The striking activity of cisplatin in an otherwise fatal disease, testicular cancer, has been established by 30 years of clinical experience. However, acquired and intrinsic resistance limits its application to a relatively narrow range of tumor types. To broaden the anticancer spectrum of this platinum agent, thousands of structural analogues have been tested. Cisplatin analogues with two amine ligands, such as carboplatin and nedaplatin (approved in Japan), are cross-resistant with cisplatin (42
). Analogues with different ligands display more diverse activity profiles (3
). Notably, oxaliplatin, with DACH in place of the two amine ligands, in combination with 5-FU/leucovorin, produced response rates twice that of 5-FU/leucovorin regimens alone in the treatment of colorectal cancer (43
), against which cisplatin is inactive (5
). Efforts to understand the differences in oxaliplatin versus cisplatin antitumor activity have focused mainly on the cellular processing of cisplatin-DNA and oxaliplatin-DNA adducts (14
). Defects in mismatch repair cause modest to moderate resistance to cisplatin but not to oxaliplatin (44
). Differences in the mechanism(s) controlling cellular uptake and efflux of these platinum compounds, although rarely investigated, may also contribute to their disparate activities considering the nature of their chemical structures.
In the present study, we observed that the influx transporters OCT1 and OCT2 play a critical role in the cellular uptake and consequent cytotoxicity of oxaliplatin (; ). In contrast, the two transporters were relatively unimportant in mediating the uptake and cytotoxicity of cisplatin and carboplatin (). Overexpression of OCT1 and, more strikingly, OCT2 in transfected cells not only increased the rate of cellular platinum accumulation but also elevated the level of platinum-DNA adducts after oxaliplatin exposure (Figs. and ). These effects were blocked by known OCT inhibitors. The data strongly suggest that oxaliplatin is an excellent substrate of human OCT1 and OCT2, and the cellular uptake of platinum mediated by these transporters has ready access to the key pharmacologic target (DNA). These results are in contrast to platinum uptake mediated by human Ctr1, which seems to sequester the drug in some intracellular compartment, rendering it inaccessible to the pharmacologic target (18
). It should be noted that a slight or modest increase in cisplatin and carboplatin uptake () was observed in MDCK-hOCT1 and HEK-hOCT2 cells compared with the corresponding MOCK cells, suggesting that cisplatin and carboplatin may be very weak substrates of human OCT1 and OCT2. A more significant interaction of cisplatin with OCT2 was obtained in a previous report (28
) possibly due to higher OCT2 expression levels in the transfected cells used in that study. In contrast to the present observation, this work (28
) concluded that oxaliplatin does not interact with human OCT2 based on the observation that oxaliplatin at 100 μmol/L could not inhibit the initial uptake of 4-[4-(dimethylamino)styryl]-N
-methylpyridinium (a substrate of OCT2) in the OCT2-transfected HEK 293 cells. As discussed below, we showed that the chemical forms of oxaliplatin that actually interact with OCTs are most likely monoaquated species carrying one positive charge. These charged species represent only a minor fraction of all the species formed when oxaliplatin is dissolved in culture medium or PBS. Therefore, the failure to observe an oxaliplatin-OCT2 interaction in the previous study (28
) may have been because the concentration(s) of charged species was too low to inhibit OCT2. Alternatively, oxaliplatin may interact with a binding site on OCT2 that is distinct from the N
-methylpyridinium site; therefore, competitive inhibition may not have occurred.
It is noteworthy that expression of OCT1 or OCT2, even at low levels, may play a significant role in the cytotoxicity of oxaliplatin. We consistently observed a >3-fold increase (3.18-fold) in the IC50 value of oxaliplatin (), but not of cisplatin or carboplatin (data not shown), in HEK-MOCK cells in the presence of the OCT inhibitor cimetidine. This result is most likely due to the inhibition of intrinsic OCT1 and/or OCT2 activity in HEK 293 cells by the OCT inhibitor. Both transporters were detected in HEK-MOCK cells in PCR studies using a cycle number of 40 (data not shown). Furthermore, cimetidine consistently produced a significant decrease in the cellular uptake of oxaliplatin, but not of cisplatin or carboplatin, in HEK-MOCK and HEK-hOCT3 cells (oxaliplatin is not a substrate of OCT3; ). The possibility that cimetidine reacts with the platinum compounds and inactivates them was checked by in vitro studies, which revealed no binding (data not shown). Moreover, this explanation is unlikely to be of primary importance because we would have expected to observe similar effects of cimetidine on the cellular uptake and cytotoxicity of cisplatin and carboplatin. Taken together, the data suggest that even low levels of expression of OCT1 and OCT2 play a significant role in sensitizing cells to oxaliplatin.
Structure-activity relationship studies revealed that the nature of the amine ligand bound to platinum is important for interaction with OCTs, with an organic component being required for effective interaction. On the other hand, the structure of the leaving ligand seems to be unimportant. Our work suggests that a monoaqua derivative of oxaliplatin, specifically the monoaqua/monochloride species and not a divalent diaqua complex, is likely to be the preferred substrate of OCT1 (). These results are probably applicable to OCT2 as well, and they are consistent with previous work showing that OCTs interact with small molecular weight monovalent organic cations (19
). These studies establish a basis for the design of additional platinum complexes to facilitate the discovery of an even more detailed structure-activity relationship, which could be used to predict and optimize cellular internalization through the OCTs. We anticipate the potential to target platinum complexes for therapy against tumors that express OCT1 and OCT2.
Our structure-activity relationship studies further suggest that OCTs do not play a major role in determining the cytotoxicity of platinum compounds with two ammine ligands, such as cisplatin, carboplatin, and nedaplatin. In contrast, OCTs may be important for mediating cytotoxicity of platinum compounds with organic amine ligands (). Cell lines that are resistant to cisplatin are cross-resistant to the bis(ammine) complexes carboplatin and nedaplatin but not to the DACH compounds oxaliplatin and tetraplatin, which share a similar activity profile (3
). The contrasting activity profiles of these compounds parallel the differences in their interaction with OCTs, suggesting that interactions with OCT1 and OCT2 may explain, at least in part, disparities in the activities and tumor specificities of platinum complexes.
It is likely that the activity of oxaliplatin in colorectal cancer can be explained, at least in part, by the selective uptake via OCTs. In this study, we detected OCT1 expression in all 20 human colon cancer tissue samples and OCT2 expression in 11 of 20 tissue samples (; Supplementary Table S2). Similar levels of OCT1 were also detected in the six tested human colon cancer cell lines, although OCT2 was not observable (; Supplementary Table S2). However, both OCT1 and OCT2 expressions have been detected in another human colon cancer cell line, Caco-2 (23
). The marked differences in OCT2 expression among these tumor samples do not seem to be related to gene amplification or differences in methylation of CpG rich sequences in the promoter region.4
As has been observed previously (3
), sensitivity to oxaliplatin was greater than to cisplatin in each of the six colon cancer cell lines (). The higher activity of oxaliplatin compared with that of cisplatin in these colon cancer cells is probably a consequence of the selective uptake of oxaliplatin mediated by the intrinsic OCT1 in these cells because similar activities of oxaliplatin and cisplatin were observed in these cells when OCTs were blocked by cimetidine ().
Based on the expression of OCT1 and OCT2 in the colon cancer tissue samples and the OCT-dependent activity of oxaliplatin in the cell lines, it is reasonable to speculate that these transporters are important determinants of oxaliplatin activity in colorectal cancer. In addition, it is possible that variable expression of OCTs, especially OCT2, may account for the variability in response to oxaliplatin treatment. Further studies are required to determine whether expression levels of OCT1 and OCT2 may be used as markers for the rational selection of oxaliplatin-based versus irinotecan-based or other combination therapies for treatment of individuals with colorectal cancer. Such selection is now primarily based on side effect profiles or clinical experience (46
). Oxaliplatin-based therapy may be a better choice for patients with high levels of OCT1 and OCT2 in their tumor samples. In addition, genotyping for nonfunctional and reduced function polymorphisms of OCT1 and OCT2 may be incorporated in the decision-making process (30
Currently, platinum-based therapies are used in the treatment of a variety of tumors, including testicular cancer, ovarian cancer, small cell lung cancer, and head and neck cancers (42
). In these therapies, cisplatin is often the drug of choice because other platinum compounds, such as oxaliplatin, are not superior. However, our studies suggest that when OCT1 or OCT2 is expressed in the tumor, oxaliplatin may be a better choice. Our studies also suggest that, in addition to efflux transporters (48
), influx transporters may play a significant role in determining tumor sensitivity/resistance to anticancer agents (49
). Recently, OCT1 and OCT2 expression has been observed in several human cancer cell lines (26
), suggesting that these transporters may be expressed in the corresponding tumors. The results of this study clearly suggest the need for further investigations to determine whether expression of OCTs can provide a basis for the rational selection of platinum-based therapies.