Functional Interactions of Cu-ATPase ATP7B with Cisplatin and the Role of ATP7B in the Resistance of Cells to the Drug

Abstract

Cisplatin is a widely used chemotherapeutic agent for treatment of ovarian, testicular, lung, and stomach cancers. The initial response to the drug is robust; however, tumor cells commonly develop resistance to cisplatin, which complicates treatment. Recently, overexpression of the Cu-ATPase ATP7B in ovary cells was linked to the increased cellular resistance to cisplatin; and the role for Cu-ATPases in the export of cisplatin from cells was proposed. Our results support functional interactions between cisplatin and ATP7B but argue against the active transport through the copper translocation pathway as a mechanism of drug resistance. In hepatocytes, we observed no correlation between the levels of endogenous ATP7B and the resistance of cells to cisplatin. Unlike copper, cisplatin does not induce trafficking of ATP7B in hepatoma cells, neither does it compete with copper in a transport assay. However, cisplatin binds to ATP7B and stimulates catalytic phosphorylation with EC 50 similar to that of copper. Mutations of the first five N-terminal copper-binding sites of ATP7B do not inhibit the cisplatin-induced phosphorylation of ATP7B. In contrast, the deletion of the first four copper-binding sites abolishes the effect of cisplatin on the ATP7B activity. Thus, cisplatin binding to ATP7B and/or general changes in cellular copper homeostasis are likely contributors to the increased resistance to the drug. The link between changes in copper homeostasis and cisplatin resistance was confirmed by treating the Huh7 cells with copper chelator and increasing their resistance to cisplatin. Cisplatin, cis-diamminedichloroplatinum (DDP),3 is a common anti-tumor agent that is used to treat many types of cancer. It is especially prescribed for testicular, ovarian, bladder, liver, lung, and stomach cancers (1-5). DDP mediates its cytotoxic effects by binding to DNA, forming the intrastrand crosslinks and thus causing an inhibition of DNA synthesis and repair with eventual cell death (6, 7). The initial tumor response to the treatment with DDP is robust; however, the efficacy of treatment decreases with longer and repetitive therapy cycles. The resistance arises rapidly and is sufficient to cause a failure of DDP therapy (8). The mechanisms by which cells develop resistance to DDP are not fully understood. Detoxification of DDP, enhanced repair and tolerance of DNA adducts, inhibition of apoptosis, impaired uptake and increased efflux of the drug may contribute to the acquired resistance of cells (9 -13).Recently, an unexpected connection was discovered between the resistance of cells to DDP and cellular copper metabolism (14 -16). Either down-regulation of CTR1 (49), a transporter responsible for the uptake of copper, and/or up-regulation of the copper-transporting ATPases (Cu-ATPases) responsible for copper efflux were found to increase cells resistance to DDP, although correlation between the levels of CTR1 and resistance were not always observed (17). The DDP-resistant cells were shown to have a lower copper content (18), and...

Cellular copper levels determine the phenotype of the Arg 875 variant of ATP7B/Wilson disease protein

“…S1B). In vitro, both the Arg-and Gly-ATP7B variants hydrolyze ATP with the formation of a phosphorylated intermediate (5,6). This observation is consistent with the designation of c2623A/G as a polymorphism, which was made in genetic studies of the Han Chinese population (7).…”

“…Although the structural basis for this increase is not yet known, higher stability of ATP7B-Arg 875 in copper-treated cells suggests that interdomain interactions impart extra rigidity to the protein. In vitro, copper binding to copper-ATPases stabilizes a distinct protein conformation (5,19). Thus, we speculate that copper-induced restriction of conformational mobility allows the ATP7B-Arg 875 to exit from the ER, the initial key step in overcoming the disease phenotype.…”

“…Finally, we tested whether drugs that mimic the effect of copper on ATP7B can influence ATP7B-Arg 875 localization and activity. We have previously found that a chemotherapeutical drug, DDP, binds to N-ATP7B (16,17) and stabilizes the copperbound-like state of ATP7B without competing with copper for transport sites (5). Therefore, we examined whether DPP would trigger ATP7B-Arg 875 trafficking to the TGN and restore copper delivery to tyrosinase in the absence of copper additions.…”

Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

“…The relationship between cisPt and Cu transporters is underscored by recent observations that cisPt therapy combined with siRNA-mediated ATP7B silencing significantly reduces tumor growth in a mouse ovarian cancer model (42). Recent in vitro data have suggested that there are direct interactions between cisPt and the metalbinding domains of ATP7B (8,22) but molecular details are lacking. Two different crystal structures of apo-Atox1 mixed with cisPt were reported last year, implying that Pt binds to the Cu site cysteines (24).…”

“…Previous work has shown that apoand Cu-loaded forms of Atox1 have similar ferredoxin-like folds apart from increased conformational dynamics in the Cu-binding loop in the holoform (21). Last year, using bacterial and hepatocytes/hepatoma cell assays, two studies reported that cisPt could bind to the MBDs of ATP7B, although it was unclear where in the polypeptide the binding site(s) were located (8,22).…”