Organic cation transporters are determinants of oxaliplatin cytotoxicity

Abstract

Although the platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin have similar DNA-binding properties, only oxaliplatin is active against colorectal tumors. The mechanisms for this tumor specificity of platinum-based compounds are poorly understood but could be related to differences in uptake. This study shows that the human organic cation transporters (OCT) 1 and 2 (SLC22A1 and SLC22A2) markedly increase oxaliplatin, but not cisplatin or carboplatin, accumulation and cytotoxicity in transfected cells, indicating that oxaliplatin is an excellent substrate of these transporters. The cytotoxicity of oxaliplatin was greater than that of cisplatin in six colon cancer cell lines [mean +/- SE of IC(50) in the six cell lines, 3.9 +/- 1.4 micromol/L (oxaliplatin) versus 11 +/- 2.0 micromol/L (cisplatin)] but was reduced by an OCT inhibitor, cimetidine, to a level similar to, or even lower than that of, cisplatin (29 +/- 11 micromol/L for oxaliplatin versus 19 +/- 4.3 micromol/L for cisplatin). Structure-activity studies indicated that organic functionalities on nonleaving groups coordinated to platinum are critical for selective uptake by OCTs. These results indicate that OCT1 and OCT2 are major determinants of the anticancer activity of oxaliplatin and may contribute to its antitumor specificity. They also strongly suggest that expression of OCTs in tumors should be investigated as markers for selecting specific platinum-based therapies in individual patients. The development of new anticancer drugs, specifically targeted to OCTs, represents a novel strategy for targeted drug therapy. The results of the present structure-activity studies indicate specific tactics for realizing this goal.

Figure 1

Chemical structures of platinum compounds.

Figure 2

Cytotoxicity of oxaliplatin in cells stably transfected with human OCTs. The cytotoxicity of oxaliplatin (7 hours of drug exposure) in OCT1-transfected (A), OCT2-transfected (B), and OCT3-transfected (C) cells (○) and in the corresponding MOCK cells (●) was determined as described in Materials and Methods. In addition, the cytotoxicity of oxaliplatin in OCT1-transfected (D) and OCT2-transfected (E) cells (○ and □) and in the corresponding empty vector–transfected cells (MOCK cells; ● and ■) in the presence (□ and ■) or absence (○ and ●) of an OCT inhibitor (disopyramide for OCT1 and cimetidine for OCT2) was also simultaneously determined in a similar fashion. When the OCT inhibitors were used, disopyramide (150 μmol/L) or cimetidine (1.5 mmol/L) was added to the incubation medium immediately before the addition of oxaliplatin. Lines, predicted data obtained by fitting the observed data using WinNonlin. Data from a typical experiment. Three to six independent experiments were done, and similar results were obtained. For clarity, the bars (SD) in (D) and (E) were eliminated.

Figure 3

Cellular accumulation rates of platinum after 2-hour exposure to cisplatin, carboplatin, and oxaliplatin. The cellular accumulation rates of platinum in OCT1-transfected (A), OCT2-transfected (B), and OCT3-transfected (C) cells and in the corresponding MOCK cells after incubation with cisplatin, carboplatin, and oxaliplatin in the presence (white columns) or absence (black columns) of an OCT inhibitor (disopyramide for OCT1 and cimetidine for OCT2 and OCT3) were determined as described in Materials and Methods. Briefly, MDCK cells (A) were incubated in the antibiotic-free medium containing cisplatin (6 μmol/L), carboplatin (20 μmol/L), or oxaliplatin (6 μmol/L) at 37°C and 5% CO₂ for 2 hours. For the inhibitor studies, the incubation medium also contained disopyramide (150 μmol/L). B, HEK 293 cells were incubated in the antibiotic-free medium containing cisplatin (0.3 μmol/L), carboplatin (10 μmol/L), or oxaliplatin (0.3 μmol/L) at 37°C and 5% CO₂ for 2 hours. For the inhibitor studies, the incubation medium also contained cimetidine (1.5 mmol/L). C, the study was done similarly as in (B), except that the concentrations of cisplatin, carboplatin, and oxaliplatin in the incubation medium were 2, 10, and 2 μmol/L, respectively. Columns, mean of six measurements for OCT1 and OCT2 and of three measurements for OCT3; bars, SD.

Figure 4

Platinum-DNA adduct formation after 2-hour exposure to oxaliplatin. The content of platinum bound to DNA after 2-hour exposure to oxaliplatin in the presence (white columns) or absence (black columns) of an OCT inhibitor (disopyramide for OCT1 and cimetidine for OCT2) was determined as described in Materials and Methods. Briefly, MDCK cells (A) were incubated in the antibiotic-free medium containing oxaliplatin (10 μmol/L) with or without disopyramide (150 μmol/L). B, HEK 293 cells were incubated in the antibiotic-free medium containing oxaliplatin (0.6 μmol/L) with or without cimetidine (1.5 mmol/L). After incubation at 37°C and 5% CO₂ for 2 hours, the cells were washed and pelleted. The platinum content associated with genomic DNA was determined and normalized for DNA content. Columns, mean of typical experiment done in triplicate; bars, SD. Two independent experiments were conducted, and similar results were obtained.

Figure 5

Platinum-DNA adduct formation after incubation with oxaliplatin or [Pt(R,R-DACH)(H₂O)₂]²⁺ in PB-Cl or PB-SO₄ buffer. MDCK cells were incubated with oxaliplatin (20 μmol/L) or [Pt(R,R-DACH)(H₂O)₂]²⁺ (1 μmol/L) in PB-Cl or PB-SO₄ buffer at 37°C and 5% CO₂ for 25 minutes. Oxaliplatin was freshly prepared and added to PB-SO₄ buffer immediately and to PB-Cl buffer at least 0.5 hour before cell incubation. Columns, mean of six measurements; bars, SD.

Figure 6

Expression of OCT1 and OCT2 in colon cancer cell lines and colon tissue samples. Total RNA was isolated from colon cancer cells and normal or cancerous colon tissues. The expression of OCT1 and OCT2 in these samples was detected by RT-PCR as described in Materials and Methods. Forty and 30 cycles were used for amplifying OCTs and GAPDH, respectively.