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 CTR1 alleles 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 (64)Cu and [(14)C]carboplatin (CBDCA) accumulation by gamma and scintillation counting.

Results: Deletion of CTR1 reduced 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.

Fig. 1

Relative CTR2 mRNA and protein levels in parental and knockdown cells measured by qRT-PCR andWestern blot analysis. Relative CTR2 mRNA (A) and protein (B) levels in CTR1^+/+/CTR2^+/+ and CTR1^+/+/CTR2^kd cells. Relative CTR2 mRNA (C) and protein (D) levels in CTR1^−/−/CTR2^+/+ and CTR1^−/−/CTR2^kd cells. Bars, SE.

Fig. 2

Inhibition of growth as a function of concentration. A, DDP. B, CBDCA. C, Cu. Bars, SE.

Fig. 3

Whole-cell accumulation of DDP in CTR1^+/+/CTR2^+/+, CTR1^+/+/CTR2^kd, CTR1^−/−/CTR2^+/+, and CTR1^−/−/CTR2^kd cells. Whole-cell Pt accumulation following 5-min (A) and 1-h (B) exposure to 30 μmol/L DDP as measured by ICP-MS. C, whole-cell [¹⁴C]CBDCA accumulation following 1-h exposure to 50 μmol/L [¹⁴C]CBDCA as measured by scintillation counting. D, whole-cell ⁶⁴Cu accumulation following 1-h exposure to 2 μmol/L ⁶⁴CuSO₄ as measured by γ counting. Bars, SE.

Fig. 4

Pt accumulation in DNA and vesicles following exposure to DDP. DNA Pt (A) and vesicle accumulation (B) following 1-h exposure to 30 μmol/L DDP as measured by ICP-MS. C, Pt content as a function of efflux time. Bars, SE.

Fig. 5

Relationship between CTR2 expression and DDP sensitivity in ovarian carcinoma cell lines. A, Western blot analysis of CTR2 in A2780, HEY, KF 28, 2008, and IGROV-1 cells. B, correlation between CTR2 mRNA levels and DDP IC₅₀ of the six ovarian carcinoma cell lines as measured by qRT-PCR (r² = 0.97, P = 0.0003). C, correlation between CTR2 protein levels and IC₅₀ of the six ovarian carcinoma cell lines as determined byWestern blot analysis (r² = 0.71, P < 0.04).