Structure and function of an active efflux pump for cisplatin

从人表皮样癌KB-3-1细胞中分离的耐顺铂的KCP-4细胞中发现了顺铂的主动外排泵。在KCP-4细胞制备的膜泡中，发现谷胱甘肽-S共轭输出泵(GS-X泵)的内源性底物白三烯C_4(LTC_4)的转运依赖于腺苷三磷酸。多药耐药相关蛋白(MRP)也被认为是一种依赖于ATP的LTC_4转运蛋白。为了验证在KCP-4细胞中表达的GS-X泵是否是MRP，我们研究了MRP在KCP-4细胞中的表达，并比较了在KCP-4细胞中表达的GS-X泵与MRP的LTC_4转运活性。免疫印迹法检测KB-3-1和KCP-4细胞的膜泡均未检测到MRP。KCP-4和C-A120囊泡对[~3H]LTC_4的转运依赖于ATP，而KB-3-1和KB-C2囊泡的转运较弱。LTC_4在KCP-4和C-A120囊泡中的转运呈饱和动力学，表观Km分别为0.445和1.55µM。与C-A120囊泡相比，2，4-二硝基苯基-S-谷胱甘肽(DNP-SG)、双(谷胱甘肽)-铂(II)络合物(GS-白金)和谷胱甘肽二硫化物(GS-SG)对KCP-4囊泡LTC_4转运的抑制作用更强，而LTD_4的抑制作用更小。在KCP-4囊泡中表达的LTC4转运蛋白的性质与MRP相似但不完全相同。我们的结果表明，在顺铂耐药的KCP-4细胞中存在不同于MRP的谷胱甘肽S偶联输出泵。

An active efflux pump for cis-diamminedichloroplatinum (II) (cisplatin) has been identified in cisplatin-resistant KCP-4 cells isolated from human epidermoid carcinoma KB-3-1 cells. The adesnosine triphosphate (ATP) -dependent transport of leukotriene C_4 (LTC_4) , an endogenous substrate for the glutathione S-conjugate export pump (GS-X pump), has been found in membrane vesicles prepared from KCP-4 cells. Multidrug resistance-associated protein (MRP) has also been identified as an ATP-dependent LTC_4 transporter. To examine whether the GS-X pump expressed in KCP-4 cells is MRP,we investigated the expression of MRP in KCP-4 cells and compared the LTC_4 transporting activity of GS-X pump expressed in KCP-4 cells with that of MRP.The level of MRP gene expression in KCP-4 cells was low and similar to that in KB-3-1 cells. MRP was not detected in membrane vesicles prepared from KB-3-1 and KCP-4 cells by immunoblotting analysis. The ATP-dependent transport of [^3H]LTC_4 was apparent in KCP-4 amd C-A120 vesicles but only slight in KB-3-1 and KB-C2 vesicles. The ATP-dependent transport of LTC_4 in KCP-4 and C-A120 vesicles showed saturable kinetics with an apparent Km of 0.445muM and 1.55muM,respectively. [^3H]LTC_4 transport in KCP-4 vesicles was more inhibited by 2,4-dinitrophenyl-S-glutathione (DNP-SG), bis- (glutathionato) -platinum (II) (GS-platinum) complex and glutathione disulfide (GS-SG) and less by LTD_4 compared with that in C-A120 vesicles. The character of the LTC_4 transporter expressed in KCP-4 vesicles is similar but not identical to that of MRP.Our results suggest that a glutathione S-conjugate export pump which is different from MRP exists in cisplatin-resistant KCP-4 cells.