Developing multidrug-resistant cells and exploring correlation between BCRP/ABCG2 over-expression and DNA methyltransferase

Abstract

Expression of breast cancer resistance protein/ATP-binding cassette sub-family G member 2 (BCRP/ABCG2) is the major cause of chemotherapy failure. It is important to establish and characterize the multidrug resistance cells and to investigate the mechanism of multidrug resistance. Multidrug-resistant cells expressing BCRP/ABCG2 based on human breast cancer MCF-7/wt cells were developed by gradually increasing application of low concentration of mitoxantrone. Real-time quantitative PCR, western blot, and immunofluorescence assay were employed to analyze BCRP mRNA and protein expression. Drug accumulation in the cells was measured by flow cytometry and DNA methyltransferases were analyzed by western blot. The results indicated that the inhibitory ratio of cell proliferative growth exhibited an exponential relation with the concentration of mitoxantrone. The IC₅₀ of MCF-7/wt cells to mitoxantrone was found to be 0.42 μM. 3-(4,5-Dimethylthlthiazol-2-YI)-2,5-Diphenyltetrazolium Bromide assay indicated that the mitoxantrone-resistant cells at different stages exhibited cross-resistance to adriamycin and taxol. BCRP/ABCG2 mRNA and protein levels in the mitoxantrone-resistant cells at different stages increased with increasing concentration of mitoxantrone. Intracellular accumulation of mitoxantrone in the cells decreased with the increase of the BCRP/ABCG2 expression levels. DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3a (DNMT3a) expressions in the cells at different stages decreased slightly, whereas DNA methyltransferase 3b (DNMT3b) expression decreases significantly. BCRP/ABCG2 overexpression and its drug-efflux function in the drug-resistant cells are the main factors to produce multidrug resistance. Our results suggest that multidrug resistance is related to overexpression of BCRP/ABCG2 and the decrease of DNA methyltransferases, especially DNMT3b.