高三尖杉酯碱对人黑色素瘤A375细胞增殖的影响及机制研究

Effect of homoharringtonine on proliferation of human melanoma A375 cells and its mechanisms

目的探究高三尖杉酯碱(homoharringtonine,HHT)对人黑色素瘤A375增殖的影响及可能的作用机制。方法采用CCK-8法检测HHT对A375细胞增殖的影响;Hoechst 33258染色法观察染色质凝集情况;细胞克隆形成实验检测HHT对A375细胞克隆形成能力的影响;流式细胞术(Flow cytometry,FCM)检测HHT对A375细胞凋亡和周期影响;Western blot检测Bcl-2、Bax、Cleaved-caspase 3、Cleaved-PARP、CyclinB1和CDK1蛋白的表达情况。结果CCK-8结果显示,随着药物的剂量(0、0.05、0.1、0.2、0.4、0.8μmol·L^-1)和作用时间(24、48、72 h)增加,HHT对A375细胞的抑制作用逐渐增强;Hoechst 33258染色和FCM结果显示,HHT可使A375细胞染色质呈凋亡形态改变,促进细胞凋亡,将A375的细胞周期阻滞于G2/M期;克隆形成实验显示,HTT可以抑制A375细胞的克隆形成能力;Western blot结果表明HHT可下调CyclinB1、CDK1和Bcl-2蛋白水平,上调Bax,Cleaved-caspase 3和Cleaved-PARP蛋白表达水平。结论HHT使A375细胞阻滞于G2/M期,抑制A375细胞增殖;HHT可激活线粒体凋亡信号通路,诱导黑色素瘤A375细胞凋亡。

Aim To explore the effectof homoharringtonine(HHT)on cell proliferation in A375 cellsandits mechanisms.Methods The effect of HHT on proliferation of A375 cells was measured by Cell Counting Kit-8 assay(CCK-8 assay).The morphology of the cell nuclei was detected by Hoechst33258 fluorescent staining.Cell proliferation ability after treating with HHT was examined by clonal formation assay.Apoptosis and cell cycle distribution of A375 cells were determined by flow cytometry(FCM).The protein levels of Bcl-2,Bax,Cleaved caspase-3,Cleaved-PARP,Cyclin B1 and CDK1 proteins were ascertained by Western blot.Results CCK-8 results showed that with the increase of HHT concentration(0,0.05,0.1,0.2,0.4,0.8μmol·L^-1)and duration of action(24,48,72 h),the inhibitory effect of HHT on A375 cells was remarkably enhanced.Hoechest33258 staining and FCM showed that HHT induced apoptosis and cell-cycle arrest in G2/M phase in A375 cells,which had typical apoptotic morphological changes in HHT-treated group.Clonal formation assay revealed that HHT inhibited colony formation in A375 cells.Western blot results showed that the protein level of Cyclin B1,CDK1 and Bcl-2 were down-regulated,while Bax,Cleaved caspase-3 and Cleaved PARP proteins were up-regulated after treating with HHT.Conclusions HHT can inhibit the proliferation of A375 cells by inducing cell cycle arrest at the G2/M phase.HHT promotes A375 apoptosis through the activation of mitochondrial-mediated apoptotic signaling pathway.