Copper Transporter 2 Regulates the Cellular Accumulation and Cytotoxicity of Cisplatin and Carboplatin

Abstract

Purpose: Copper transporter 2 (CTR2) is known to mediate the uptake of Cu +1 by mammalian cells. Several other Cu transporters, including the influx transporter CTR1 and the two efflux transporters ATP7A and ATP7B, also regulate sensitivity to the platinum-containing drugs. We sought to determine the effect of CTR2 on influx, intracellular trafficking, and efflux of cisplatin and carboplatin. Experimental Design: The role of CTR2 was examined by knocking down CTR2 expression in an isogenic pair of mouse embryo fibroblasts consisting of a CTR1 +/+ line and a CTR1 -/-line in which both CTR1alleles had been deleted. CTR2 levels were determined by quantitative reverse transcription-PCR and Western blot analysis. Cisplatin (DDP) was quantified by inductively coupled plasma mass spectrometry and Results: Deletion of CTR1reduced the uptake of Cu, DDP, and CBDCA and increased resistance to their cytotoxic effects by 2-to 3-fold. Knockdown of CTR2 increased uptake of Cu only in the CTR1 +/+ cells. In contrast, knockdown of CTR2 increased whole-cell DDP uptake and DNA platination in both CTR1+/+ and CTR1 -/-cells and proportionately enhanced cytotoxicity while producing no effect on vesicular accumulation or efflux. A significant correlation was found between CTR2 mRNA and protein levels and sensitivity to DDP in a panel of six ovarian carcinoma cell lines. Conclusions: CTR2 is a major determinant of sensitivity to the cytotoxic effects of DDP and CBDCA. CTR2 functions by limiting drug accumulation, and its expression correlates with the sensitivity of human ovarian carcinoma cell lines to DDP.Platinum-based chemotherapeutic agents have been among the most widely used and effective anticancer agents since the 1970s (1). However, patients often develop resistance during the course of treatment (2 -7) and the mechanisms that account for such resistance have not been fully identified. Changes in drug influx and efflux, deficiencies in the mismatch repair pathway, and down-regulation of the apoptotic cascade have all been proposed (8 -12). To complicate matters, little is known about the intracellular metabolism of platinum-based agents. It is believed that these drugs kill cells through formation of adducts on the purine bases of nuclear DNA (9,13,14); however, it is not clear how the drugs traffic through the cell to reach the nucleus. There is a strong correlation between drug sensitivity and drug accumulation, with DDPresistant cells uniformly accumulating less drug (4, 15 -21).Several studies have provided evidence that the platinumcontaining drugs are taken up, shuttled around the cell, and exported by transporters and chaperones belonging to the Cu homeostasis system. Cells selected for resistance to cisplatin (DDP) are cross-resistant to . CTR1 is a high-affinity plasma membrane Cu transporter. Cu associates with the hCTR1 metal binding motif in the NH 2 -terminal domain and is transported into the cytosol through a channel formed by the hCTR1 trimer (26,27). Cu is then handed off to the various chaperones to...

CBP501-Calmodulin Binding Contributes to Sensitizing Tumor Cells to Cisplatin and Bleomycin

“…The samples were then diluted with aqua regia (4% v/v), and the platinum concentration was measured by inductively coupled plasma mass spectroscopy analysis. To detect the concentration of DNA-platinum adducts, genomic DNA was isolated from the cell lines by methods previously described (14).…”

Hyperthermic potentiation of cisplatin by magnetic nanoparticle heaters is correlated with an increase in cell membrane fluidity

“…[14][15][16][17] Although some groups report distinct molecular behaviors of the different members of the hCTR family, which appear to be cell-dependent, all reports agree that copper, due to its affinity with the hCTR receptor, affects receptormediated entry of cDDP negatively, resulting in improved cytotoxicity. 9,[18][19][20][21][22][23] Although copper receptors have a critical role in the uptake of cisplatin, one of the first studies that focused on the mechanisms of thermal enhancement of cDDP reported an increase in cell membrane fluidity, which in turn augmented the uptake of cDDP. 17,24,25 Therefore, the synergism of cDDP and hyperthermic treatment can potentially be explained in the context of membrane fluidity.…”

“…[9][10][11][12][13][14][19][20][21][22][23][24] It is widely accepted as the primary mechanism of influx of this platinum-based drug in cells. 10,11 To assess the effect of extracellular copper on this form of drug transport, the surviving fraction and viability ratio of Caco-2 cells was studied by exposing cells to a mixture of 5 µM copper and increasing concentrations of cDDP for 2.5 hours.…”

Sec61β Controls Sensitivity to Platinum-Containing Chemotherapeutic Agents through Modulation of the Copper-Transporting ATPase ATP7A

“…CTR1 and CTR2 regulate uptake, whereas ATP7A and ATP7B are involved in intracellular sequestration and drug export. Chaperones such as ATOX1 may transport the drugs between intracellular sites (Holzer et al, 2003;Samimi et al, 2004a;Safaei et al, 2007;Blair et al, 2009).…”