Fujii R, Mutoh M, Sumizawa T, Chen Z S, Yoshimura A, Akiyama S
Department of Cancer Chemotherapy, Faculty of Medicine, Kagoshima University, Japan.
J Natl Cancer Inst. 1994 Dec 7;86(23):1781-4. doi: 10.1093/jnci/86.23.1781.
Cisplatin accumulation is decreased in many cisplatin-resistant cells. An active efflux pump for cisplatin exists in cisplatin-resistant human epidermoid carcinoma cells (called KB cells). A previous study has suggested that the adenosine triphosphate (ATP)-dependent glutathione S-conjugate export pump (GS-X pump), which exports the bis-(glutathionato)-platinum (II) (GS-platinum) complex, could contribute to cellular resistance to cisplatin.
In this study, we examined whether the active efflux pump for cisplatin in the cisplatin-resistant KB cells is the GS-X pump and tested its activity by using an endogenous substrate, [3H]leukotriene C4 ([3H]LTC4).
Membrane vesicles were prepared from KB-3-1 (clone from parental KB cells) cells and from cisplatin-resistant KCP-4 (a mutant clone derived from KB-3-1 cells) cells. Using a filtration technique, we measured the uptake and transport of [3H]LTC4, a substrate for the GS-X pump, into membrane vesicles at 37 degrees C.
The uptake of [3H]LTC4 in the membrane vesicles from both the KB-3-1 and KCP-4 cells was ATP-dependent. In contrast, the ATP-dependent transport of [3H]LTC4 was observed only in KCP-4 membrane vesicles but not in KB-3-1 membrane vesicles. The ATP-dependent transport was vanadate sensitive and was inhibited by GS-platinum complex but only marginally by cisplatin and glutathione and not by vincristine or verapamil. The nucleotide triphosphates, guanosine triphosphate, cytidine triphosphate, uridine triphosphate, and deoxythymidine triphosphate could be substituted for ATP but were less efficient. A nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene) triphosphate, was not effective.
The transport of LTC4 in membrane vesicles prepared from KCP-4 cells was facilitated by an ATP-dependent pump that appeared very similar to the GS-X pump.
Our study suggests that the GS-X pump is involved in the decreased accumulation of cisplatin in KCP-4 cells.
在许多顺铂耐药细胞中，顺铂的蓄积减少。在顺铂耐药的人表皮样癌细胞（称为KB细胞）中存在一种顺铂的活性外排泵。先前的一项研究表明，依赖三磷酸腺苷（ATP）的谷胱甘肽S-共轭物输出泵（GS-X泵）可输出双（谷胱甘肽）铂（II）（GS-铂）复合物，这可能导致细胞对顺铂产生耐药性。
在本研究中，我们检测了顺铂耐药KB细胞中顺铂的活性外排泵是否为GS-X泵，并通过使用内源性底物[3H]白三烯C4（[3H]LTC4）来测试其活性。
从KB-3-1（亲本KB细胞的克隆）细胞和顺铂耐药的KCP-4（源自KB-3-1细胞的突变克隆）细胞制备膜囊泡。采用过滤技术，我们在37℃下测量了GS-X泵的底物[3H]LTC4进入膜囊泡的摄取和转运。
KB-3-1细胞和KCP-4细胞的膜囊泡中[3H]LTC4的摄取均依赖于ATP。相比之下，仅在KCP-4膜囊泡中观察到[3H]LTC4的ATP依赖性转运，而在KB-3-1膜囊泡中未观察到。ATP依赖性转运对钒酸盐敏感，且受到GS-铂复合物的抑制，但仅受到顺铂和谷胱甘肽的轻微抑制，不受长春新碱或维拉帕米的抑制。三磷酸核苷酸，即三磷酸鸟苷、三磷酸胞苷、三磷酸尿苷和三磷酸脱氧胸苷可替代ATP，但效率较低。一种不可水解的ATP类似物，腺苷5'-（β，γ-亚甲基）三磷酸无效。
KCP-4细胞制备的膜囊泡中LTC4的转运由一种ATP依赖性泵促进，该泵似乎与GS-X泵非常相似。
我们的研究表明，GS-X泵参与了KCP-4细胞中顺铂蓄积的减少。
