Mechanism Of Disulfiram Chelated With Copper Reverses Taxol Resistance Of Breast Cancer MCF-7 Cells

Background:Breast cancer is one of the leading causes of cancer-related deaths in women worldwide and chemotherapy plays a very important role in it’s comprehensive treatment.As one of the basic chemotherapy drugs for breast cancer,paclitaxel(Taxol)has very good antitumor effect.With the widespread application of Taxol,more and more patients develop acquired resistance.Therefore,it is great significant to study the mechanism and find ways to reverse Taxol acquired resistance to improve the efficacy of chemotherapy for breast cancer.As an ancient anti-alcoholism drug,disulfiram(DSF)chelated with cuprum(Cu)namely(DSF-Cu)can enhance the toxicity of chemotherapeutics and reverse drug resistance.MCF-7 cells are subtypes of breast cancer cells that express hormone receptor(HR)positively.Presently,the effect and mechanism of DSF-Cu on Taxol-resistant breast cancer MCF-7/Taxol cells have not been reported.Purposes:This study will investigate the changes in molecular indicators related to apoptosis and autophagy of MCF-7 cells under Taxol intervention and MCF-7/Taxol cells under DSF-Cu intervention.The results will clarify mechanism of MCF-7 cells Taxol acquired resistance and inhibition effect of DSF-Cu on MCF-7/Taxol cells and provide a more solid theoretical basis for the better application of Taxol and DSF in clinical treatment of breast cancer.Methods:1.Culturing MCF-7 and MCF-7/Taxol cells,observing the morphology and growth status of the two cells by microscope;CCK-8 was used to analyze the Taxol resistance of MCF-7/Taxol cells.The qRT-PCR was used to measure the geneexpression of Bcl-2,Bax and Beclin-1 in MCF-7 and MCF-7/Taxol cells.Western Blot was used to measure the protein expression of Bcl-2,Bax and Beclin-1 in MCF-7and MCF-7/Taxol cells.2.CCK-8 was used to measure the inhibition rate to study the relationship between DSF-Cu concentration and intervention time on MCF-7/Taxol cells.When MCF-7/Taxol cells were treated with Taxol and DSF-Cu monotherapy or combination therapy,observing the cells growth status under the microscope.Scratch test was used to compare the effect of DSF-Cu on migration of MCF-7/Taxol cells.Flow cytometry was used to measure the apoptosis rate of MCF-7/Taxol cells to study the relationship between DSF-Cu concentration and intervention time.3.Western Blot was used to measure the expression of Bcl-2,Bax,Beclin-1,Caspase-3 and Cyclin D1 in MCF-7/Taxol cells after Taxol and DSF-Cu monotherapy or combination therapy.Results:1.The CCK-8 results showed with the increased concentrations of Taxol,the proliferation inhibition effect on MCF-7 cells and MCF-7/Taxol cells was enhanced gradually,in which,MCF-7 cells had stronger inhibitory effect than MCF-7/Taxol cells(P<0.05),the difference was statistically significant.The qRT-PCR results showed the gene expression of Bcl-2 and Beclin-1 of MCF-7/Taxol cells were increased(P<0.05),but the Bax was decreased(P<0.05),the difference was statistically significant.Western Blot results showed the protein expression of Bcl-2and Beclin-1 of MCF-7/Taxol cells were increased(P<0.05),but the Bax was decreased(P<0.05),the difference was statistically significant.2.CCK-8 results showed that with the increase of DSF-Cu concentration and prolonged action time,the cell proliferation inhibition rate was gradually increased(P<0.05),the cell viability was gradually decreased(P<0.05),the difference was statistically significant.Observing the cells growth status by microscope showed that MCF-7/Taxol cells had the worst growth status after combination therapy of Taxol and DSF-Cu.The Scratch test results showed that DSF-Cu can slow down the healing speed of MCF-7/Taxol cells(P<0.05),the difference was statistically significant.Flow cytometry results showed that after DSF-Cu intervention,with the increased concentrations of DSF-Cu,the MCF-7/Taxol cells apoptosis rate gradually increased(P<0.05),with the increase of intervention time,the apoptosis rate gradually increased(P<0.05),the difference was statistically significant.3.Western Blot results showed that in MCF-7/Taxol cells after the combination therapy of DSF-Cu and Taxol,the expressions of Bax and Caspase-3 was gradually increased(P<0.05),but the Bcl-2,Beclin-1 and Cyclin D1 were gradually decreased(P<0.05),the difference was statistically significant.Conclusions:1.The acquired resistance to Taxol in MCF-7 cells is related to the uncoupling of Beclin-1/Bcl-2 and the formation of Bcl-2/Bax heterodimers,which make the expression of Beclin-1 and Bcl-2 increased but the Bax expression decreased,thereby inhibiting apoptosis and inducing protective autophagy,eventually leading to MCF-7cells producing Taxol acquired resistance.2.DSF-Cu can inhibit the proliferation of MCF-7/Taxol cells and it’s inhibitory capacity is directly proportional to DSF-Cu concentration and intervention time.In addition,DSF-Cu can inhibit migration and promote apoptosis of MCF-7/Taxol cells.3.DSF-Cu combined with Taxol can inhibit Beclin-1-mediated autophagy,arrest G1/S,induce tumor cell apoptosis through Bcl-2/Bax pathway and eventually reverse the Taxol acquired qqresistance of MCF-7 cells.