Inhibitory Effects Of Disulfiram Combined With Copper On Human Adrenocortical Carcinoma Cells

Objective:To investigate the effects of disulfiram（DSF）combined with copper（Cu）on the proliferation,apoptosis,cell invasion and migration ability of human adrenocortical carcinoma cells H295R and SW-13,and to explore preliminarily the related mechanisms.Methods:H295R and SW-13 cells were cultured in vitro.Cells were divided into blank control group（containing 0.1%dimethyl sulfoxide solvent）,Cu group(0.5μM Cu²⁺),DSF group and DSF/Cu group(the concentrations of DSF were 0.1,0.2,0.4,0.8 and 1.6μM,respectively;and the concentration of Cu²⁺was 0.5μM).After treatment of drug intervention for 12 hours,24 hours and 48 hours,the inhibition of cell proliferation was tested by MTT assay.The inverted microscope was used to observe the changes of cell morphology.Annexin V-APC/7-AAD double staining flow cytometry was applyed to detect the apoptosis rate of cells in Cu group and DSF/Cu group for 24 hours.Transwell assay and scratch wound healing assay were respectively performed to evaluate the invasion and migration ability of H295R cells treated by DSF/Cu.Western blot assay was applied to examine the changes of protein expressions of Bcl-2,Bax,and MMP-2.Results:1.The results of MTT showed that DSF/Cu inhibited proliferation of H295R and SW-13 cells,and was affected by different intervention time and DSF concentration（P<0.05）.However,the proliferation inhibition rate of cells in Cu or DSF group had no statistical significance compared with the blank control group（P>0.05）.The 50%inhibitory concentration of H295R and SW-13cells after DSF/Cu intervention for 24 hours was 0.27μM and 0.69μM,respectively.2.After treated with DSF（0.2,0.4,0.8μM）combined with 0.5μM Cu²⁺for 24 hours,the number of apoptosis in H295R cells was higher than that in Con group（P<0.05）.However,when DSF≥0.4μM,the apoptosis rate of DSF/Cu group significantly increased compared with blank control group in SW-13 cells（P<0.05）.No significant change was observed in cell apoptosis between Cu group and blank control group（P>0.05）.3.Transwell results showed that the invasiveness of H295R cells in DSF/Cu group was significantly lower than that in Con group（P<0.01）.There was no inhibitory effect on cell invasion in Cu group or DSF group（P>0.05）.The results of cell scratch wound healing assay showed that the migration ability of H295R cells in DSF/Cu group was lower than that in blank control group（P<0.05）.There was no significant difference of migration ability in Cu group compared with the blank control group（P>0.05）.4.The results of Western blot showed that the expression of Bax was increased and the expression of Bcl-2 and MMP-2 was down-regulated compared with the blank control group after the treatment of DSF/Cu（P<0.05）.However,in comparison with the control group,the expression of Bax,Bcl-2and MMP-2 was not significantly different by 0.5μM Cu²⁺intervention（P>0.05）.Conclusion:1.DSF/Cu can inhibite the proliferation of human adrenal cortical carcinoma cell lines H295R and SW-13 in a time-and dose-dependent manner,while Cu or DSF have no significant inhibitory effect.2.DSF/Cu can induce apoptosis of human adrenal cortical carcinoma cells lines H295R and SW-13 which may be involved in the apoptotic pathway regulated by decreased expression of Bcl-2 and increased expression of Bax.3.DSF/Cu can inhibit the ability of invasion and migration in human adrenocortical carcinoma cells,and its mechanism may be related to the regulation of MMP-2.