Bladder Cancer BIU-87 Cells Drug Resistance Induction And Experimental Study Of Verapamil Reversal

Objective:This essay is mainly to study the drug resistance induction,through the interval-drug-use method to induce the bladder cancer BIU-87/HCPT cell of hydroxycamptothecin-resistance,then the relevant protein detect of multidrug resistance test and cross-drug resistance test are done.Finally,the drug resistance of BIU-87/HCPT cell through verapamil reverse is also tested.Methods:Using hydroxycamptothecin to induce BIU-87 cells by interval-drug-use method,drug resistance on hydroxycamptothecin will be made,and then drug resistance index is determined and drug cross-resistance on commonly-used antineoplastic by MTT method.The cell immunohistochemical staining assay to detect resistance-associated proteins of P-gp,MRP1,Bcl-2 and Survivin is used. Through MTT method is to determine cell-toxicity of verapamil.Then applying MTT assay and flow cytomeytry is to determine the reversed effect of drug resistance of verapamil on the BIU-87/HCPT cells.Results:After five months of drug resistance induction,the drug resistance index of BIU-87/HCPT cell increase by 55 times.Drug resistance of BIU-87/HCPT cells to vincristine,mitomycin and cisplatin are enhanced to different degrees.Particularly vincristine increases by 26.88 times to maximum level.However drug resistance to adriamycin is not improved obviously.By the cell immunohistochemical staining assay,the positive rate of P-gp and MRP1 are + +,however Bcl-2 and Survivin are + + +.By MTT assay,the concentration of verapamil in 10μg/ml is low toxicity, non-toxic when in 5μg/ml.When the concentration of verapamil is lower than 10μg/ml,the reversal efficiency is directly proportional to the concentration.When the concentration of verapamil is 10μg/ml,the reversal efficiency is 3.14 times to the maximum reversal efficiency.Results measured by flow cytometry are well matched with the MTT assay.However,by comparing with the reversal efficiency and sensitive control groups,there are a big gap on cell death rate.Conclusion:The improved interval drug-induced assay can enhance the drug resistance index of drug-resistant cells greatly within a relatively short period of time. The BIU-87/HCPT cells,induced by HCPT with drug-resistant cells have cross-resistance on other antineoplastic drugs and increased expression of resistance-associated protein P-gp,MRP1,Bcl-2 and Survivin are detected.But the reversal efficiency of Verapamil to BIU-87/HCPT cells is lower than reported in the Documents.That may be corresponded with the complex multidrug resistance mechanisms of tumor cells and the P-gp expression of BIU-87/HCPT cell relatively not high,therefore further study is needed.