蛇葡萄素通过核转录因子-κB通路调控黑色素瘤A375细胞周期和凋亡

Ampelopsin Induces Melanoma A375 Cells Apoptosis and Cell Cycle Arrest by Regulating Nuclear Factor-κB Signal Pathway

目的研究蛇葡萄素对A375黑色素瘤细胞增殖,细胞周期和凋亡作用,并探讨其潜在机制。方法利用八肽胆囊收缩素(CCK-8)实验测定细胞活性;用Hoechst33258染色、流式细胞术检测细胞凋亡和细胞周期;相关蛋白的表达利用蛋白质印迹法(WB)检测。结果经不同浓度蛇葡萄素(0μmol/L、25μmol/L、50μmol/L、100μmol/L)处理48 h后,人黑色素瘤A375细胞增殖明显减弱;蛇葡萄素能显著抑制黑色素瘤A375细胞核转录因子-κB(NF-κB)通路的活化;蛇葡萄素以浓度依赖的方式通过下调B细胞淋巴瘤-2(Bcl-2)蛋白的表达,下调Bax蛋白和Cleavedcaspase-3蛋白的表达,促进人黑素瘤A375细胞凋亡;Hoechst33258染色法显示蛇葡萄素诱导的A375细胞凋亡的形态学变化;蛇葡萄素能下调细胞周期调控蛋白Cyclin D1和CDK4的表达,将A375细胞周期阻滞于G_(0)/G_(1)期;但NF-κB通路活化剂佛波醇脂(PMA)处理A375细胞后抑制了蛇葡萄素对A375细胞NF-κB信号通路,细胞增殖与凋亡,细胞周期的作用。结论蛇葡萄素能通过调节NF-κB通路抑制A375细胞的增殖,诱导细胞凋亡,阻滞细胞周期于G_(0)/G_(1)期。

Objective In order to study the effects of Ampelopsin on proliferation,apoptosis and cell cycle in human melanoma A375 cells line by regulating nuclear factor-κB(NF-κB)signal pathway.Methods The cell viability was evaluated by cholecystokinin octapeptide(CCK-8)assay;and apoptotic cell morphology was assessed by Hoechst 33258 staining.The functions of PolyphylinⅠin the cell cycle and apoptosis were determined by flow cytometry with annexin V-FITC/PI.The protein expressions were detected by Western blot assay.Results Forty-eight hours after A375 cells were treated with 0μmol/L,25μmol/L,50μmol/L,100μmol/L Ampelopsin,Ampelopsin suppressed the A375 cells proliferation in a dose-dependent manner.Ampelopsin treatment attenuated significantly the activation of NF-κB signal pathway in A375 cells.Ampelopsin promoted remarkably apoptosis of A375 cells by decreasing the level of Bcl-2 and elevating the levels of Bax and cleaved-caspase 3 in a dose-dependent manner.The ampelopsin-induced morphological changes were observed in apoptotic A375 cells via Hoechst 33258 staining.Ampelopsin treatment triggered A375 cells cycle blockage in G_(0)/G_(1) stage,and alleviated the expressions of Cyclin D1 and CDK4.However,treatment with NF-κB signal pathway activator PMA attenuated these ampelopsin-mediated effects on NF-κB signal pathway,cell proliferation,cell apoptosis and cell cycle.Conclusion Ampelopsin inhibits cell proliferation,promotes cell apoptosis,arrests cell cycle in G_(0)/G_(1) phase in human melanoma A375 cells via inhibiting NF-κB signal pathway.