Regulation of the high-affinity copper transporter (hCtr1) expression by cisplatin and heavy metals
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Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.
KeywordsCisplatin High-affinity copper transporter Sp1 Copper homeostasis Platinum drug resistance
Cytochrome c oxidase
High-affinity copper transporter 1
Human high-affinity copper transporter 1
RNase protection assay
Small cell lung cancer
Small interfering RNA
Superoxide dismutase 1
Platinum-based drugs have been the mainstay of cancer chemotherapy for a broad spectrum of human malignancies for the last three decades [1, 2]. However, resistance to these drugs has been an obstacle to their effective use [3, 4, 5]. Although many mechanisms have been described for platinum drug resistance, a well-recognized and important mechanism of resistance is the reduced transport or enhanced efflux (or both) of cellular platinum drugs [6, 7].
Multiple mechanisms are involved in platinum drug transport. Cisplatin—cis-[PtCl2(NH3)2]—may enter cells by means of passive diffusion or endocytosis [3, 8, 9] and by high-affinity copper transporter 1 (Ctr1). The involvement of Ctr1 in cisplatin transport was initially demonstrated using yeast genetics which showed that deletion of CTR1 resulted in impaired cisplatin transport and cisplatin resistance [10, 11]. In a Ctr1-knockout murine embryonic fibroblast model, although Ctr1−/− cells accumulated only 5.7 % of the amount of copper that Ctr1+/+ cells accumulated during 1 h exposure to 2 μM copper, the amount of cisplatin accumulated in these Ctr1−/− cells was 35–36 % of that accumulated in Ctr1+/+ cells . Moreover, cisplatin-resistant human cell lines exhibited reduced cisplatin contents, and the resistance was restored when CTR1 was introduced into these cells [12, 13, 14]. These results demonstrated that Ctr1 plays an important role in cisplatin resistance.
In clinical studies, the human Ctr1 (hCtr1) expression level in tumor tissue specimens has been positively correlated with the treatment outcome of patients who had undergone platinum-based cancer chemotherapy [14, 15, 16]. Using cultured cell models, we recently demonstrated that copper chelators could upregulate the hCtr1 level to a greater extent in cisplatin-resistant cells than in cisplatin-sensitive cells, leading to resensitization of the resistant cells to cisplatin . These findings provided a mechanistic basis for the first study in humans using a copper chelator to overcome platinum resistance in ovarian cancer patients .
The observation that copper chelation enhances hCtr1 expression was part of our previous investigation into the mechanisms of mammalian copper homeostasis regulation. We demonstrated that copper chelation induces the expression of transcription factor Sp1, which binds the promoters of SP1 and CTR1, thereby upregulating their expression, whereas copper overload shuts down expression of SP1 and CTR1 by dissociating Sp1 from their promoters. Thus, mammalian copper homeostasis is transcriptionally regulated within a loop consisting of Sp1, hCtr1, and copper in a three-way mutually regulated manner [6, 18]. Posttranslational regulation which involves the internalization and subcellular processing of hCtr1 in response to extracellular copper availability has also been reported ,
Although much has been learned on the regulation of hCtr1 expression by copper bioavailability, whether hCtr1 is regulated by cisplatin and other metal ions is not known. In this communication, we demonstrate that cisplatin, Ag(I), Zn(II), and Cd (II) can also regulate hCtr1 expression through interference with copper homeostasis, thus revealing a regulatory mechanism of copper homeostasis by cisplatin and heavy metal ions.
Materials and methods
Cell lines and reagents
Human ovarian cancer cell lines (IGROV1, SKOV-3, 59M, and OVCAR-3) were obtained from Gordon Mills (MD Anderson Cancer Center). The small cell lung cancer (SCLC) cell line was obtained from N. Savaraj (University of Miami School of Medicine, Miami, FL, USA). Polyclonal anti-hCtr1 antibody obtained using the extracellular 50 amino acid residues of hCtr1 as the immunogen was previously described . Sp1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cisplatin was purchased from Sigma-Aldrich (St Louis, MO, USA). Other chemicals were of chemical grade.
Cell culture and determination of hCtr1 and Sp1 messenger RNA and protein expression by the RNase protection assay and Western blotting
Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum at 37 °C in a 5 % CO2 atmosphere. Cells at the exponential growth stage were treated with cisplatin, CuSO4, AgNO3, zinc acetate (ZnAc2) or cadmium acetate (CdAc2). Procedures for RNA extraction and determination of hCtr1 messenger RNA (mRNA) and Sp1 mRNA levels by the RNase protection assay (RPA) using the isoform-specific probes were described previously [12, 13]. Procedures for Western blotting were described previously [12, 13]. Owing to the constraint of the copper homeostasis regulatory loop , the magnitudes of hCtr1 and Sp1 regulation by cisplatin and heavy metals were low at the mRNA and protein levels. Thus, the conditions for the RPA and Western blotting needed to be optimized.
Images were taken only under exponential exposure conditions. Images were scanned in grayscale at a resolution of 600 dpi. The band intensities were measured with ImageJ  and normalized using the intensity of tubulin for Western blots or 18S for RPA as references.
All statistical analyses were conducted from at least three measurements using the two-tailed t test, and the results were expressed as the mean ± the standard deviation (SD); p < 0.05 was considered as statistically significant.
Copper and cisplatin transport assays, determination of drug sensitivity, and measurement of Km and Vmax
For copper transport analyses, 2 × 105 cells per well were plated in six-well plates. After 12 h, fresh medium containing various concentrations of CuSO4 was added and cultured for various time intervals. Cells were washed four times with phosphate-buffered saline and then lysed in 400 μl of lysis buffer . Cellular copper content was measured using atomic absorption spectroscopy. For cisplatin transport measurement, 5 × 106 cells per well were treated with various concentrations of cisplatin. Cells were harvested and lysed in 50 μl of benzethonium hydroxide at 50 °C for 16 h . The lysates were acidified with 200 μl of 0.3 N HCl, and the platinum content was determined by a Zeeman atomic absorption spectrometer (AA240Z) equipped with a GTA12 graphite atomizer according to the procedure described previously . The results were from at least three measurements and are given as the mean ± the SD.
Drug sensitivity tests were performed according to the procedure described previously . In brief, cells were grown in 96-well plates (104 cells per well) and were treated with various concentrations of cisplatin, CuSO4, or CdAc2, and cell sensitivity was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Experiments were performed with eight replicates at each dose. Values represent the mean ± the SD.
Measurements of the Vmax and Km values were done according to the procedures previously described . Vmax and Km were calculated according to the Michaelis–Menten equation: 1/V = 1/Vmax + Km/(Vmax × [S]), where [S] is the copper or cisplatin concentration, and V is the copper or platinum concentration inside the cells at a given time point according to the procedures previously described .
Upregulation of hCtr1 and Sp1 expression by cisplatin
We also determined the expression levels of Sp1 in these cell extracts by using a commercial antibody. Anti-Sp1 antibody detected only a 98-kDa signal, consistent with the molecular mass of Sp1. The signals were also increased in cisplatin-treated SCLC cells (Fig. 2b), and in CTR1-transfected SCLC cells owing to expression of the dominant-negative hCtr1 recombinant, which acts much like a copper-lowering agent [14, 18].
Protein and mRNA levels of hCtr1 and Sp1 in SCLC cells treated with 10 μM cisplatin for various times were determined by the RPA (Fig. 2d, top) and Western blotting (Fig. 2d, bottom). Upregulation of hCtr1 and Sp1 mRNA occurred between 0.5 and 2 h after the treatment and plateaued 6–8 h later with approximately fourfold to sevenfold increases. Sp1 and hCtr1 mRNA and protein induced by cisplatin followed the same kinetics, suggesting that the induction is coordinated. These results demonstrated that the regulation of hCtr1 and Sp1 expression by cisplatin is mainly at the mRNA level.
We observed a concentration-dependent induction of hCtr1 and Sp1 expression in IGROV1 cells (Fig. 2e) and SKOV-3 cells (Fig. 2f) treated with cisplatin for 16 h. Induction of hCtr1 and Sp1 expression could be seen at 2.5 μM, a concentration relevant to the therapeutic dose. Induction of hCtr1 and Sp1 expression was generally correlated at the mRNA and protein expression levels. Thus, from the investigation of five cell lines, we concluded that hCtr1 and Sp1 are coordinately upregulated by cisplatin in human cancer cells.
Induction of hCtr1 by cisplatin is regulated by Sp1
Suppression of cisplatin-induced hCtr1 and Sp1 expression by copper
We hypothesized that induction of hCtr1 and Sp1 by cisplatin was due to competition for hCtr1-mediated copper transport by cisplatin resulting in reduced cellular copper levels. Under this scenario, cisplatin functions like a copper-lowering agent for Sp1/hCtr1 induction. Although reduced copper levels can be verified by reduced activities of copper-dependent enzymes, such as superoxide dismutase 1 (SOD1) and mitochondrial cytochrome c oxidase (CCO) [24, 25], we considered that these biomarkers are inadequate for determining cellular copper status in the cisplatin-treated cells, because SOD1 and CCO are redox-sensitive enzymes  and cisplatin is a potent redox-generating agent . Consistent with this notion, we found that the antitumor drug elesclomol (STA4783), which is a strong pro-oxidant copper chelator , induced hCtr1 expression but that SOD1 and CCO activities were also increased (our unpublished data). Ceruloplasmin, another frequently used copper biomarker [24, 25], would not be suitable because cisplatin can also bind ceruloplasmin .
To determine whether cisplatin treatment would reduce the steady-state levels of cellular copper levels, we treated IGROV1 and SKOV-3 cells with various concentrations (5–80 μM) of cisplatin for 4 h. Figure 4b shows that reduced cellular copper contents correspond to increased concentrations of cisplatin in both cell lines. These results suggest that cisplatin treatment resulted in reduced copper accumulation.
To more precisely determine how cisplatin affects hCtr1-mediated Cu(I) transport and vice versa, we measured the kinetic constants Km and Vmax in IGROV1 cells. Km for copper uptake was 5.31 ± 0.31 μM (Fig. 4c), which is in agreement with the values reported for Ctr1-mediated Cu(I) transporters in a variety of organisms (between 1 and 6 μM) [13, 30, 31]. In the presence of cisplatin, however, Km increased to 11.02 ± 1.08 μM (about a twofold increase), whereas Vmax reduced from 17.93 ± 1.33 to 7.76 ± 0.84 pmol/mg protein/min (more than twofold reduction), indicating that cisplatin interferes with hCtr1-mediated Cu(I) transport. Km for cisplatin uptake was 13.29 ± 1.14 μM, which is also in agreement with the previous result (11–13 μM) . In the presence of CuSO4, Km for cisplatin uptake was 18.20 ± 1.14 μM (a 37 % increase), whereas Vmax decreased from 24.48 ± 1.31 to 21.58 ± 11.18 pmol/mg protein/min, only 11 % reduction. These results further suggest that cisplatin and Cu(I) mutually interfere with each other in their transport  and strongly support the notion that cisplatin is a weak substrate of hCtr1.
We next investigated whether inhibition of cisplatin-induced hCtr1 expression by copper would reduce cisplatin’s cell-killing capacity. We measured the survival rates of IGROV1 and SKOV-3 cells treated with 10 μM cisplatin plus increased concentrations of CuSO4 (like the treatment shown in Fig. 5a), the same concentration range of CuSO4 without cisplatin, or 10 μM cisplatin alone for 16 h. Treating IGROV1 and SKOV-3 cells with 10 μM cisplatin alone resulted in survival rates of 92.9 ± 4.7 and 95.3 ± 4.8 %, respectively. The survival rates decreased in a concentration-dependent manner in IGROV1 and SKOV-3 cells treated with 10 μM cisplatin plus 200 μM CuSO4, reaching 72.9 ± 4.1 and 75.9 ± 5.2 %, respectively, whereas they were 81.8 ± 4.1 and 85.7 ± 5.2 %, respectively, for treatment with 200 μM CuSO4 alone (Fig. 5c).
Treating IGROV1 and SKOV-3 cells with 200 μM CuSO4 for 3 days resulted in survival rates of 25.9 ± 0.6 and 50.8 ± 0.16 %, respectively. In the presence of 10 μM cisplatin, the survival rates correspondingly reduced to 5.1 ± 0.12 and 15.9 ± 0.12 %, respectively (Fig. 5c). These results demonstrated that despite the suppression of cisplatin-induced hCtr1 mRNA expression by copper as shown in Fig. 5a, no corresponding reduction of cellular toxicity was found.
Regulation of hCtr1 and Sp1 expression by metal ions
The major finding of this study is that cisplatin induces hCtr1 expression through the co-induced expression of Sp1, which functions as a positive regulator for hCtr1 expression. This finding reveals a new role of hCtr1 in the cisplatin pharmacology and provides important insights into how cisplatin may use hCtr1 in eliciting its antitumor activity in cancer chemotherapy.
Induction of hCtr1 by cisplatin treatment could be seen at the mRNA and protein levels. Although the magnitudes of induction were small, the results were highly reproducible. Changes of hCtr1 expression were demonstrated in concentration- and time-dependent manners. The small magnitudes of the changes reflect the tightly controlled copper homoeostasis regulation mechanism . Our ability to detect small changes relied on the use of appropriate assay tools: an isoform-specific RPA probe for mRNA and a well-characterized anti-hCtr1 antibody in Western blotting. Polyclonal anti-hCtr1 antibodies reported by different laboratories detected different molecular masses of hCtr1 monomer, i.e., 28 kDa , 24 kDa , 35 kDa , and 24 and 36 kDa . The 23–24-kDa protein corresponds to the unmodified hCtr1 monomer, which consists of 190 amino acid residues. The 35–36-kDa protein is considered to be glycosylated hCtr1 [24, 38]. Although the roles of glycosylated hCtr1 and native hCtr1 in copper transport remain to be critically investigated, we demonstrated in this and previous communications  that the expression levels of 23-kDa hCtr1 are well correlated with hCtr1 mRNA levels within the context of overall copper homeostasis regulation. However, the antibody that recognizes 35-kDa hCtr1 failed to detect hCtr1 changes in human embryonic kidney cells that overexpressed hCtr1 by transfection . Because our antibody recognizes unmodified hCtr1 monomer, the signal shown in the Western blot is discrete, whereas the 35-kDa signal is general is more diffuse because of different degrees of protein modification. The discrete signal can be readily quantified as demonstrated in this study.
The demonstration that cisplatin regulates hCtr1 expression as an integral part of mammalian copper homeostasis regulation lends further support for the importance of hCtr1 in cisplatin transport. The controversy surrounding cisplatin as a substrate for hCtr1 relates to its molecular size and charge . Although the molecular size of cisplatin is greater than the “narrowest opening” of trimeric hCtr1, recent studies suggest that prior to entering a cell, cisplatin is activated by interacting with the extracellular methionine clusters of hCtr1, resulting in release of the carrier ligand [41, 42, 43] and formation of the [Pt(Met)Cl(NH3)2]+ intermediate. The radius of this platinum intermediate (2.4 Å)  is smaller than the radius of the narrowest opening (4 Å) of hCtr1 . Moreover, we previously demonstrated that cisplatin, like copper, can induce conformational stabilization of trimeric hCtr1 . The dynamic nature of the interactions between cisplatin and its transporter has to be taken into consideration when modeling the molecular basis of hCtr1-mediated transport. Extracellular copper exists in an oxidized form (Cu2+) but is reduced to Cu+ by the membrane-bound cupric reductases  before transport. Thus, the activated cisplatin intermediate also shares a similar charge content as in Cu+. It has been proposed that the activated cisplatin may use methionine-based intermolecular sulfur–sulfur exchange along the axis of trimeric hCtr1 to facilitate its transport , much like the mechanism underlying Cu(I) or Ag(I) transport by the CusA efflux pump in bacteria . This mechanism is consistent with the recently proposed model describing how Cu(I) ions pass through the transporter [7, 48]. Given that hCtr1-mediated cisplatin transport shares many similarities with transport of copper, including most of the proposed binding sites , it is likely that the proposed model could be shared by cisplatin transport.
Upregulated hCtr1 and accumulation of cellular cisplatin were seen in cisplatin-treated cells (see Fig. 4a, left). However, it is difficult to assign the extent by which upregulated hCtr1 contributed to the overall cellular platinum accumulation, because the level of cisplatin (which is a substrate) was also increased in the system. Induction of hCtr1 expression by a copper-lowering agent takes several hours to reach a plateau, but requires 72–96 h to return to its basal level after the agent is removed . We also showed that treating cells with CuSO4 resulted in accumulation of cellular copper content (see Fig. 4a, right), whereas expression of hCtr1 was downregulated in cells treated with copper [18, 49]. A plausible explanation to account for these results is increased hCtr1 transport activity, because expression of copper efflux transporters (ATP7A and ATP7B) is not regulated by acute environmental copper changes [6, 14]. These results demonstrated that the capacities of cellular copper or cisplatin accumulations cannot be simply related to the abundance of hCtr1 when cells are exposed to its substrates; rather, they are controlled by the overall copper homeostasis system, which involves interregulatory components consisting of copper, hCtr1, and Sp1 . Consistent with this model, we showed that although cisplatin treatment induced hCtr1 expression, cellular copper levels were reduced (Fig. 4b), suggesting that cisplatin is a competitor for hCtr1-mediated copper transport.
This argument may explain why addition of copper suppressed cisplatin-induced hCtr1 expression yet no reduction in overall cytotoxicity was found. Furthermore, both cisplatin and copper are potent pro-oxidants with many cellular lethality targets. The complexity of the cytotoxic mechanisms of cisplatin and copper, especially when in combination, cannot be explained by the expression of hCtr1 alone.
We found that Ag(I) and Zn(II) can also induce Sp1/hCtr1 expression, which can be explained by the same mechanism in that these metals may compete against hCtr1-mediated copper transport, resulting in reduced cellular copper levels and thereby upregulated Sp1/hCtr1. Because the major influx transporter for Zn(II) is the SLC39 family , the physiological implication of hCtr1 upregulation by Zn(II) remains to be evaluated. However, it has been reported that zinc supplement can enhance the treatment efficacy of platinum-based chemotherapy in nasopharyngeal carcinoma . Likewise, several organs in silver-fed mice exhibited rates of cisplatin uptake different from those in normal-diet-fed mice , suggesting that Ag(I) may affect the pharmacodynamics of platinum drugs in cancer chemotherapy. In contrast, we found that Cd(II) suppresses Sp1 expression (and therefore hCtr1 expression), most likely owing to Cd(II)-induced damage to the DNA-binding zinc finger motifs of Sp1 . Cd(II) is a potent environmental poison, as are Ag(I) and Zn(II) to lesser extents. The current study provides important insights into how these heavy metals may affect copper metabolism through interventions in copper homeostasis regulation.
This research was supported in part by grants CA149260 (M.T.K.) and CA16672 (MD Anderson Core Facilities) from the National Cancer Institute, and by the National Science Council, Taiwan (NSC99-2314-B-006-038, H.H.W.C.). We thank Elizabeth Hess, Department of Scientific Publications, MD Anderson Cancer Center, for editorial assistance with the manuscript.
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