Interactions of cisplatin and the copper transporter CTR1 in human colon cancer cells
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There is much interest in understanding the mechanisms by which platinum-based anticancer agents enter cells, and the copper transporter CTR1 has been the focus of many recent studies. While there is a clinical correlation between CTR1 levels and platinum efficacy, cellular studies have provided conflicting evidence relating to the relationship between cisplatin and CTR1. We report here our studies of the relationship between cisplatin and copper homeostasis in human colon cancer cells. While the accumulation of copper and platinum do not appear to compete with each other, we did observe that cisplatin perturbs CTR1 distribution within 10 min, a far shorter incubation time than commonly employed in cellular studies of cisplatin. Furthermore, on these short time-scales, cisplatin caused an increase in the cytoplasmic labile copper pool. While the predominant focus of studies to date has been on CTR1, these studies highlight the importance of investigating the interaction of cisplatin with other copper proteins.
KeywordsAnticancer drug Cisplatin Copper Metal transport
Antioxidant 1 copper chaperone
Copper chaperone for superoxide dismutase
Copper chaperone for cytochrome C oxidase
Copper transporter 1
Human colon adenocarcinoma
Divalent metal transporter 1
Graphite furnace atomic absorption spectroscopy
Green fluorescent protein
Human embryonic kidney
Inductively coupled plasma mass spectrometry
The discovery of cisplatin has revolutionised the treatment of ovarian, testicular, lung and other cancers, and the field of medicinal inorganic chemistry . However, it presents key disadvantages including dose-limiting side effects such as kidney damage, and the development of resistance during treatment. It is widely accepted that these platinum complexes function by aquation of their labile ligands, interaction with nucleophilic DNA bases, and induction of apoptosis in cancer cells . However, research is still ongoing to understand the full mechanism of cisplatin activity, with only 1% of cellular platinum reaching the nucleus .
There has been a recent focus on the role of the copper homeostasis system in cancer and in the cellular uptake, distribution and elimination of platinum complexes . The copper transport protein, CTR1 has been linked to platinum uptake and efficacy, with cell knockout models in yeast and mice displaying resistance and reduced platinum accumulation . Investigations of cisplatin-resistant cancer cell lines also confirm a correlation between CTR1 levels and platinum uptake [6, 7]. In animal studies as well as in a clinical setting, prognosis after platinum chemotherapy positively correlates with tumour CTR1 expression in ovarian and non-small-cell lung cancer [8, 9, 10]. It has therefore been proposed that CTR1 is a major route of cisplatin uptake, though not all evidence is consistent with this role . Overexpression of CTR1 in ovarian cancer cells increases copper uptake, but has a smaller effect on platinum accumulation and none on cytotoxicity . Fewer studies have silenced CTR1 in human cancer cell lines, and these have produced contrasting results [13, 14, 15].
The poor correlation between platinum accumulation and cytotoxicity has also been observed in some overexpression studies, whereby platinum accumulation increased by 50%, but had little effect on cytotoxicity and levels of DNA-bound platinum . This also highlights a weakness of current techniques to study platinum drug uptake, which primarily rely on graphite furnace atomic absorption spectroscopy (GF-AAS) and inductively coupled plasma mass spectrometry (ICP-MS). These techniques measure the total platinum content of a sample, with no information on the oxidation state or coordination sphere. Hence, the readings do not just represent the levels of active cisplatin, but also encompass all protein-bound and deactivated platinum: this should be kept in mind when interpreting such data.
At present, there remains some uncertainty over the exact role of CTR1 and other copper handling proteins in platinum uptake and activity. In addition, there has been little focus on whether platinum complexes are simply a substrate for these proteins, or if they have a direct effect on protein function. Liang et al.  demonstrated that treatment with 10 µM cisplatin for 4 h lowered basal copper levels, and that platinum and copper interfered with each other in their uptake. They postulated that cisplatin could compete with copper for CTR1-mediated transport, but did not further investigate the consequences on copper homeostasis or potential modification of CTR1. Platinum coordination may also alter conformation or block necessary amino acids for native Cu(I) interactions. If so, dysregulation of copper homeostasis may be involved in the undesirable side effects of platinum chemotherapy, or even the chemotherapeutic mechanism.
In this paper, we have explored the relationship between cisplatin and copper homeostasis in human colon cancer cells. This was achieved by analysing the effect of cisplatin on basal copper levels, and copper uptake and bioavailability. In addition, we examined whether increasing CTR1 levels in colon cancer cells affected sensitivity to cisplatin. Finally, we studied the interaction between cisplatin and CTR1 in live cells.
Reagents and cell culture
Cisplatin was prepared according to literature methods . Aliquots (2 mM) were dissolved in PBS and stored at −20 °C for up to 2 weeks. CuSO4 aliquots were dissolved in Milli-Q water (10 mM stocks). DLD-1 cells were cultured in Advanced DMEM supplemented with 2% foetal calf serum and 2.5 mM l-glutamine (ThermoFisher Scientific) and maintained at 37 °C in 5% CO2.
Metal accumulation assay
DLD-1 cells were seeded into six-well plates at 2.5 × 105 cells/well and allowed to adhere for 24 h. The cells were treated as indicated, and the media was removed and the cells were washed three times with PBS. Lysis buffer (1% Triton X-100, 2 mM EDTA) was added to each well, frozen overnight, and thawed on ice. The cell lysates were collected by scraping and stored at −80 °C until analysed.
For copper measurements, the digested samples were diluted with 0.1 M hydrochloric acid to a maximum concentration of 20% HNO3. The samples were analysed by a Varian Zeeman atomic absorption spectrometer (240Z AA) equipped with a GTA 120 graphite tube atomiser and Cu UltrAA hollow cathode lamp (Agilent Technologies). For platinum measurements, the digested samples were diluted with Milli-Q water containing 10 ppb iridium. The samples were analysed by a Perkin-Elmer NexION 350X ICP-MS. Metal concentrations were normalised against protein concentrations determined by the Bio-Rad Protein Assay (Bio-Rad, #5000006).
Cells were treated and lysed as described above, with lysis buffer containing 1% (v/v) protease inhibitor (Sigma-Aldrich, P8340) and stored at −80 °C. The lysates were centrifuged at 16,000×g at 4 °C. The supernatant was collected, and the protein concentration determined by the Bio-Rad Protein Assay (Bio-Rad, #5000006). 15–50 µg of total protein was loaded on a 15-well 4–12% Bis-Tris gel (Bolt, Invitrogen) and separated in MES SDS buffer at 150 V for 50 min. The proteins were transferred to PVDF membranes (ImmobilonP, Millipore) in 25 mM bicine, 25 mM Bis-Tris and 1 mM EDTA transfer buffer, pH 7.2 at 30 V for 60 min at room temperature. Membranes were blocked in 5% skim milk in TBST buffer, and incubated for 1 h at room temperature with anti-CCS antibodies (1:500, Santa Cruz, FL-274) anti-CTR1 (Anti-SLC31A1/CTR1 antibody [EPR7936] (ab129067)I) and anti-β-Actin (1:2000, Sigma-Aldrich, #A2228). The membranes were washed in TBST (4 × 15 min) then incubated for 1 h at room temperature with the appropriate anti-rabbit or anti-mouse secondary antibody (Cell Signalling Technology, #7076 or #7074). The blots were incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0050), and visualised and quantified with the Bio-Rad ChemiDoc Imaging system.
Cell viability assay
96-well plates were seeded with 2 × 103 cells/well DLD-1. The day after plating, the cells were incubated with varying concentrations of cisplatin for 72 h. 10 µL of resazurin solution (650 µM) was added and incubated for a further 2–4 h until a colour gradient was visible between the control and treated wells. The reduction of resazurin was measured by fluorescence (excitation 570 nm, emission 585 nm). Cell viability was calculated relative to control wells, and fitted to a non-linear ‘log(inhibitor) vs response’ equation to calculate the IC50 value using GraphPad Prism 6.
DLD-1 cells were plated at a density of 2 × 105 cells/well in a six-well plate and allowed to adhere for 24 h. The cells were transfected with control GFP or GFP-CTR1 using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturers protocols. For determination of IC50, cells were re-plated into 96-well plates and viability measured as described above.
The probe CF4 was kindly provided by Prof. Christopher Chang (UC Berkeley): this probe and its analogues are described in detail elsewhere [18, 19, 20]. DLD-1 cells were plated on Nunc™ Lab-Tek™ II Chamber Slides™ (four chambers per slide) and grown in Advanced DMEM overnight before treatments. Cells were then treated with 0.5 mL of supplemented media (glutamine and FBS) containing either vehicle control (PBS) or cisplatin (2.5 µL of a 10 mM stock in PBS for a final concentration of 10 µM). After 16 h, copper sulphate was added (10 µL of a 10 mM stock solution of copper sulphate in PBS to the final concentration of 100 µM) to each chamber at time points 5, 10, 20, and 30 min for the vehicle control and cisplatin pre-treated cells. CF4 was then added directly prior to imaging in a solution of DMEM (0.5 mL, containing glutamine only). After 15 min incubation, cells were washed three times with PBS and imaged in PBS immediately.
For live cell imaging of GFP-tagged CTR1, transfected cells were re-plated into Lab-Tek Chambered #1.0 Borosilicate Coverglass System (Thermo Fisher Scientific) at a density of 2 × 104. Cells were imaged 48 h after re-plating.
All imaging experiments were performed using an Olympus FV1000 Confocal microscope (488 nm laser) and a UPLSAPO 60X water-immersion objective lens. Images were processed and analysed using Fiji image processing software (NIH).
Results and discussion
Effects of cisplatin on CTR1 and cellular copper levels
To determine whether cisplatin affected cellular copper levels, DLD-1 cells were treated with 10 µM cisplatin for 2, 4, 8, 16 and 24 h, followed by analysis of total cellular copper content by GF-AAS (Fig. 1c). Correlating with CTR1 levels, a small, non-significant increase in copper content was observed after 2 h cisplatin treatment, but at later time-points copper levels were similar to those in untreated cells. These experiments were performed in normal cell culture media without an additional supplementation of copper. To test whether cisplatin affected copper accumulation when additional copper was supplied, cells were pre-treated with 10 µM cisplatin for 24 h followed by addition of 50 µM copper for 2 h. Supplementation with copper increased cellular copper twofold and pre-treatment with cisplatin did not significantly affect this increase (Fig. 1d), suggesting that cisplatin does not affect total copper accumulation in DLD-1 cells.
While these studies do not directly probe CTR1, these results suggest that if cisplatin interacts with CTR1, the interaction differs from that of copper, and it is unlikely that CTR1 is the main transporter of cisplatin into the cell. However, the specific effect of cisplatin on CTR1 cannot be concluded, as the contribution of CTR1 to Cu uptake in DLD-1 cells is unknown. Other cell-type specific mechanisms of Cu uptake, such as the divalent metal transporter 1 (DMT1) , may compensate for loss of CTR1. Previous reports on the effect of cisplatin on copper uptake are limited in number and conflicting. The concentration of cisplatin used in the above experiments were based on a report by Liang et al. that cisplatin reduced copper uptake by at least 50% in ovarian cancer cells at similar concentrations of copper and cisplatin . However, another study reported no difference in copper uptake in the presence of 30 µM cisplatin in mouse fibroblasts . These conflicting data may reflect differences between cell types, but also highlight the importance of studying widely varying cisplatin concentrations to ensure that key effects are not overlooked.
Despite the commonly accepted view that CTR1 mediates platinum uptake, few studies have demonstrated the effect of copper on platinum uptake in cancer cells. In human embryonic kidney (HEK) cells, platinum uptake during treatment with 10 µM cisplatin was not affected by the presence of 1 mM copper , consistent with the findings of this study. However, platinum uptake was increased in HEK cells overexpressing CTR1, and this additional uptake was lowered by supplementation with 1 mM copper.
The effect of CTR1 levels on sensitivity to cisplatin and platinum accumulation
High CTR1 expression levels have been correlated with increased sensitivity to cisplatin, and low CTR1 levels, with decreased sensitivity [14, 26, 27, 28]. Despite this, cell studies involving overexpression of CTR1 have failed to confirm the contribution of CTR1 to cisplatin accumulation and sensitivity [12, 13]. A recent study using CRISPR-Cas9 editing in HEK and ovarian cancer cells reported that deletion of the CTR1 gene did not have a significant effect on sensitivity to cisplatin .
These overexpression data suggest that CTR1 is not involved in transporting active cisplatin into DLD-1 cells. In contrast to a study by Holzer et al., we did not observe an increase in platinum accumulation when CTR1 was overexpressed . However, despite the increase in platinum accumulation in their study, the increase in cisplatin toxicity was only marginal. This suggests that in some cell systems, CTR1 may contribute to platinum uptake, but the active drug fails to reach its nuclear target. Furthermore, knockdown of CTR1 in ovarian carcinoma cells was shown to reduce copper entry but not cisplatin . Ivy et al. suggest that active platinum drugs do not substantially enter cells through a protein-mediated pathway. While in a clinical setting, CTR1 levels correlate with cisplatin sensitivity, it remains unclear whether this is directly related to a transport role of CTR1 [8, 9, 10]. It would be interesting to see the results of in vivo xenograft studies with CTR1 deleted in various cell lines, similar to the study performed by Bompiani et al. .
Effect of cisplatin on copper bioavailability
In a previous study, CCS expression in mouse fibroblasts increased up to fivefold after 48 h incubation with cisplatin . However, the authors did not further investigate the cause or consequences of CCS upregulation other than demonstrating that copper depletion did not occur within hours. It is likely that the regulation of copper varies between normal and cancerous cells, as well as different cell types. It may be of interest to compare copper regulation in these cell lines to pinpoint how cisplatin may modulate copper bioavailability.
Using protein levels as a measure of copper availability may not be sensitive enough to detect small variations and fluctuations of copper in a shorter timeframe. Fluorescent probes provide an excellent tool for measuring dynamic fluctuations of analytes in live cells . Therefore, to further assess the levels of bioavailable copper in DLD-1 cells, we utilised the fluorescent copper probe CF4 (kindly provided by Christopher Chang) which undergoes a fluorescence turn-on in response to Cu(I) . We confirmed the response of CF4 to copper supplementation and starvation in the cuvette (Fig. 4e) and in DLD-1 cells (Fig. 4f, g). Cellular fluorescence was then analysed by confocal microscopy after pre-treatment with cisplatin for 24 h followed by copper treatment for the indicated time-points. We found that pre-treatment with cisplatin-increased bioavailable copper in the cytoplasm of DLD-1 cells (Fig. 4h).
We have previously shown that cisplatin pre-treatment leads to a decreased uptake of copper into the mitochondria . Interestingly, we now demonstrate an increase in copper in the cytoplasm of cisplatin-treated cells. These results may seem contradictory at first, but could result from the ability of cisplatin to interact with multiple different copper proteins [37, 38, 39]. Cisplatin may bind to copper proteins in the cell and displace copper, thereby increasing free copper in some sub-cellular locations and disrupt copper transport pathways to other regions. For example, COX17 has been shown to bind and facilitate cisplatin transport to the mitochondria  and Atox1 interacts with cisplatin in melanoma cells .
Interaction between cisplatin and CTR1 in live cells
These images suggest the internalisation of CTR1 in response to cisplatin treatment, with the puncta observed between 8 and 12 min consistent with the endosomal-localisation of CTR1 in conditions of elevated copper . These results show that cisplatin does have an effect on CTR1, although it is not clear whether this effect is direct or indirect, and importantly this effect occurs on very short time-scales, far shorter than those commonly employed in cellular studies of cisplatin. Guo et al. demonstrated formation of a CTR1 multimeric complex after cisplatin treatment that could be reversed by treatment with a metal chelator . In the same study, the authors reported that cisplatin does not affect the localisation of CTR1 in fixed cells by immunohistochemistry. However, the authors observed the cells after 1–2 h of cisplatin treatment in contrast to our much shorter incubation times. Our results, together with the above study, support the conclusion that cisplatin and CTR1 are able to interact in vivo, but the implication of this interaction on cisplatin uptake and biological activity remain to be determined.
We have investigated the interaction between cisplatin and copper handling in DLD-1 cells. Our results suggest that pre-treatment with cisplatin does not interfere with total copper uptake when investigated on the hours timescale. Furthermore, the accumulation of copper and platinum do not appear to compete with each other. An interesting and novel finding in this study is that cisplatin increases cytoplasmic labile copper levels. Taken together, these results suggest that while copper and cisplatin are likely to enter the cell by different mechanisms, cisplatin may affect the intracellular distribution of copper, perhaps by interaction with other copper proteins. While our results were consistent with CTR1 playing no significant role in cisplatin uptake in DLD-1 cells, our observations of CTR1 relocalisation in live cells at short time-points after cisplatin treatment raises questions that will be the subject of further studies.
The authors acknowledge the support of the Australian Research Council (TWH and EJN; DP150103369), an Australian Postgraduate Award (CS) and the Westpac Bicentennial Foundation (EJN). We thank Christopher Chang (UC Berkeley) for the provision of CF4, Nicholas Proschogo for assistance with ICP-MS measurements, and acknowledge the scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis (ACMM) at the University of Sydney.