双硫仑/铜复合物联合多柔比星对人肝癌细胞HepG2增殖和凋亡的影响  

Effects of disulfiram/copper complex combined with Doxorubicin on proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells

作　　者：张弓[1] 李素新[1] 郭化虎 李路豪 李林[1] 刘兆臣 Zhang Gong;Li Suxin;Guo Huahu;Li Luhao;Li Lin;Liu Zhaochen(Department of Hepatobiliary&Pancreatic Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院肝胆胰外科,郑州450052

出　　处：《中华实验外科杂志》2024年第8期1725-1729,共5页Chinese Journal of Experimental Surgery

摘　　要：目的检测双硫仑/铜复合物(DSF/Cu)联合多柔比星对人肝癌细胞HepG2增殖和凋亡的影响并探讨其作用机制。方法以浓度5.000、2.500、1.250、0.625、0.313、0.156、0.078、0.039、0.019、0.009μmol/L的多柔比星及相同浓度梯度的DSF/Cu(Cu2+固定浓度1μmol/L)分别处理HepG2细胞,噻唑蓝(MTT)法计算多柔比星和DSF/Cu对HepG2细胞的半抑制浓度(IC50)。以上述浓度梯度的多柔比星单独及联合0.15μmol/L的DSF/Cu处理HepG2细胞,MTT法分析单用多柔比星及联合DSF/Cu对HepG2细胞增殖的影响,CompuSyn软件计算两药作用的联合指数(CI)。将HepG2肝癌细胞分为未处理组(不添加任何药物)、二甲基亚砜(DMSO)处理组(仅加入与DSF/Cu处理组等量的DMSO)、DSF/Cu处理组(IC50浓度的DSF/Cu处理)、多柔比星处理组(IC50浓度的多柔比星处理)、多柔比星+DSF/Cu处理组(IC50浓度的多柔比星及DSF/Cu联合处理),以流式细胞技术检测各组HepG2肝癌细胞中ALDH+细胞数量的改变,干细胞成球实验检测各组HepG2肝癌细胞成瘤性的改变,蛋白质印迹法(Western blot)检测各组HepG2肝癌细胞中人表皮生长因子受体2(Her-2)、性别决定区Y框蛋白9(SOX9)、c-Myc、LC3-Ⅱ/Ⅰ和裂解的聚腺苷二磷酸核糖聚合酶(cleaved PARP)蛋白的表达。组间两两比较采用配对样本t检验,组间多个均数比较采用单因素方差分析。结果多柔比星和DSF/Cu对HepG2肝癌细胞作用48 h的IC50分别为0.8698、0.5538μmol/L。多柔比星联合0.15μmol/L的DSF/Cu对HepG2肝癌细胞的增殖抑制作用明显强于单用多柔比星(t=8.930,P<0.01),CompuSyn分析显示多柔比星联合DSF/Cu对HepG2肝癌细胞的增殖抑制呈协同作用(CI<1)。流式细胞分析显示,DSF/Cu处理组[(0.886±0.082)×10^(5)]和多柔比星处理组[(1.374±0.041)×10^(5)]HepG2肝癌细胞中ALDH+细胞数量低于未处理组[(1.859±0.979)×10^(5)],差异有统计学意义(t=10.800、6.444,P<0.05)。多柔比星+DSF/Cu处理组[(0.306±0.122)×10^ObjectiveTo investigate the effects of disulfiram/copper complex(DSF/Cu)combined with Doxorubicin on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells,and to explore the underlying mechanism.MethodsThe human hepatocellular carcinoma cell line HepG2 was obtained from ATCC cell bank.HepG2 cells were treated with Doxorubicin at concentrations of 5.000,2.500,1.250,0.625,0.313,0.156,0.078,0.039,0.019,and 0.009μmol/L,as well as with DSF/Cu complexes at the same concentration gradient(with a fixed concentration of 1μmol/L Cu^(2+)).The half maximal inhibitory concentration(IC 50)of both Doxorubicin and DSF/Cu against HepG2 cells was determined using the methyl thiazolyl tetrazolium(MTT)assay.Using the aforementioned concentration gradient of Doxorubicin alone and in combination with 0.15μmol/L DSF/Cu to treat HepG2 cells,the MTT assay was used to analyze the effects of Doxorubicin alone and in combination with DSF/Cu on the proliferation of HepG2 cells.The combination index(CI)of the two drugs was calculated using CompuSyn software.HepG2 cells were then divided into the following groups:untreated,DMSO-treated,DSF/Cu-treated,Doxorubicin-treated,and Doxorubicin+DSF/Cu-treated groups.Flow cytometry was used to detect changes in the number of ALDH+cells in each group.The sphere formation assay was performed to examine alterations in the tumorigenicity of HepG2 cells.Western blotting analysis was conducted to detect the expression levels of human epidermal growth factor receptor-2(Her-2),sex determining region Y box 9(SOX9),c-Myc,LC3-Ⅱ/Ⅰ,and cleaved poly adenosine diphosphate-ribose polymerase(PARP)proteins in each group.For pairwise comparisons between groups,the paired sample t-test was used,while for comparing multiple means across groups,one-way ANOVA was employed.ResultsThe IC 50 for Doxorubicin and DSF/Cu about HepG2 cells after 48 h of treatment was 0.8698μmol/L and 0.5538μmol/L,respectively.Doxorubicin combined with 0.15μmol/L DSF/Cu significantly inhibited the proliferation of He

关 键 词：双硫仑/铜复合物 多柔比星 肝癌 增殖 凋亡 

分 类 号：R735.7[医药卫生—肿瘤]