Effects of disulfiram/copper complex combined with Doxorubicin on proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells
Objective To investigate the effects of disulfiram/copper complex(DSF/Cu)com-bined with Doxorubicin on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells,and to explore the underlying mechanism.Methods The 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 0.009 µmol/L,as well as with DSF/Cu complexes at the same concentration gradient(with a fixed concentration of 1 µmol/L Cu2+).The half maximal inhibitory concentration(IC50)of both Doxorubicin and DSF/Cu against HepG2 cells was deter-mined using the methyl thiazolyl tetrazolium(MTT)assay.Using the aforementioned concentration gradient of Doxorubicin alone and in combination with 0.15 µmol/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 altera-tions 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.Results The IC50 for Doxorubicin and DSF/Cu about HepG2 cells after 48 h of treatment was 0.8698 µmol/L and 0.5538μmol/L,respectively.Doxorubicin combined with 0.15 μmol/L DSF/Cu significantly inhibited the proliferation of HepG2 cells compared to Doxorubicin alone(t=8.930,P＜0.01).CompuSyn analysis revealed a synergistic effect of Doxorubicin combined with DSF/Cu on inhibiting HepG2 cell proliferation(CI＜1).Flow cytometry analy-sis showed that the number of ALDH+HepG2 cells was less in the DSF/Cu-treated group[0.886±0.082)× 105]and Doxorubicin-treated group[(1.374±0.041)× 105]compared to the untreated group[(1.859±0.979)× 105],with statistically significant differences(t=10.800,6.444,P＜0.05).The number of ALDH+HepG2 cells in the Doxorubicin+DSF/Cu-treated group[(0.306±0.122)× 105]was significantly less than that in the DSF/Cu-treated group and Doxorubicin-treated group,with statistically significant differences(t=5.590,11.730,P＜0.01).The sphere formation assay revealed that the num-ber of HepG2 stem cell spheres was less in the DSF/Cu-treated group(2.167±1.169)and Doxorubicin-treated group(21.170±4.875)compared to the untreated group(46.500±5.357),with statistically sig-nificant differences(t=19.800,8.567,P＜0.05).The number of HepG2 stem cell spheres in the Doxo-rubicin+DSF/Cu-treated group(0.833±0.753)was less than that in the Doxorubicin-treated group(21.170±4.875),with a statistically significant difference(t=10.100,P＜0.01).There was no statisti-cally significant difference in the number of stem cell spheres between the Doxorubicin+DSF/Cu-treated group and DSF/Cu-treated group(0.833±0.753 vs.2.167±1.169,t=2.349,P＞0.05).Western blotting results showed that the expression of Her-2,SOX9,and c-Myc proteins in HepG2 cells was signifi-cantly lower in the DSF/Cu-treated group and Doxorubicin+DSF/Cu-treated group than the untreated group(0.132±0.013 vs.0.049±0.010 vs.0.950±0.052,0.325±0.049 vs.0.311±0.038 vs.0.922±0.027,0.308±0.041 vs.0.235±0.027 vs.0.816±0.092,F=412.50,113.20,47.54,P＜0.05),while the expression of LC3-Ⅱ/Ⅰ and Cleaved PARP proteins was higher in the DSF/Cu-trea-ted group and Doxorubicin+DSF/Cu-treated group than in the untreated group(1.527±0.201 vs.2.629±0.224 vs.0.766±0.159,0.906±0.083 vs.0.834±0.058 vs.0.039±0.001,F=35.15,170.40,P＜0.05).In the Doxorubicin-treated group,the expression of Her-2 and SOX9 proteins in HepG2 cells was lower than that in the untreated group(0.275±0.018 vs.0.950±0.052,0.535±0.034 vs.0.922±0.027,t=17.440,12.680,P＜0.05),but there were no statistically significant differences in the expression of c-Myc,LC3-Ⅱ/Ⅰ,and Cleaved PARP proteins compared to the untreated group(0.914±0.097 vs.0.816±0.092,1.329±0.152 vs.0.766±0.159,0.063±0.017 vs.0.039±0.001,t=1.042,4.205,2.061,P＞0.05).In the Doxorubicin+DSF/Cu-treated group,the expression of c-Myc,Her-2 and SOX9 proteins was significantly lower than that in the Doxorubicin-treated group(0.235±0.027 vs.0.914±0.097,0.049±0.010 vs.0.275±0.018,0.311±0.038 vs.0.535±0.034,t=9.579,15.520,6.222,P＜0.05),while the expression of LC3-Ⅱ/Ⅰ and Cleaved PARP proteins was higher than that in the Doxorubicin-treated group,with statistically significant differ-ences(2.629±0.224 vs.1.329±0.152,0.834±0.058 vs.0.063±0.017,t=6.800,17.980,P＜0.05).Conclusion The combination of DSF/Cu complex and Doxorubicin can synergistically inhibit the proliferation of HepG2 cells,and its mechanism may be related to the inhibition of HCSCs and stemness gene-related proteins,as well as the induction of autophagy and apoptosis in HepG2 cells.
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