Methylation status of breast cancer resistance protein detected by methylation‐specific polymerase chain reaction analysis is correlated inversely with its expression in drug‐resistant lung cancer cells

Abstract

Breast cancer resistance protein (BCRP) functions as a drug efflux transporter that mediates drug resistance. Topoisomerase I inhibitors, including 7-ethyl-10-hydroxycamptothecin (SN-38), are substrates effluxed by BCRP. However, it remains unclear whether the overexpression of BCRP induces drug resistance during chemotherapy. The objectives of the current study were to examine a correlation of altered promoter methylation of BCRP with BCRP expression and to investigate the correlation between methylation status according to methylation-specific polymerase chain reaction (MSP) analysis and BCRP expression levels in several small cell and nonsmall cell lung cancer cells. Non-BCRP-expressing PC-6 cells, which were sensitive to SN-38, were treated with DNA methyltransferase inhibitor to induce BCRP re-expression by means of reverse transcriptase-polymersae chain reaction, Western blot, and flow cytometric analyses. Subsequently, bisulfite sequencing analysis in both PC-6 cells and SN-38-resistant PC-6/SN2-5H, highly expressing BCRP cells was performed to identify the methylated region in the BCRP promoter. Finally, the authors established an MSP method on the basis of methylated and unmethylated DNA sequences. DNA methyltransferase inhibitor treatment of PC-6 cells induced BCRP re-expression at the messenger RNA and protein levels. Bisulfite sequencing analysis revealed that both alleles at all CpG sites were methylated completely in PC-6 cells, whereas alleles at portions of CpG sites in PC-6/SN2-5H cells were unmethylated. There was an inverse correlation between promoter methylation of BCRP determined by MSP and BCRP expression in both small cell and nonsmall cell lung cancer cells. The current results indicated that demethylation of at least 1 allele is necessary for BCRP re-expression and that promoter demethylation of BCRP may be a mechanism of BCRP expression in lung cancer cells.