Choi Hoo-Kyun, Cho Kyoung Bin, Phuong Nguyen Thi Thuy, Han Chang Yeob, Han Hyo-Kyung, Hien Tran Thi, Choi Hong Seok, Kang Keon Wook
BK21 Project Team, College of Pharmacy, Chosun University , Gwangju 501-759, South Korea.
Mol Pharm. 2013 Jul 1;10(7):2517-27. doi: 10.1021/mp400287p. Epub 2013 Jun 13.
Our previous studies have shown that multidrug resistance protein 2 (MRP2) is overexpressed in tamoxifen-resistant MCF-7 breast cancer cells (TAMR-MCF-7 cells) and forkhead box-containing protein, O subfamily1 (FoxO1), functions as a key regulator of multidrug resistance 1 (MDR1) gene transcription. This study aimed to investigate the role of FoxO1 in regulating MRP2 gene expression in TAMR-MCF-7 cells. The proximal promoter region of the human MRP2 gene contains four putative FoxO binding sites, and MRP2 gene transcription was stimulated by FoxO1 overexpression in MCF-7 cells. Subcellular fractionation and immunoblot analyses revealed that basal MRP2 expression and nuclear levels of FoxO1 were enhanced in TAMR-MCF-7 cells compared to MCF-7 cells and the enhanced MRP2 gene transcription was suppressed by FoxO1 siRNA. Because nuclear localization of FoxO1 is regulated by SIRT1 deacetylase, we were further interested in whether SIRT1 is involved in MRP2 expression. Overexpression of SIRT1 with FoxO1 potentiated the gene transcriptional activity of MRP2, and the basal activity and expression of SIRT1 was increased in TAMR-MCF-7 cells. In addition, SIRT1 inhibition reduced both the nuclear FoxO1 levels and MRP2 expression and enhanced cytotoxic effects of paclitaxel and doxorubicin in TAMR-MCF-7 cells. These results suggest that FoxO1 activation via SIRT1-mediated deacetylation is closely related with up-regulation of MRP2 in TAMR-MCF-7 cells.
我们之前的研究表明，多药耐药蛋白2（MRP2）在他莫昔芬耐药的MCF-7乳腺癌细胞（TAMR-MCF-7细胞）中过表达，含叉头框蛋白O亚家族1（FoxO1）作为多药耐药1（MDR1）基因转录的关键调节因子。本研究旨在探讨FoxO1在调节TAMR-MCF-7细胞中MRP2基因表达中的作用。人MRP2基因的近端启动子区域包含四个假定的FoxO结合位点，并且在MCF-7细胞中FoxO1过表达可刺激MRP2基因转录。亚细胞分级分离和免疫印迹分析显示，与MCF-7细胞相比，TAMR-MCF-7细胞中基础MRP2表达和FoxO1的核水平增强，并且增强的MRP2基因转录被FoxO1 siRNA抑制。由于FoxO1的核定位受SIRT1脱乙酰酶调节，我们进一步感兴趣的是SIRT1是否参与MRP2表达。SIRT1与FoxO1共过表达增强了MRP2的基因转录活性，并且TAMR-MCF-7细胞中SIRT1的基础活性和表达增加。此外，SIRT1抑制降低了核FoxO1水平和MRP2表达，并增强了紫杉醇和阿霉素对TAMR-MCF-7细胞的细胞毒性作用。这些结果表明，通过SIRT1介导的去乙酰化激活FoxO1与TAMR-MCF-7细胞中MRP2的上调密切相关。
