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. 2008 Sep;74(3):697-704.
doi: 10.1124/mol.108.047969. Epub 2008 Jun 3.

Elevated glutathione levels confer cellular sensitization to cisplatin toxicity by up-regulation of copper transporter hCtr1

Helen H W Chen  1 , Im-Sook Song, Anwar Hossain, Min-Koo Choi, Yoshiaki Yamane, Zheng D Liang, Jia Lu, Lily Y-H Wu, Zahid H Siddik, Leo W J Klomp, Niramol Savaraj, Macus Tien Kuo
Affiliations

Elevated glutathione levels confer cellular sensitization to cisplatin toxicity by up-regulation of copper transporter hCtr1

Helen H W Chen et al. Mol Pharmacol. 2008 Sep.

Abstract

Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) up-regulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit. It has also been shown that many CDDP-resistant cell lines exhibit high levels of GCLC/GCLM and GSH. Because the GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into consideration that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with an expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through up-regulation of human copper transporter (hCtr) 1, which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. Although rates of copper transport were also up-regulated in the transfected cells, these cells exhibited biochemical signature of copper deficiency, suggesting that GSH functions as an intracellular copper-chelator and that overexpression of GSH can alter copper metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes the bioavailable copper pool, leading to up-regulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications in that modulation of the intracellular copper pool may be a novel strategy for improving chemotherapeutic efficacy of platinum-based antitumor agents.

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Figures

Fig. 1
Fig. 1
Measurements of sensitivities to CDDP (A) and Cu (B) and uptake of CDDP (C) and Cu (D) in SR3A and the GCLC-transfected (SR3A-13, SR3A-14, and SR3A-15) cell lines. Measurements of sensitivities to CDDP and CuSO4, and rates of uptakes of CDDP and 64Cu are described in Experimental procedures. Each bar represents mean ± S.D. *: P<0.01, significantly different from SR3A cells when tested by Student’ t-test.
Fig. 2
Fig. 2
Analyses of the expression of various copper transporters in SR3A and GCLC-transfected variants. Messenger RNA (A) and protein (B, C, D) levels of hCtr1, ATP7A, and ATP7B were determined by the RNase protection assay and by immunoblots using 18S RNA and β-actin as loading controls, respectively. Numbers underneath are fold increased estimated by phosphimager analyses. Panel D, Analysis of hCtr1 expression in AdE1.tTA.GCLC-transduced SR3A cells. The cells were treated with recombinant adenovirus at 50 MOI for 24 and 48 hr as indicated either in the presence (+) or absence (-) of 1 μg/ml tet. Total cell lysates were prepared and probed by using anti-GCLC, anti-hCtr1, and anti-β-actin antibodies.
Fig. 3
Fig. 3
hCtr1 knockdown decreases sensitivity against CDDP. A. Cell extracts prepared from cells treated with hCTR1-specific siRNA (100 nM), or mock, scrambled siRNA, and were subjected to 12% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and immunoblotted by anti-hCtr1-specific antibody. Anti-β-actin antibody was used as a loading control. Bands were visualized with SuperSignal West Femto western-blot detection kit. B. Reduction of sensitivity of GCLC-transfected cells to CDDP by knockdown of hCtr1. All results are expressed as the means ± SD of the results of at least three experiments.
Fig. 4
Fig. 4
Measurements of biochemical signature for Cu availability in the GCL- transfected cells either in the presence of absence of BSO. (A) SOD1 activities; (B) CCO activities; and (C) western blotting analysis of ceruloplasmin. Numbers in Panel C denote the relative band intensity determined by phosphoimerger. *: P<0.05, significantly different from SR3A cells when tested by Student’ t-test.
Fig. 5
Fig. 5
Determinations of the effects of BSO treatments on the cytotoxicity of CDDP (A) and Cu (B), and rates of uptake of CDDP (C) and Cu (D), expression of hCtr1, ATP7A, and ATP7B by western blots (E) and hCtr1. For cytotoxicity assays, cells grown in 96-well plates (104 cells/well) were continuously exposed to various concentrations of copper and CDDP in the presence or absence of 100 μM BSO. After 72 hr incubation, cytotoxicity was measured by MTT assay. The IC50 value (μM) was calculated by the Hill plot method with linear regression. * p<0.01, significantly different from SR3A cells. #: p<0.01, significantly different from SR3A cell in the presence of BSO.
Fig. 6
Fig. 6
Immunofluorescence staining in SR3A and GCLC-transfected cells. SR3A, SR3A-13, SR3A-14, SR3A-15 treated with or without BSO were stained with anti-hCtr1 anti-hCtr1 antibody and counterstained with DAPI for nucleus. Fluorescence signals were viewed by an Nikon Eclipse TE2000 Confocal Microscope.
Fig. 7
Fig. 7
Schematic illustration showing the effects of GCLC overexpression on cellular Cu metabolism. Overexpression of GCLC, which catalyzes the ligation of cysteine (Cys) and glutamate (Glu), results in increased GSH levels. Excess GSH functions as a Cu depletor, as evidenced by the reduction of CCO and SOD activity, and holo-ceruloplasmin (Cu-Cp) contents. Intracellular Cu deficiency upregulates hCtr1 expression resulting in elevated sensitivity to CDDP treatment. CCS, HAH1, and COX17 are Cu chaperones that shuffle Cu to their respective targets as indicated by arrows.
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