DSF/Cu诱导氧化应激和下调PTEN/Akt信号通路促进肝细胞癌细胞死亡

DSF/Cu promotes cell death of hepatocellular carcinoma by inducing oxidative stress and downregulating PTEN/Akt signaling pathway

目的探讨双硫仑/铜(DSF/Cu)对肝细胞癌(HCC)细胞活力和增殖的影响及其可能机制。方法使用不同浓度DSF/Cu 0、0.25、0.50和1.00μmol/L处理HCC细胞系HUH-7,采用CCK-8法检测细胞活力,免疫荧光分析细胞增殖,流式细胞术和比色法分别检测活性氧和丙二醛(MDA)含量,Western blot法检测磷酸化组蛋白(γ-H2AX)、人第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)、Akt、磷酸化Akt(p-Akt)蛋白表达。另用DSF/Cu 0.50μmol/L分别联合还原型谷胱甘肽(GSH)1.0 mmol/L、米托蒽醌甲磺酸盐(MitoQ)1.0μmol/L处理HCC细胞,观察其对细胞活力、增殖及活性氧含量的影响。结果DSF/Cu可显著抑制HUH-7细胞活力和增殖,增加活性氧和MDA含量,上调γ-H2AX和PTEN蛋白表达,下调p-Akt蛋白表达(P<0.05)。而加入抗氧化剂GSH或MitoQ后,可降低HUH-7细胞内活性氧含量,增加细胞活力和增殖(P<0.05)。结论DSF/Cu通过促进细胞氧化应激反应,下调PTEN/Akt信号通路的表达,从而抑制HCC细胞活力和增殖。

Objective To investigate the effect and underlying mechanism of disulfiram/copper(DSF/Cu)on the cell viability and proliferation of hepatocellular carcinoma(HCC).Methods HCC cell line HUH-7 was treated with different concentrations of DSF/Cu 0,0.25,0.50 and 1.00μmol/L,and the cell viability was measured by CCK-8 method.The cell proliferation was observed by immunofluorescence.The contents of reactive oxygen species(ROS)and malondialdehyde(MDA)were determined by flow cytometry and colorimetry,respectively.The protein expressions ofγ-H2AX,PTEN,Akt and p-Akt were detected by Western blot.Then HCC cells were treated with the combination of DSF/Cu 0.50μmol/L and GSH 1.0 mmol/L or MitoQ 1.0μmol/L,and the cell viability,proliferation and content of ROS were observed.Results DSF/Cu could significantly inhibit the viability and proliferation of HUH-7 cells,increase the contents of ROS and MDA,upregulate the protein expressions ofγ-H2AX and PTEN,and downregulate the protein expression of p-Akt(P<0.05).The content of ROS was decreased,and the cell viability and proliferation were increased after the treatment of antioxidant GSH or MitoQ(P<0.05).Conclusion DSF/Cu can inhibit the viability and proliferation of HCC cells by promoting the oxidative stress response and downregulating the PTEN/Akt signaling pathway.