Resistance to the cytotoxic effect of DDP is closely linked to reduced drug accumulation in human tumor cell lines (4
). This decrease in accumulation must be the result of either impaired drug influx, reduced intracellular sequestration, enhanced efflux, or a combination of these. Previous studies have documented that the Cu transporters can regulate all of these processes. Elimination of CTR1 reduces initial influx and results in substantial degrees of DDP resistance when measured both in vitro
and in vivo
). Increased expression of the Cu efflux transporters ATP7A and ATP7B enhances resistance to DDP (43
). ATP7A seems to function primarily to sequester DDP intracellularly (43
), whereas ATP7B mediates platinum drug efflux (44
) via a process that involves its transport into vesicles involved in the secretory pathway (45
). The results of the current study indicate that CTR2 is also an important determinant of both sensitivity to the cytotoxic effect of DDP and its intracellular pharmacology.
To study the effect of CTR2 on the cellular pharmacology of the platinum drugs, we took advantage of a very powerful model and knocked down the expression of CTR2 in both CTR1+/+
mouse embryo fibroblasts. Elimination of CTR1 resulted in a substantial increase in resistance to DDP and CBDCA, as we have previously reported (30
). Whereas CTR2 mRNA levels were reduced by 82% to 88%, this resulted in a more modest 33% to 55% reduction in the CTR2 protein level. These sublines were selected in a medium that did not contain additional Cu, and it is possible that a more severe reduction in CTR2 is lethal, particularly in the absence of CTR1 function. These relatively modest reductions in CTR2 protein level led to a 2.0- to 3.2-fold increase in sensitivity to DDP irrespective of the presence or absence of CTR1. A similar result was observed for CBDCA. Thus, the effect of reducing CTR2 expression was not dependent on the CTR1 status of the cells. This is in contrast to its effect on sensitivity to the cytotoxic effect of Cu. Elimination of the expression of CTR1 produced the anticipated decrease in sensitivity to Cu; however, knockdown of CTR2 in the CTR1−/−
cells had no further effect on sensitivity. These results support two conclusions. First, in the case of the platinum drugs, the effect of knocking down CTR2 seems to be independent of the status of CTR1. Second, CTR2 functions differently with respect to Cu and the platinum drugs.
To explore the mechanism by which loss of CTR2 increased cell sensitivity to the platinum drugs, we measured whole-cell drug accumulation at 5 minutes and 1 hour, the extent of DNA adduct formation, and the amount of DDP associated with the vesicle fraction of the cells by ICP-MS. Consistent with our prior studies (30
), deletion of both alleles of CTR1 reduced the influx of DDP when measured at both 5 minutes and 1 hour, and this was accompanied by a proportional decrease in DNA adduct formation. Reduction of CTR2 expression had the opposite effect. Knockdown of CTR2 led to a ~2.1 to 3.5-fold increase in whole-cell Pt accumulation and DNA adduct formation, and it did so irrespective of whether CTR1 was expressed or not. The increase in whole-cell platinum accumulation and DNA adduct formation was similar to the magnitude of the change in cytotoxicity, suggesting that the hypersensitivity caused by loss of CTR2 was directly linked to increased accumulation. Knockdown of CTR2 produced a very similar change in cytotoxicity and drug accumulation for CBDCA, indicating that, despite the differences in the structure of DDP and CBDCA and their rates of aquation and reaction with nucleophilic targets, these drugs are affected similarly by CTR2.
As noted with respect to cytotoxicity, the knockdown of CTR2 had somewhat different effects on the cellular accumulation of DDP and CBDCA versus Cu. Several points are noteworthy. First, complete loss of CTR1 expression only reduced whole-cell Cu accumulation at 1 hour by 31%, indicating that there is another route of Cu accumulation other than just CTR1. Second, unlike the situation for DDP, the effect of knocking down CTR2 on Cu accumulation was dependent on the status of CTR1. Knockdown of CTR2 increased Cu accumulation only when CTR1 was also expressed. In the absence of CTR1 expression, the reduction in CTR2 produced only a small additional increase in uptake. Interestingly, the increase in DDP accumulation that accompanied knockdown of CTR2 was associated with an increase in cytotoxicity, whereas this was not true for Cu. These observations indicate that CTR2 interacts differently with DDP than with Cu.
Knockout of CTR1 has been shown to reduce the rate of DDP accumulation in both yeast and mammalian cells (28
). Because a reduction in uptake was observed at the earliest measurable time (5 minutes), this was interpreted as indicating an effect on influx. However, the detailed measurements of efflux made using ICP-MS in the current study reveal that there is a very rapid phase of DDP efflux, an observation that confirms results obtained using less sensitive atomic absorption spectroscopic measurements many years ago (46
). The observation that loss of CTR1 resulted in a substantial increase in the rate of initial efflux indicates that CTR1 functions to retain DDP in the cell and raises the question of whether the impaired influx observed in CTR1−/−
cells is actually due to an increased rate of efflux.
How can the effects of knocking down CTR2 on Cu uptake be explained in light of what is already known about the function of CTR1 and CTR2 in mammalian cells? CTR2 is primarily localized to the late endosome and lysosome compartments (36
) where, by inference from the results obtained in yeast, it is thought to participate in the export of Cu to the cytoplasm. However, in Cu-starved COS-7 cells transfected with a CTR2 expression vector, some CTR2 was observed at the plasma membrane and these cells exhibited enhanced Cu uptake but no change in Cu efflux (36
), indicating that either CTR2 transports Cu across the plasma membrane or it enhances intracellular sequestration. We observed that knockdown of CTR2 in the CTR1+/+
cells increased Cu accumulation by 78% at 1 hour, indicating that CTR2 normally functions to limit the accumulation of Cu. The fact that this was not observed when CTR2 was knocked down in the CTR1−/−
cells indicates that it was specifically dependent on CTR1. This suggests that CTR2 either regulates the level of CTR1 or its ability to mediate Cu influx. CTR2 has been reported to partially colocalize with CTR1 on intracellular vesicles of mammalian cells (37
), consistent with possible regulatory role for the former by the latter.
The results reported here provide only an outline of how CTR1 and CTR2 modulate the accumulation of DDP and CBDCA. Because even DDP, the smaller of these two drugs, is too large to fit through the pore that trimeric CTR1 has been reported to form in the plasma membrane (47
), and because DDP seems to trigger rapid macropinocytosis of CTR1 (48
), our current hypothesis is that DDP and CBDCA bind to the extracellular domain of CTR1 and enter via an endocytotic process that delivers them to intracellular vesicles. One possibility is that CTR2 functions sequentially with CTR1 in the drug influx process by transporting DDP out of vesicles and into the cytoplasm. However, there are several problems with this model. First, one would expect knockdown of CTR2 to have little effect when CTR1 was not expressed and that a limited amount of DDP would enter the cell by endocytosis, whereas the actual observation was that the CTR2 knockdown increased DDP uptake in both CTR1+/+
cells. Second, knockdown of CTR2 had no effect (at all) on the absolute amount of DDP that accumulated in intracellular vesicles; changes in the fraction of DDP in the vesicular compartment were due to changes in uptake in other parts of the cell. Finally, how CTR2 might export DDP from intracellular vesicles is uncertain. Like CTR1, CTR2 may also form trimers (37
) whose pore size is likely to be too small to accommodate DDP so that one would have to hypothesize some other mechanism by which CTR2 transfers DDP out of vesicles. The finding that knockdown of CTR2 enhanced the accumulation of DDP, at even just 5 minutes, suggests that CTR2 is restraining initial influx in some manner either through regulation of the uptake at the level of the plasma membrane or by regulating trafficking to intracellular sites. The fact that CTR2 has no effect on drug efflux lends credence to the possibility that CTR2 acts to mediate DDP influx rather than to restrain efflux.
Given that small changes in CTR2 expression produced relatively large changes in sensitivity due to the cytotoxic effect of DDP, we were interested in whether differences in CTR2 expression in human tumor cell lines were linked to differences in intrinsic sensitivity to DDP. In a panel of six human ovarian carcinoma cell lines, we found a significant correlation between DDP sensitivity and both CTR2 mRNA level (r2 = 0.97) and CTR2 protein level (r2 = 0.71). This provides the impetus for further studies focused on how CTR2 levels change during the acquisition of resistance that accompanies repeated exposure to DDP both in vitro and in vivo. The fact that modest reductions in the level of CTR2 protein were accompanied by quite large increases in sensitivity to both DDP and CBDCA suggests that CTR2 expression may also be a useful biomarker of initial responsiveness to therapy with these drugs in patients with ovarian cancer and that strategies directed at selectively modulating the expression of this transporter may be therapeutically useful.
Cisplatin (DDP) and carboplatin (CBDCA) are two of the most widely used chemotherapeutic agents. Despite years of clinical use, the mechanism by which these drugs enter tumor cells, are distributed within, and exported from cells remains poorly understood. We and others have previously documented that the copper transporters CTR1, ATP7A, and ATP7B and the intracellular chaperone ATOX1 have major effects on the cellular pharmacology of DDP and CBDCA. In this study, we show that another copper transporter, copper transporter 2 (CTR2), also regulates accumulation and cytotoxicity of these drugs. The magnitude of the effect is quite large. This result provides further evidence that the platinum-containing drugs use the copper homeostasis proteins to enter tumor cells and access key targets. Our findings also provide the basis for enhancing the efficacy of DDP and CBDCA through pharmacologic regulation of CTR2 expression.