Differential transport of platinum compounds by the human organic cation transporter hOCT2 (hSLC22A2)

Abstract

Background: Solute carriers (SLCs), in particular organic cation transporters (OCTs), have been implicated in the cellular uptake of platinum-containing anticancer compounds. The activity of these carriers may determine the pharmacokinetics and the severity of side effects, including neuro- and nephrotoxicity of platinum-based chemotherapy. As decreased drug accumulation is a key mechanism of platinum resistance, SLCs may also contribute to the development of resistance. Here, we define the role of hSLC22A2 (OCT2) in the cellular uptake of platinum compounds.

Experimental approach: Human embryonic kidney (HEK) 293 cells stably expressing the hSLC22A2 gene (HEK293/hSLC22A2) were used in platinum accumulation studies. Following a 2 h exposure to various platinum compounds (100 microM), intracellular platinum levels were determined by flameless atomic absorption spectrometry.

Key results: HEK293/hSLC22A2 cells, compared with HEK293/Neo control cells, displayed significant increases in oxaliplatin (28.6-fold), Pt[DACH]Cl(2) (20.6-fold), ormaplatin (8.1-fold), tetraplatin (4.5-fold), transplatin (3.7-fold) and cisplatin (1.3-fold), but not carboplatin. SLC22A2-mediated transport could be inhibited by 1-methyl-4-phenylpyridinium. Furthermore, hSLC22A2-mediated oxaliplatin and cisplatin accumulation was time- and concentration-dependent, but non-saturable. Expression of hSLC22A2 in HEK293 cells resulted in enhanced sensitivity to oxaliplatin (12-fold) and cisplatin (1.8-fold). Although, hSLC22A2 mRNA expression was frequently found in ovarian cancer cell lines, its expression in clinical ovarian cancer specimens (n= 80) was low and did not correlate with the treatment outcome of platinum-based regimens.

Conclusions and implications: The hSLC22A2 drug transporter is a critical determinant in the uptake and cytotoxicity of various platinum compounds, particularly oxaliplatin.

Figure 1

Characterization of human embryonic kidney (HEK) 293/hSLC22A2 and HEK293/Neo control cells. (A) Relative hSLC22A2 mRNA expression was determined by real-time RT-PCR using TaqMan chemistry and expressed in arbitrary units (a.u.). Insert shows the duplicate hSLC22A2 amplification plots of HEK293/hSLC22A2 and Neo control cells. (B) Analysis of 4-[4-(diethylamino)styryl]-N-methylpyridiniumiodide (ASP⁺) accumulation in HEK293/hSLC22A2 and HEK293/Neo control cells. Cells were incubated with 1 µM ASP⁺ for 20 min at 37°C and ASP⁺ uptake was measured by FACScan flow cytometry. ASP⁺ was significantly increased in the hSLC22A2-overexpressing cells compared with the Neo control cells. (C) SLC22A2-mediated accumulation of ASP⁺ was inhibited by addition of 1-methyl-4-phenylpyridinium (MPP⁺) (30 µM). ASP⁺ uptake in HEK293/hSLC22A2 is shown in the absence of MPP⁺ and in the presence of MPP⁺.

Figure 2

Differential intracellular accumulation of platinum compounds in human embryonic kidney (HEK) 293/hSLC22A2 cells and HEK293/Neo control cells. Cisplatin (A), carboplatin (B), oxaliplatin (C), PtCL2[R,R-DACH] (D), ormaplatin (E), tetraplatin (F) and transplatin (G). Columns and error bars represent mean values ± SD of duplicate measurements of at least three independent accumulation experiments. The asterisk (*) indicates a significant difference in accumulation (P < 0.05; Student's t-test)

Figure 3

Intracellular accumulation of oxaliplatin (A–C), cisplatin (D), PtCL2[R,R-DACH] (E) and transplatin (F) in human embryonic kidney (HEK) 293/hSLC22A2 cells (solid columns) and HEK293/Neo control cells (open columns) after 2 h exposure to the different drugs (100 µM) in the presence (+) and absence (−) of 100 µM 1-methyl-4-phenylpyridinium (MPP⁺) (A,E,D,F), 100 µM tetraethylammonium (TEA) (B) or 300 µM cimetidine (C). Columns and error bars represent means ± SD of triplicate measurements and the asterisk (*) indicates a significant difference in accumulation (P < 0.05; Student's t-test).

Figure 4

Dose-dependent intracellular accumulation of oxaliplatin (A) and cisplatin (B) in human embryonic kidney (HEK) 293/hSLC22A2 cells and HEK293/Neo control cells after 2 h exposure to these platinum drugs. Values and error bars represent means ± SD of triplicate measurements.

Figure 5

Platinum sensitivity of human embryonic kidney (HEK) 293/hSLC22A2 cells compared with HEK293/Neo cells. Cytotoxicity of oxaliplatin (A) and cisplatin (B) was measured by the sulphorhodamine B assay. The curves presented were derived from typical and representative experiments and values and error bars represent means ± SD of quadruplicate measurements.

Figure 6

SLC22A2 expression in NCI-60 panel of human cancer cell lines. Relative hSLC22A2 mRNA expression was determined by real-time RT-PCR using TaqMan chemistry and expressed in arbitrary units (a.u.).