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DNA-based diagnosis on responsibility for anticancer drugs by methylation-specific PCR for BCRP

通过 BCRP 甲基化特异性 PCR 进行基于 DNA 的抗癌药物责任诊断

基本信息

批准号：
16590437
负责人：
TSUKAMOTO Kazuhiro
金额：
$ 2.3万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590437/
关键词：
Breast cancer resistance protein : BCRP Methylation Methylation-specific PCR Prediction for responsibility of anticancer drugs DNA-based diagnosis Irinotecan : CPT-11 Lung cancer Colorectal cancer methylation-specific PCR(MSP)塩酸イリノテカンCP

项目摘要

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 … More 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

乳腺癌耐药蛋白（BCRP）作为一种药物外排转运蛋白，介导耐药性。拓扑异构酶I抑制剂包括SN-38（伊立替康的活性代谢产物）是BCRP外排的底物。获得耐药性过程中BCRP过度表达的潜在机制尚不清楚。然后，用DNA甲基转移酶抑制剂5-氮杂-2 '-脱氧胞苷处理不表达BCRP的小细胞肺癌细胞PC-6，诱导BCRP在mRNA和蛋白水平上重新表达。亚硫酸氢盐测序分析和随后在BCRP启动子区CpG位点处亚硫酸氢盐修饰和未修饰DNA序列之间的比较显示，PC-6中所有89个CpG位点处的两个等位基因均完全甲基化，而表达BCRP的耐药小细胞肺癌细胞PC-6/SN 2 - 5 H中79个CpG位点处的等位基因未甲基化。这些结果表明BCRP启动子区的去甲基化和BCRP再表达之间存在相关性， ...更多信息其次，根据BCRP甲基化和甲基化区域的序列，建立了BCRP甲基化特异性PCR（MSP）检测方法。MSP法显示BCRP基因启动子区甲基化状态与BCRP mRNA表达在几种肺癌细胞系中呈负相关（体外）和手术切除的肺癌和结肠直肠癌（体内）。使用MSP检测肺癌患者化疗前活检标本中BCRP启动子区甲基化状态与抗癌药物的相关性，考察以DNA为基础的诊断敏感性为83.3%，特异性为77.8%。这项研究表明，它是非常有用的，作为一种新的生物标志物，在预测耐药性和抗癌药物的责任，检查甲基化状态的BCRP启动子区使用MSP不仅肺癌，但结直肠癌，胃癌，乳腺癌，卵巢肿瘤，绒毛膜癌。少