Structure and function of an active efflux pump for cisplatin

顺铂主动外排泵的结构和功能

• 批准号: 06807015
• 负责人: AKIYAMA Shinichi
• 金额: $ 1.22万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06807015/
• 关键词: Cisplatin-resistance; Leukotriene C4; GS-X pump; MRP; GS-X pump; 多剤耐性; 重金属; ロイコトリエンC4; GS-Xポンプ

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.

在从人表皮样癌KB-3-1细胞中分离的顺铂耐药KCP-4细胞中发现了顺式二氨二氯铂（II）（顺铂）的主动外排泵。在KCP-4细胞制备的膜囊泡中发现了三磷酸腺苷（adesnosine triphosphate，ATP）依赖的白三烯C_4（leukotriene C_4，LTC_4）转运。多药耐药相关蛋白（MRP）也被鉴定为ATP依赖的LTC_4转运蛋白。为了检测KCP-4细胞中表达的GS-X泵是否为MRP，我们研究了MRP在KCP-4细胞中的表达，并比较了KCP-4细胞中表达的GS-X泵与MRP的LTC_4转运活性。免疫印迹分析显示，在KB-3-1和KCP-4细胞制备的膜囊泡中未检测到MRP。[^3H]LTC_4在KCP-4和C-A120囊泡中有明显的ATP依赖性转运，而在KB-3-1和KB-C2囊泡中只有轻微的ATP依赖性转运。LTC_4在KCP-4和C-A120囊泡中的ATP依赖性转运呈饱和动力学，表观Km分别为0.445 μ M和1.55 μ M。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）（GS-platinum）络合物和谷胱甘肽二硫化物（GS-SG）对KCP-4囊泡中[^3H]LTC_4转运的抑制作用更强，而LTD_4对KCP-4囊泡中[^3H]LTC_4转运的抑制作用更弱。KCP-4囊泡中表达的LTC_4转运体的性质与MRP相似但不完全相同，提示顺铂耐药KCP-4细胞中存在不同于MRP的谷胱甘肽S-结合物输出泵。

项目成果

Fujii,R: "Adenosine triphosphate-dependent transport of leukotriene C4 by membrane vesicles prepared from cisplatin-resistant human eqidermoid carcinome tumor cells" J.Natl.Cancer Inst.86. 1781-1784 (1994)

Fujii,R：“由顺铂耐药的人类皮样癌肿瘤细胞制备的膜囊泡对白三烯 C4 的腺苷三磷酸依赖性转运”J.Natl.Cancer Inst.86。

Matsumoto,T.: "Effect of newly synthesized indole derivatives on multidrug resistance in human cancer cells." Anti-Cancer Drug Design. 9. 251-261 (1994)

Matsumoto,T.：“新合成的吲哚衍生物对人类癌细胞多药耐药性的影响。”

Tomoyuki Sumizawa: "Non-P-glycoprotein-mediated multidrug-resistant human KB cells selected in medium containing adriamycin, cepharanthine and mezerein." Somat.Cell.Mol.Genet.20. 423-435 (1994)

Tomoyuki Sumizawa：“在含有阿霉素、千金藤素和 mezerein 的培养基中选择的非 P-糖蛋白介导的多重耐药人 KB 细胞。”

Isamu Sugawara: "Preferential expression of the multidrug-resistance associated protein (MRP) in adenocarcinoma of the lung." Int.J.Cancer. 64. 322-325 (1995)

Isamu Sukawara：“多药耐药相关蛋白（MRP）在肺腺癌中优先表达。”

Sumizawa,T.: "Non-P-glycoprotein-mediated multidrug resistant human KB cells selected in medium containing adriamycin,cepharanthine,and mezerein." Somat.Cell Mol.Genet.20. 423-435 (1994)

Sumizawa,T.：“在含有阿霉素、千金藤素和 mezerein 的培养基中选择的非 P-糖蛋白介导的多重耐药人 KB 细胞。”