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
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
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DOI：
10.1002/cncr.23285
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发表时间：
2008-03-01
期刊：
影响因子：
6.2
通讯作者：
Tsukamoto, Kazuhiro
中科院分区：
文献类型：
--
作者：
Nakano, Hirofumi;Nakamura, Yoichi;Tsukamoto, Kazuhiro
背景乳腺癌耐药蛋白（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7-乙基-10-羟基喜树碱（SN-38），是BCRP外排的底物。然而，目前尚不清楚BCRP的过度表达是否会导致化疗期间的耐药性。本研究的目的是检测BCRP启动子甲基化改变与BCRP表达的相关性，并研究甲基化特异性聚合酶链反应（MSP）分析的甲基化状态与几种小细胞和非小细胞肺癌细胞中BCRP表达水平之间的相关性。对SN-38敏感的不表达BCRP的PC-6细胞用DNA甲基转移酶抑制剂处理，通过逆转录酶聚合酶链反应、Western印迹和现在的细胞计数分析诱导BCRP重新表达。随后，在PC-6细胞和SN-38耐药PC-6/SN 2 - 5 H、高表达BCRP细胞中进行亚硫酸氢盐测序分析，以鉴定BCRP启动子中的甲基化区域。最后，作者建立了一个MSP方法的基础上甲基化和非甲基化的DNA序列。PC-6细胞的DNA甲基转移酶抑制剂处理诱导BCRP在信使RNA和蛋白质水平上重新表达。亚硫酸氢盐测序分析显示，在PC-6细胞中，所有CpG位点的两个等位基因均完全甲基化，而在PC-6/SN 2 - 5 H细胞中，部分CpG位点的等位基因未甲基化。MSP检测的BCRP启动子甲基化与BCRP在小细胞肺癌和非小细胞肺癌中的表达呈负相关。目前的研究结果表明，至少1个等位基因的去甲基化是BCRP重新表达所必需的，BCRP的启动子去甲基化可能是BCRP在肺癌细胞中表达的一种机制。
BACKGROUND. Breast cancer resistance protein (BCRP) functions as a drug efflux transporter that mediates drug resistance. Topoisomerase I inhibitors, including 7-ethyl-10-hydroxycampto thecin (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.METHODS. 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 now 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.RESULTS. 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.CONCLUSIONS. 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.