甜菜碱抑制PI3K/AKT/NF-κB通路诱导C4-2B前列腺癌细胞凋亡

Betaine induces apoptosis of C4-2B prostate cancer cells via inhibiting PI3K/AKT/NF-κB signaling pathway

目的探讨甜菜碱(BET)对C4-2B前列腺癌细胞的抑制作用及可能机制.方法采用(0、100、200、400、600、800)mmol/LBET处理C4-2B前列腺癌细胞,CCK-8法检测细胞增殖活性的变化,显微镜下观察细胞形态的改变;平板克隆实验检测细胞集落形成能力变化,流式细胞术检测细胞凋亡率;Western blot法检测B细胞淋巴瘤因子2(Bcl2)、Bcl2相关蛋白X(BAX)、裂解型胱天蛋白酶3(c-caspase-3)、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(AKT)、核因子κB p65(NF-κB p65)等蛋白的表达水平变化,给予NF-κB信号通路抑制剂BAY11-7082处理,进一步验证BAX、Bcl2、c-caspase-3、NF-κB p65蛋白表达的改变.结果BET抑制前列腺癌C4-2B细胞增殖并呈浓度依赖性,刺激48 h后半数抑制浓度(IC_(50))为422.7 mmol/L.与对照组(0 mmol/L BET处理组)比较,(200、300、400)mmol/L BET处理组细胞数量减少、形状改变、集落形成能力降低,细胞凋亡率增加.与对照组比较,(300、400)mmol/L BET处理组BAX、c-caspase-3蛋白水平升高,Bcl2、PI3K、AKT、NF-κB p65蛋白水平降低;单纯给予BAY11-7082处理后,Bcl2、BAX、c-caspase-3、NF-κB p65等蛋白表达变化趋势与300 mmol/L BET处理组一致;BET联合BAY11-7082处理组Bcl2、NF-κB p65蛋白表达水平更低,BAX、c-caspase-3蛋白表达水平更高.结论BET抑制C4-2B前列腺癌细胞增殖并促进其凋亡,可能与抑制PI3K/AKT/NF-κB信号通路有关.

Objective To investigate the inhibitory effect of betaine(BET)on the proliferation of C4-2B prostate cancer cells and its possible mechanism.Methods C4-2B cells were treated with 0,100,200,400,600,800 mmol/L BET.CCK-8 assay was used to assess the cell proliferation,plate cloning formation assay to detect clone formation ability and flow cytometry to evaluate cell apoptosis,and the cell morphological alteration was observed by microscopy.The protein expression of BAX,Bcl2,cleaved caspase 3(c-caspase-3),phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),and NF-κB p65 were detected by Western blotting,and the changes of BAX,Bcl2,c-caspase-3,and NF-κB p65 proteins were further verified after the cells were treated with NF-κB pathway inhibitor BAY11-7082.Results BET inhibited the proliferation of C4-2B cells in a dose-dependent manner.The 50%inhibitory concentration(IC_(50))was 422.7 mmol/L after the cells were treated with BET for 48 hours.Compared with the control group(0 mmol/L BET treatment),the proliferation of C4-2B cells was inhibited along with morphological changes,decreased clone formation ability and increased apoptosis rate in 200,300,400 mmol/L BET treated groups.Meanwhile,the protein expression of BAX and c-caspase-3 were up-regulated and Bcl2,PI3K,AKT and NF-κB p65 were down-regulated in 300,400 mmol/L BET groups as compared with the control group.After BAY11-7082 treatment alone,Bcl2,BAX,c-caspase-3,NF-κB p65 protein expression trend was consistent with that of the 300 mmol/L BET treated group,and Bcl2,NF-κB p65 protein expression levels were lower and BAX and c-caspase-3 protein expression levels were higher in BET combined with BAY11-7082 treated group.Conclusion BET can inhibit C4-2B cell proliferation and induce its apoptosis by blocking PI3K/AKT/NF-κB signaling pathway.