Platinum anticancer therapy remains among the most effective in the treatment of various solid tumors. The distinct spectrum of activity of earlier-generation platinums, cisplatin and oxaliplatin, was attributed to their difference in DNA adduct formation accompanied by differences in the ability of nucleotide excision repair machinery to repair the platinum DNA adducts. However, in recent studies, we and others showed that oxaliplatin is an excellent substrate of OCTs, whereas cisplatin and carboplatin are not (10
). The studies also showed that OCTs were highly expressed in colorectal cancer cell lines and tumor samples, suggesting that interaction with OCTs may be a contributory factor for the efficacy of oxaliplatin against colon cancer.
Due to the structural similarity of oxaliplatin and picoplatin, we hypothesized that OCTs may also play a role in enhancing the antitumor effects of picoplatin. Our initial efforts sought to understand the interaction between picoplatin and OCTs. The reduction in the IC50 of picoplatin in HEK293 cells transfected with OCT1 (6.74-fold) and OCT2 (10.7-fold) compared with empty vector-transfected cells showed the potential role of these transporters in picoplatin cytotoxicity. The maximum degree of selectivity for the transporter of interest was achieved by drug exposure for 7 hours compared with 24 or 48 hours at which a slight decrease in the modification factor (selectivity) was observed. Furthermore, a dramatically higher uptake of picoplatin in these cell lines established it as an excellent substrate of OCT1 and OCT2. Results of DNA adduct formation experiments agreed well with our cytotoxicity experiments and proved that the intracellular picoplatin is available for reaction with its cellular target—DNA. These effects were blocked by known OCT inhibitors. Further mechanistic investigation of picoplatin uptake in OCT1-transfected cells suggests that picoplatin itself, along with the positively charged monoaqua complex of picoplatin and not the diaqua complex, is likely to be transported by OCT1 (Supplementary Fig. S1
). OCT3 had little effect on picoplatin cytotoxicity, consistent with its small effect in enhancing picoplatin-DNA adduct formation (). Although the exact mechanism for the low OCT3-mediated picoplatin binding to DNA is not known, it may be due to binding of picoplatin to intracellular histamine, the endogenous substrate of OCT3. Histamine-platinum complexes have been described (17
) and would reduce the availability of intracellular picoplatin for interaction with DNA. Such effects would not be observed for OCT1 and OCT2 because histamine is a poorer substrate for these transporters.
We also determined the interaction of picoplatin with Ctr1, MATE1, and MATE2K. Ctr1 has been previously shown to be responsible for cisplatin accumulation and cytotoxicity. Furthermore, cisplatin and oxaliplatin have been indicated to be good substrates for MATE1 and MATE2K, respectively (18
). All of these transporters, in our in vitro experiments, failed to show any significant contribution to picoplatin cytotoxicity (Supplementary Table S3
). Our mechanistic studies involving DNA unwinding and transcription inhibition studies have shown a slightly reduced DNA binding potency of picoplatin over cisplatin but with significantly reduced glutathione reactivity (data not shown). Cisplatin reacts with cellular thiols glutathione and metallothionine, which limit the amount of drug available to bind to its biological target, DNA. This reactivity is considered to be one of the mechanisms of resistance to cisplatin therapy. Improved safety profile of picoplatin over cisplatin, along with its reduced reactivity toward glutathione, although compromised DNA reactivity, makes it a promising clinical candidate.
Picoplatin has shown activity in a variety of solid tumors, including lung, ovarian, colorectal, and hormone-refractory prostrate cancers. Clinical data show its activity in platinum-sensitive, platinum-resistant, and platinum-refractory disease. Results of phase II clinical trials of picoplatin have shown survival benefit in ovarian, NSCLC, and SCLC patients (20
). The chemotherapeutic treatment of NSCLC by platinum agents has been hampered by rapid development of resistance (21
). Consideration of picoplatin as a potential treatment for NSCLC led us to determine the role of OCT-mediated uptake in its lung cancer efficacy. The expression of OCT1 and OCT2 was detectable in normal and tumor lung tissue samples and lung cancer cell lines, with the expression of OCT2 being the lowest (; Supplementary Table S1
). Our Western blot studies showed that OCT1 was also present in lung cancer cell lines. Reduction in cytotoxic potency of picoplatin but not that of cisplatin and carboplatin, in the presence of an OCT inhibitor in the lung cancer cell lines, corroborates the involvement of OCTs in picoplatin lung cancer efficacy. Cisplatin is effectively transported by OCT2, but its negligible expression in the lung cancer cell lines tested resulted in only minimal inhibition of its cytotoxicity.
OCT1 and OCT2 genes are highly polymorphic, and the reduced/nonfunctional polymorphic variants of OCT1 and OCT2 identified in our previous studies (13
) had significant effect on picoplatin uptake and DNA adduct formation. In particular, the common OCT1 variants 420D, G465R, and R61C showed significant reduction in cellular accumulation and DNA adduct formation of picoplatin when compared with the reference OCT1 transporter. The common OCT2 variant, A270S, exhibited increased uptake of picoplatin compared with the reference OCT2 transporter. Similar results were obtained previously in our laboratory with metformin (13
) for interaction with the same variants of OCT1 and OCT2, respectively, when compared with the reference transporter. All the variants of OCTs tested had similar levels of mRNA expression (13
). However, the levels of the proteins on the plasma membranes from some of the variants of OCT1, including R61C, are reduced presumably due to poor trafficking or altered stability of the transporter, thus leading to functional differences. Higher capacity of OCT2 variant A270S to transport metformin was alluded to be due to higher membrane protein expression compared with the reference OCT2 transporter, which might be the case with picoplatin (22
). However, data from our laboratory are not consistent with recent data (23
) and may be related to differences in the OCT2 cell lines being used between laboratories. The presence of OCT1 in lung tumor samples at high levels and the highly polymorphic nature of this gene led us to look further into its effect on picoplatin pharmacokinetics and in vivo tumor efficacy.
In vivo pharmacokinetic experiments did not reveal any statistically significant differences in the pharmacokinetic profiles of picoplatin in Oct1−/− and Oct1/2−/− mice; however, the knockout mice exhibited a trend toward higher mean plasma concentration when compared with the wild-type mice (Supplementary Table S2
; Supplementary Fig. S2
). The tissue distribution studies suggest a role of OCTs in platinum uptake in the liver, kidney, and heart (Supplementary Fig. S3
). These three tissues express substantial levels of OCT1 (liver), OCT2 (kidney), and OCT3 (heart). Our findings that picoplatin accumulation is highest in liver, kidney, and heart are consistent with the high expression levels of OCTs in the three organs. However, it is possible that species differences in the interaction of picoplatin between mouse and human orthologs of OCT1 may exist and that the effect of OCT1 on picoplatin pharmacokinetics may be more pronounced in humans. Further, we observed compensatory upregulation of OCT2 and OCT3 in the liver and kidney of Oct1−/− mice (Supplementary Fig. S4
), which may have obscured the effects of OCT1 on the tissue distribution and pharmacokinetics of picoplatin. Due to similar compensatory upregulation mechanisms pertaining to OCT2 (2- to 2.5-fold) and OCT3 (1.4- to 1.7-fold), our in vitro experiments in HCT-116 cell line using small interfering RNA (siRNA) against OCT1 failed to yield a conclusive result. However, the combination of OCT1-siRNA with an OCT inhibitor, cimetidine, and/or OCT2-siRNA indeed resulted in □ 40% reduction in picoplatin uptake (data not shown).
To establish the link between OCT1 expression and antitumor efficacy of picoplatin, we prepared xenografts of HEK293 cells stably transfected with hOCT1 and the corresponding empty vector. At initial time points of 5 and 8 days, picoplatin treatment was associated with a statistically significant reduction in the volume of OCT1 xenografts compared with control saline-treated xenografts (), which was not observed in the case of the empty vector xenografts. Similar results were obtained with oxaliplatin, a well-established substrate of OCT1. Although this trend was evident also on days 14 and 18 of picoplatin treatment initiation, it did not reach statistical significance at those time points, probably due in part to a reduction in OCT1 expression in the HEK-hOCT1 xenografts (data not shown). However, there was a trend toward reduction in tumor volume by picoplatin in the HEK-EV xenografts, suggesting that OCT1 contributes significantly to, but is not essential for, the antitumor efficacy of picoplatin.
The results of our in vitro studies and preliminary in vivo experiments highlight the significance of OCTs in picoplatin antitumor efficacy. A phase III clinical trial of picoplatin is currently under way for lung cancer. The variable expression of OCTs in tumors along with their genetic variants could contribute to any observed variation in response to picoplatin treatment. Our study has provided a basis for consideration of OCTs as possible markers for picoplatin efficacy. Development of new platinum-based anticancer agents with modification in picoplatin structure would be an interesting avenue to explore to obtain a clinical candidate with improved safety and efficacy profiles. Our data suggest that consideration of picoplatin in the treatment of colorectal cancer may be justified, as well as consideration for other cancers that express OCTs. Our data suggest that OCTs could serve as biomarkers in tumors for susceptibility to picoplatin, thus justifying thorough experimentation pertaining to the evaluation of picoplatin against OCT-expressing tumors.