Holzer Alison K, Katano Kuniyuki, Klomp Leo W J, Howell Stephen B
Department of Medicine and the Rebecca and John Moores Cancer Center, University of California at San Diego, La Jolla, California 92093-0058, USA.
Clin Cancer Res. 2004 Oct 1;10(19):6744-9. doi: 10.1158/1078-0432.CCR-04-0748.
Cisplatin (DDP)-resistant cells commonly exhibit reduced drug accumulation. Previous studies have shown that the major copper (Cu) influx transporter CTR1 controls the uptake of DDP in yeast and mammalian cells. The goal of this study was to examine the effect of Cu and DDP on the level and subcellular localization of hCTR1 protein in human ovarian carcinoma cells.
Cultured human ovarian carcinoma A2780 cells were exposed to DDP and Cu, and the effect on hCTR1 was determined using Western blot analysis and confocal digital deconvolution microscopy.
Loss of hCTR1 was triggered by DDP exposure in a concentration and time-dependent manner. Exposure to 0.5 micromol/L DDP for 5 minutes reduced hCTR1 levels and exposure to DDP concentrations > or =2 micromol/L caused almost complete disappearance. The loss of hCTR1 was observed within 1 minute of the start of exposure to 2 micromol/L DDP. Treatment of cells with 100 micromol/L Cu for 5 minutes produced a smaller effect. Pretreatment of cells with 2 micromol/L DDP for 5 minutes resulted in a 50% decrease in 64Cu uptake, demonstrating that the DDP-induced loss of hCTR1 detected by Western blot analysis and imaging was functionally significant.
DDP down-regulated the amount of its major influx transporter in cultured human ovarian carcinoma cells in a concentration- and time-dependent manner. The effect was observed at DDP concentrations within the range found in the plasma of patients being treated with DDP, and it occurred very quickly relative to the half-life of the drug.
顺铂（DDP）耐药细胞通常表现出药物蓄积减少。既往研究表明，主要的铜（Cu）流入转运体CTR1控制酵母和哺乳动物细胞中DDP的摄取。本研究的目的是检测铜和DDP对人卵巢癌细胞中hCTR1蛋白水平和亚细胞定位的影响。
将培养的人卵巢癌A2780细胞暴露于DDP和铜，使用蛋白质印迹分析和共聚焦数字去卷积显微镜确定对hCTR1的影响。
DDP暴露以浓度和时间依赖性方式触发hCTR1缺失。暴露于0.5 μmol/L DDP 5分钟可降低hCTR1水平，暴露于≥2 μmol/L DDP浓度可导致几乎完全消失。在开始暴露于2 μmol/L DDP后1分钟内观察到hCTR1缺失。用100 μmol/L铜处理细胞5分钟产生较小影响。用2 μmol/L DDP预处理细胞5分钟导致64Cu摄取减少50%，表明蛋白质印迹分析和成像检测到的DDP诱导的hCTR1缺失具有功能意义。
DDP以浓度和时间依赖性方式下调培养的人卵巢癌细胞中其主要流入转运体的量。在接受DDP治疗患者血浆中发现的DDP浓度范围内观察到该效应，并且相对于药物半衰期而言发生非常迅速。
