| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
Breast cancer resistant protein (BCRP) functions as a drug efflux transporter that mediates drug resistance. Topoisomerase I inhibitors including SN-38 (an active metabolite of irinotecan) are substrates effluxed by BCRP. The underlying mechanisms for overexpression of BCRP during acquisition of drug resistance remain unclear. Then, treatment of non-BCRP-expressing small-cell lung cancer cells, PC-6, with DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, induced BCRP re-expression at the mRNA and protein levels. Bisulfite sequencing analysis and subsequent comparisons between bisulfite-modified and unmodified DNA sequences at CpG sites in the promoter region of BCRP revealed that both alleles at all 89 CpG sites were completely methylated in PC-6, while those at 79 CpG sites in BCRP-expressing resistant small-cell lung cancer cells, PC-6/SN2-5H, were unmethylated. These results indicate a correlation between demethylation of the promoter region of BCRP and BCRP re-expression, an
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d suggest one of mechanisms for SN-38 drug resistance.
Next, a methylation-specific PCR (MSP) method for BCRP was established based on the sequences of BCRP in the methylated and methylated regions. MSP method revealed an inverse correlation between methylation status of the promoter region of BCRP and expression of BCRP mRNA in several lung cancer cell lines (in vitro) and lung and colorectal cancers resected on operation (in vivo).
As a cohort study, an association of methylation status of the promoter region of BCRP using MSP for biopsy specimens of lung cancer patients before chemotherapy with the responsibility for anticancer drugs was examined. This DNA-based diagnosis revealed that the sensitivity was 83.3% and the specificity was 77.8%. This study implies that it is very useful as a new biomarker in predicting drug resistance and responsibility for anticancer drugs to examine methylation status of the promoter region of BCRP using MSP for not only lung cancers, but also colorectal cancers, gastric cancers, breast cancers, ovarian tumors, and choriocarcinomas. Less
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