Elevated Glutathione Levels Confer Cellular Sensitization to Cisplatin Toxicity by Up-Regulation of Copper Transporter hCtr1

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 CDDPresistant 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 upregulation 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 platinumbased antitumor agents.

Cellular glutathione plays a key role in copper uptake mediated by human copper transporter 1

“…4B). Most of the reduction in the rate of copper uptake was observed with 25 M BSO, which reduced GSH to ϳ350 M. Contrary to a previous report (12), BSO treatment did not change the level of hCTR1 in the plasma membrane. In five independent experiments, plasma membrane levels of hCTR1 were unaltered by BSO treatment (Fig.…”

What do we know about the reduction of Pt(IV) pro-drugs?

“…Therefore, chloride ligands bound to Pt(II) are readily substituted by cysteines and methionines but require activation by aquation to bind to amines. passive diffusion but more recently it was shown that the copper transport hCtr1 receptor facilitates cisplatin uptake and oxaliplatin and pyriplatin, cis-[Pt(NH 3 ) 2 (py)Cl] + , can enter cells via the organic cation transporters [13][14][15][16][17][18]. In the bloodstream where the chloride levels are high (100 mM) cisplatin does not undergo aquation and the chlorido ligands remain bound to the platinum.…”

Mechanistic Comparison of Human High-Affinity Copper Transporter 1-Mediated Transport between Copper Ion and Cisplatin

“…The cross-linking reaction was incubated at 22°C for 30 min and stopped by the addition of 1 M Tris-HCl, pH 7.5, at a final concentration of 20 to 50 mM. After incubating for an additional 15 min, the protein was extracted for Western blotting analyses using anti-HA antibody or anti-hCtr1 antibody Cu and CDDP,IC 50 , and RNase protection assay using RNase RPA III Ribonuclease Protection Assay Kit (Applied Biosystems/Ambion, Austin, TX) have been described previously (Song et al, 2004Chen et al, 2008).…”