Smitherman Pamela K, Townsend Alan J, Kute Timothy E, Morrow Charles S
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
J Pharmacol Exp Ther. 2004 Jan;308(1):260-7. doi: 10.1124/jpet.103.057729. Epub 2003 Oct 20.
Our previous studies have shown that the glutathione S-transferases (GSTs) can operate in synergy with the efflux transporter multidrug resistance protein 1 (MRP1, ABCC1) to confer resistance to the cyto- and genotoxicities of some anticancer drugs and carcinogens. The current study was designed to determine whether the alternative efflux transporter, MRP2 (ABCC2), can also potentiate GST-mediated detoxifications in HepG2 cells. HepG2 cells, which express high-level MRP2 but not MRP1, were stably transduced with GST expression vectors under tetracycline-repressible transcriptional control. MRP2 was able to support GSTA1-1-mediated resistance to chlorambucil (CHB) cytotoxicity in HepG2 cells. Resistance was GST isozyme-specific in that GSTP1a-1a and GSTM1a-1a failed to confer protection from CHB toxicity. Moreover, inhibition of MRP2 with sulfinpyrazone completely reversed GSTA1-1-associated resistance, indicating that MRP2-efflux function is required to potentiate GSTA1-1-mediated resistance. Relative transport by MRP1 versus MRP2 of monoglutathionyl-CHB (CHB-SG) was examined using inside-out plasma membrane vesicles derived from MCF7 cells transduced with MRP1 or MRP2 expression vectors. Both MRP1 and MRP2 transported CHB-SG efficiently, at the levels of protein expressed, with similar Vmax and with Km of 0.39 and 10 microM, respectively. We conclude that detoxification of CHB by GSTA1-1 requires the removal of the glutathione conjugate formed and that either MRP1 or MRP2 can serve this efflux function. These findings have implications for the role of MRP2 in detoxification of alkylating agents in the apical epithelium of liver and kidney where it is highly expressed as well as the role of MRP2 in the emergence of alkylating drug resistance in cancer cells.
我们之前的研究表明,谷胱甘肽S-转移酶(GSTs)可与外排转运蛋白多药耐药相关蛋白1(MRP1,ABCC1)协同作用,赋予细胞对某些抗癌药物和致癌物的细胞毒性及遗传毒性的抗性。本研究旨在确定另一种外排转运蛋白MRP2(ABCC2)是否也能增强HepG2细胞中GST介导的解毒作用。HepG2细胞高水平表达MRP2但不表达MRP1,在四环素可抑制的转录控制下用GST表达载体进行稳定转导。MRP2能够支持GSTA1-1介导的HepG2细胞对苯丁酸氮芥(CHB)细胞毒性的抗性。抗性具有GST同工酶特异性,因为GSTP1a-1a和GSTM1a-1a未能赋予对CHB毒性的保护作用。此外,用磺吡酮抑制MRP2可完全逆转与GSTA1-1相关的抗性,表明MRP2的外排功能是增强GSTA1-1介导的抗性所必需的。使用由转导了MRP1或MRP2表达载体的MCF7细胞衍生的外翻质膜囊泡,检测了MRP1与MRP2对单谷胱甘肽化-CHB(CHB-SG)的相对转运。在表达的蛋白水平上,MRP1和MRP2均能高效转运CHB-SG,Vmax相似,Km分别为0.39和10 microM。我们得出结论,GSTA1-1对CHB的解毒作用需要去除形成的谷胱甘肽共轭物,并且MRP1或MRP2均可发挥这种外排功能。这些发现对MRP2在肝脏和肾脏顶端上皮中高表达的烷基化剂解毒作用中的作用以及MRP2在癌细胞中烷基化药物抗性出现中的作用具有重要意义。