没食子酸诱导ROS积蓄介导黑色素瘤B16-F10细胞内源性凋亡及周期阻滞

Endogenous apoptosis and cycle arrest of melanoma B16-F10 cells mediated by ROS accumulation induced by gallic acid

目的:探究没食子酸诱导活性氧(reactive oxygen species,ROS)积蓄介导黑色素瘤B16-F10细胞凋亡及周期阻滞的作用机制。方法:以梯度浓度的没食子酸作用于黑色素瘤B16-F10细胞,采用MTT法检测没食子酸对细胞生长的影响,平板克隆技术检测细胞克隆形成率,Transwell实验检测细胞迁移及侵袭能力,ROS Assay Kit检测B16-F10细胞内ROS水平,线粒体膜电位检测试剂盒检测细胞膜电位的变化,Hoechst 33258荧光染色法进行细胞形态学检测,流式细胞术检测细胞凋亡及细胞周期阻滞水平,Western blot检测细胞内相关蛋白水平的变化。结果:结果显示,没食子酸明显抑制B16-F10细胞的生长,且具有浓度依赖性。没食子酸作用后,B16-F10细胞增殖、迁移及侵袭能力明显下降,细胞内ROS水平明显升高,线粒体膜电位明显下降;细胞数减少,细胞凋亡率增加,G_(0)/G_(1)期细胞数明显增多;细胞内凋亡相关蛋白Bax、Cytochrome C、Caspase-9以及Caspase-3表达量增加,而Bcl-2表达量减少;周期相关蛋白Chk2、p53、p21表达量明显增加,CyclinE1、CDK2表达量减少。结论:没食子酸作用于黑色素瘤B16-F10细胞后,通过诱导ROS积蓄,引起B16-F10细胞内源性凋亡及周期阻滞。

Objective:To explore the mechanism of apoptosis and cycle arrest of melanoma B16-F10 cells mediated by ROS accumulation induced by gallic acid.Methods:B16-F10 cells were treated with gradient concentration of gallic acid.MTT was used to detect the effects of gallic acid on cell growth.Colony formation rate was detected by plate cloning technique.Cell migration and invasion were detected by Transwell assay.ROS assay kit was used to detect the level of ROS in B16-F10 cells.Mitochondrial membrane potential detection kit was used to detect the changes of cell membrane potential.Hoechst 33258 fluorescence staining was used to detect cell morphology.The levels of apoptosis and cell cycle arrest were detected by flow cytometry.The changes of intracellular related proteins were detected by Western blot.Results:The results showed that gallic acid inhibited the growth of B16-F10 cells in a dose-dependent manner.After gallic acid treatment,the proliferation,migration and invasion ability of cells were significantly decreased.Intracellular ROS level significantly increased.Mitochondrial membrane potential decreased significantly.Cell number decreased,cell apoptosis rate and G_(0)/G_(1) phase cell number increased significantly.The expression of apoptosis related proteins Bax,Cytochrome C,Caspase-9 and Caspase-3 increased,while the expression of Bcl-2 decreased.The expression of cycle associated proteins Chk2,p53 and p21 increased significantly,while the expression of CyclinE1 and CDK2 decreased.Conclusion:It is speculated that gallic acid induced endogenous apoptosis and cycle arrest of B16-F10 cells by inducing ROS accumulation.