Roohangiz Safaei

Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells

Previous work has shown that cisplatin (CDDP) becomes concentrated in lysosomes, and that acquired resistance to CDDP is associated with abnormalities of protein trafficking and secretion. The lysosomal compartment in CDDP-sensitive 2008 human ovarian carcinoma cells was compared with that in CDDP-resistant 2008/C13*5.25 subline using deconvoluting imaging and specific dyes and antibodies. The lysosomal compartment in CDDP-resistant cells was reduced to just 40% of that in the parental CDDP-sensitive cells (P < 0.002). This was accompanied by a reduced expression of the lysosome-associated proteins 1 and 2 (LAMP1 and LAMP2) as determined by both microscopy and Western blot analysis. The CDDP-resistant cells released more protein as exosomes and Western blot analysis revealed that these exosomes contained substantially more LAMP1 than those released by the CDDP-sensitive cells. Following loading of the whole cell with CDDP, the exosomes released from 2008/C13*5.25 cells contained 2.6-fold more platinum than those released from sensitive cells. Enhanced exosomal export was accompanied by higher exosomal levels of the putative CDDP export transporters MRP2, ATP7A, and ATP7B. Expression profiling identified significant increases in the expression of several genes whose products function in membrane fusion and vesicle trafficking. This study shows that the lysosomal compartment of human ovarian carcinoma cells selected for stable resistance to CDDP is markedly reduced in size, and that these cells abnormally sort some lysosomal proteins and the putative CDDP transporters into an exosomal pathway that also exports CDDP.

Copper Transporters and the Cellular Pharmacology of the Platinum-Containing Cancer Drugs

Multiple lines of evidence indicate that the platinum-containing cancer drugs enter cells, are distributed to various subcellular compartments, and are exported from cells via transporters that evolved to manage copper homeostasis. The cytotoxicity of the platinum drugs is directly related to how much drug enters the cell, and almost all cells that have acquired resistance to the platinum drugs exhibit reduced drug accumulation. The major copper influx transporter, copper transporter 1 (CTR1), has now been shown to control the tumor cell accumulation and cytotoxic effect of cisplatin, carboplatin, and oxaliplatin. There is a good correlation between change in CTR1 expression and acquired cisplatin resistance among ovarian cancer cell lines, and genetic knockout of CTR1 renders cells resistant to cisplatin in vivo. The expression of CTR1 is regulated at the transcriptional level by copper via Sp1 and at the post-translational level by the proteosome. Copper and cisplatin both trigger the down-regulation of CTR1 via a process that involves ubiquitination and proteosomal degradation and requires the copper chaperone antioxidant protein 1 (ATOX1). The cisplatin-induced degradation of CTR1 can be blocked with the proteosome inhibitor bortezomib, and this increases the cellular uptake and the cytotoxicity of cisplatin in a synergistic manner. Copper and platinum(II) have similar sulfur binding characteristics, and the presence of stacked rings of methionines and cysteines in the CTR1 trimer suggest a mechanism by which CTR1 selectively transports copper and the platinum-containing drugs via sequential transchelation reactions similar to the manner in which copper is passed from ATOX1 to the copper efflux transporters.

Increased Expression of the Copper Efflux Transporter ATP7A Mediates Resistance to Cisplatin, Carboplatin, and Oxaliplatin in Ovarian Cancer Cells

Purpose: The goal of this study was to determine the effect of small changes in ATP7A expression on the pharmacodynamics of cisplatin, carboplatin, and oxaliplatin in human ovarian carcinoma cells. Experimental Design: Drug sensitivity and cellular pharmacology parameters were determined in human 2008 ovarian carcinoma cells and a subline transfected with an ATP7A-expression vector ATP7A (2008/MNK). Drug sensitivity was determined by clonogenic assay, platinum (Pt) levels were measured by inductively coupled plasma mass spectroscopy, copper (Cu) accumulation was quantified with 64Cu, and the subcellular distribution of ATP7A was assessed by confocal digital microscopy. Results: The 1.5-fold higher expression of ATP7A in the 2008/MNK cells was sufficient to alter Cu cellular pharmacokinetics but not confer Cu resistance. In contrast, it was sufficient to render the 2008/MNK cells resistant to cisplatin, carboplatin, and oxaliplatin. Resistance was associated with increased rather than decreased whole-cell Pt drug accumulation and increased sequestration of Pt into the vesicular fraction. Cu triggered relocalization of ATP7A away from the perinuclear region, whereas at equitoxic concentrations the Pt drugs did not. Conclusions: A small increase in ATP7A expression produced resistance to all three of the clinically available Pt drugs. Whereas increased expression of ATP7A reduced Cu accumulation, it did not reduce accumulation of the Pt drugs. Under conditions where Cu triggered ATP7A relocalization, the Pt drugs did not. Thus, although ATP7A is an important determinant of sensitivity to the Pt drugs, there are substantial differences between Cu and the Pt drugs with respect to how they interact with ATP7A and the mechanism by which ATP7A protects the cell.

The role of the mammalian copper transporter 1 in the cellular accumulation of platinum-based drugs.

The mammalian copper transporter 1 (CTR1) is responsible for the uptake of copper from the extracellular space. In this study, we used an isogenic pair of CTR1(+/+) and CTR1(-/-) mouse embryo fibroblasts to examine the contribution of CTR1 to the influx of cisplatin (DDP), carboplatin (CBDCA), oxaliplatin (L-OHP), and transplatin. Exposure to DDP triggered the rapid degradation of CTR1, suggesting that its contribution to influx was likely to be on the initial phase of drug entry. Loss of CTR1 decreased the initial binding of DDP to cells and reduced influx measured over the first 5 min of drug exposure by 81%. Loss of CTR1 almost completely eliminated the initial influx of CBDCA and reduced the initial uptake of L-OHP by 68% but had no effect on the influx of transplatin. Loss of CTR1 rendered cells resistant to even high concentrations of DDP when measured in vitro, and re-expression of CTR1 in the CTR1(-/-) cells restored both DDP uptake and cytotoxicity. The growth of CTR1(-/-) tumor xenografts in which CTR1 levels were restored by infection with a lentivirus expressing wild-type CTR1 was reduced by a single maximum tolerated dose of DDP in vivo, whereas the CTR1(-/-) xenografts failed to respond at all. We conclude that CTR1 mediates the initial influx of DDP, CBDCA, and L-OHP and is a major determinant of responsiveness to DDP both in vitro and in vivo.

Copper Transporter 2 Regulates the Cellular Accumulation and Cytotoxicity of Cisplatin and Carboplatin

Purpose: Copper transporter 2 (CTR2) is known to mediate the uptake of Cu+1 by mammalian cells. Several other Cu transporters, including the influx transporter CTR1 and the two efflux transporters ATP7A and ATP7B, also regulate sensitivity to the platinum-containing drugs. We sought to determine the effect of CTR2 on influx, intracellular trafficking, and efflux of cisplatin and carboplatin. Experimental Design: The role of CTR2 was examined by knocking down CTR2 expression in an isogenic pair of mouse embryo fibroblasts consisting of a CTR1+/+ line and a CTR1−/− line in which both CTR1 alleles had been deleted. CTR2 levels were determined by quantitative reverse transcription-PCR and Western blot analysis. Cisplatin (DDP) was quantified by inductively coupled plasma mass spectrometry and 64Cu and [14C]carboplatin (CBDCA) accumulation by γ and scintillation counting. Results: Deletion of CTR1 reduced the uptake of Cu, DDP, and CBDCA and increased resistance to their cytotoxic effects by 2- to 3-fold. Knockdown of CTR2 increased uptake of Cu only in the CTR1+/+ cells. In contrast, knockdown of CTR2 increased whole-cell DDP uptake and DNA platination in both CTR1+/+ and CTR1−/− cells and proportionately enhanced cytotoxicity while producing no effect on vesicular accumulation or efflux. A significant correlation was found between CTR2 mRNA and protein levels and sensitivity to DDP in a panel of six ovarian carcinoma cell lines. Conclusions: CTR2 is a major determinant of sensitivity to the cytotoxic effects of DDP and CBDCA. CTR2 functions by limiting drug accumulation, and its expression correlates with the sensitivity of human ovarian carcinoma cell lines to DDP.

Transport of Cisplatin by the Copper Efflux Transporter ATP7B

ATP7B is a P-type ATPase that mediates the efflux of copper. Recent studies have demonstrated that ATP7B regulates the cellular efflux of cisplatin (DDP) and controls sensitivity to the cytotoxic effects of this drug. To determine whether DDP is a substrate for ATP7B, DDP transport was assayed in vesicles isolated from Sf9 cells infected with a baculovirus that expressed either the wild-type ATP7B or a mutant ATP7B that was unable to transport copper as a result of conversion of the transmembrane metal binding CPC motif to CPA. Only the wild-type ATP7B-expressing vesicles exhibited copper-dependent ATPase activity, copper-induced acyl-phosphate formation, and ATP-dependent transport of copper. The amount of DDP that became bound was higher for vesicles expressing either type of ATP7B than for those not expressing either form of ATP7B, but only the vesicles expressing wild-type ATP7B mediated ATP-dependent accumulation of the drug. At pH 4.6, the vesicles expressing the wild-type ATP7B exhibited ATP-dependent accumulation of DDP with an apparent Km of 1.2 ± 0.5 (S.E.M.) μM and Vmax of 0.03 ± 0.002 (S.E.M.) nmol/mg of protein/min. DDP also induced the acyl-phosphorylation of ATP7B but at a much slower rate than copper. Copper and DDP each inhibited the ATP-dependent transport of the other. These results establish that DDP is a substrate for ATP7B but is transported at a much slower rate than copper.

Confocal Microscopic Analysis of the Interaction between Cisplatin and the Copper Transporter ATP7B in Human Ovarian Carcinoma Cells

Some cisplatin (DDP)-resistant cells overexpress the copper export transporter ATP7B, and cells molecularly engineered to overexpress ATP7B are resistant to DDP. The interaction of Cu with ATP7B normally triggers its relocalization from the perinuclear region to more peripheral vesicles. To investigate the interaction of DDP with ATP7B, we examined the effect of DDP on the subcellular localization of ATP7B using human ovarian carcinoma cells expressing a cyan fluorescent protein (ECFP)-tagged ATP7B (2008/ECFP-ATP7B). ATP7B expression was confirmed in 2008/ECFP-ATP7B cells by Western blotting, and its functionality was documented by showing that it rendered the cells 1.9-fold resistant to CuSO4 and 4.1-fold resistant to DDP and also reduced the accumulation of both drugs. There was greater sequestration of Pt into intracellular vesicles in the 2008/ECFP-ATP7B cells than in the 2008/ECFP cells. Confocal digital microscopy revealed that ECFP-ATP7B localized in the perinuclear region in absence of drug exposure and that both Cu and DDP triggered relocalization to more peripheral vesicular structures. A fluorescein-labeled form of DDP that retained cytotoxicity and was subject to the same mechanisms of resistance as DDP colocalized with ECFP-ATP7B in the 2008/ECFP-ATP7B cells, whereas the same fluorochrome lacking the DDP moiety did not. These results provide evidence that DDP directly interacts with ATP7B to trigger its relocalization and that ATP7B mediates resistance to DDP by sequestering it into vesicles of the secretory pathway for export from the cell.

Effects of the loss of Atox1 on the cellular pharmacology of cisplatin

Previous work has demonstrated that the copper (Cu) transporters Ctr1, Atp7a and Atp7b regulate the cellular pharmacology of cisplatin (CDDP) by mediating its uptake and efflux. It was also shown that, in the process of uptake by Ctr1, CDDP triggers the rapid proteasomal degradation of its own transporter. The current study examined the role of the metallochaperone Atox1 in the regulation of uptake, efflux and subcellular distribution of CDDP by using a pair of fibroblast cell lines established from Atox1(+/+) and Atox1(-/-) mice. Atox1 is a metallochaperone that is known to play a central role in distributing Cu within the cells and was recently shown to act as a Cu-dependent transcription factor. Loss of Atox1 increased Cu accumulation and reduced efflux. In contrast, loss of Atox1 reduced the influx of CDDP and subsequent accumulation in vesicular compartments and in DNA. Loss of Atox1 was found to block the CDDP-induced down regulation of Ctr1. Ctr1 was found to be polyubiquitinated in an Atox1-dependent manner during CDDP exposure. In conclusion, Atox1 is required for the polyubiquitination of Ctr1 and the Ctr1-mediated uptake of CDDP.

Copper Transporter 2 Regulates Endocytosis and Controls Tumor Growth and Sensitivity to Cisplatin In Vivo

Copper transporter 2 (CTR2) is one of the four copper transporters in mammalian cells that influence the cellular pharmacology of cisplatin and carboplatin. CTR2 was knocked down using a short hairpin RNA interference. Robust expression of CTR2 was observed in parental tumors grown in vivo, whereas no staining was found in the tumors formed from cells in which CTR2 had been knocked down. Knockdown of CTR2 reduced growth rate by 5.8-fold, increased the frequency of apoptotic cells, and decreased the vascular density, but it did not change copper content. Knockdown of CTR2 increased the tumor accumulation of cis-diamminedichloroplatinum(II) [cisplatin (cDDP)] by 9.1-fold and greatly increased its therapeutic efficacy. Because altered endocytosis has been implicated in cDDP resistance, uptake of dextran was used to quantify the rate of macropinocytosis. Knockdown of CTR2 increased dextran uptake 2.5-fold without reducing exocytosis. Inhibition of macropinocytosis with either amiloride or wortmannin blocked the increase in macropinocytosis mediated by CTR2 knockdown. Stimulation of macropinocytosis by platelet-derived growth factor coordinately increased dextran and cDDP uptake. Knockdown of CTR2 was associated with activation of the Rac1 and cdc42 GTPases that control macropinocytosis but not activation of the phosphoinositide-3 kinase pathway. We conclude that CTR2 is required for optimal tumor growth and that it is an unusually strong regulator of cisplatin accumulation and cytotoxicity. CTR2 regulates the transport of cDDP in part through control of the rate of macropinocytosis via activation of Rac1 and cdc42. Selective knockdown of CTR2 in tumors offers a strategy for enhancing the efficacy of cDDP.

The role of the methionines and histidines in the transmembrane domain of mammalian copper transporter 1 in the cellular accumulation of cisplatin.

Mammalian copper transporter 1 (CTR1) is a high-affinity copper influx transporter that also mediates the uptake of platinum-containing chemotherapeutic agents including cisplatin (cDDP). Methionines 150, 154, and histidine 139 have been proposed to form a series of stacked rings in the pore formed by the CTR1 homotrimer, each of which is required for maximal copper transport. To examine the mechanism by which hCTR1 also transports cDDP, variant forms of hCTR1 in which methionines 150 and 154 were converted to isoleucines or in which histidine 139 was converted to alanine were re-expressed in cells in which both alleles of CTR1 had been knocked out. Each of these conversions disabled copper transport and increased cellular resistance to the cytotoxic effect of copper. In contrast, conversion of the methionines increased the uptake and cytotoxicity of cDDP well above that attained with wild-type hCTR1. Conversion of His139 to alanine did not impair cDDP uptake and actually enhanced cytotoxicity. Thus, although Met150 and Met154 facilitate the movement of copper through the pore, they serve to obstruct the passage of cDDP. None of the modifications altered the ability of cDDP to trigger the degradation of hCTR1, indicating that cDDP must interact with hCTR1 at other sites as well. Although both copper and cDDP may rely on a series of transchelation reactions to pass through the hCTR1 trimeric complex, the details of the molecular interactions must be different, which provides a potential basis for selective pharmacological modulation of copper versus cDDP cytotoxicity.