylcysteine (NAC).
    RESULTS L-mimosine significantly inhibited the proliferation of A375 and B16 cells and significantly promoted cell apoptosis. Western blot analysis showed that L-mimosine could significantly increase the content of Apaf-1 and Cyt C in the cytoplasm, up-regulate the expression of Cleaved PARP, Cleaved Caspase-3, Cleaved Caspase-9, Bax, Bim-EL and Bad, and down-regulate the expression of anti-apoptotic protein Bcl-2. Further experiments showed that L-mimosine significantly induced the accumulation of ROS in A375 cells and activated the JNK/c-Jun signaling pathway, which was manifested by the significant increase of p-JNK, p-c-Jun, p-MKK4 and p-ASK1. Additionally, the antioxidant NAC could significantly antagonize the ROS accumulation induced by L-mimosine, thereby reversing the pro-apoptotic effects of L-mimosine on melanoma cells. In vivo experiments, NAC also significantly antagonized the tumor growth inhibition induced by L-mimosine, as evidenced by increased tumor volume and reversed expression levels of apoptosis-related proteins.
    CONCLUSION L-mimosine induces apoptosis in melanoma cells by increasing ROS levels and activating the JNK/c-Jun signaling pathway.

     

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