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. 2013 Jul 2;110(27):11199-204.
doi: 10.1073/pnas.1305321110. Epub 2013 Jun 17.

Oxaliplatin-induced neurotoxicity is dependent on the organic cation transporter OCT2

Jason A Sprowl  1 , Giuliano Ciarimboli, Cynthia S Lancaster, Hugh Giovinazzo, Alice A Gibson, Guoqing Du, Laura J Janke, Guido Cavaletti, Anthony F Shields, Alex Sparreboom
Affiliations

Oxaliplatin-induced neurotoxicity is dependent on the organic cation transporter OCT2

Jason A Sprowl et al. Proc Natl Acad Sci U S A. .

Abstract

Oxaliplatin is an integral component of colorectal cancer therapy, but its clinical use is associated with a dose-limiting peripheral neurotoxicity. We found that the organic cation transporter 2 (OCT2) is expressed on dorsal root ganglia cells within the nervous system where oxaliplatin is known to accumulate. Cellular uptake of oxaliplatin was increased by 16- to 35-fold in cells overexpressing mouse Oct2 or human OCT2, and this process was associated with increased DNA platination and oxaliplatin-induced cytotoxicity. Furthermore, genetic or pharmacologic knockout of Oct2 protected mice from hypersensitivity to cold or mechanical-induced allodynia, which are established tests to assess acute oxaliplatin-induced neurotoxicity. These findings provide a rationale for the development of targeted approaches to mitigate this debilitating toxicity.

Keywords: chemotherapy; neuropathy; solute carriers.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Human and murine OCT2 expression as a mediator of oxaliplatin uptake and cytotoxicity. (A–C) Transport of oxaliplatin by human OCT2 (hOCT2) (A) mouse Oct2 (mOct2) (B) and mouse Oct1 (mOct1) (C) transfected in HEK293 cells (500 µM; 30-min incubations). (D) Platinum (Pt)-DNA adduct formation in HEK293 cells transfected with hOCT2 following exposure to oxaliplatin (500 µM; 30-min incubations). (E) Cell growth inhibition induced by various concentrations of oxaliplatin in HEK293 cells transfected with hOCT2 or VC. The inset shows the estimated concentration associated with 50% growth inhibition (IC50) and the associated 95% confidence intervals (95% CIs). (F) Inhibition of cellular uptake of oxaliplatin (500 µM; 30-min incubations) with cimetidine (1 mM; white bars) in transfected HEK293 cells. (G) Inhibition of 14C-oxaliplatin (2 µM) uptake with various concentrations of cimetidine in HEK293 cells transfected with hOCT2 or mOct2 (30-min incubations). The inset shows the estimated concentration associated with 50% inhibition of uptake (IC50) and the associated 95% CIs. (H) Concentration-dependence of oxaliplatin transport by mOct2 and hOCT2 transfected into HEK293 cells. (I) Concentration-dependence of cisplatin transport by hOCT2 transfected into HEK293 cells (16). Data represent the net difference in uptake observed in cells with or without the transporter. Km denotes the Michaelis–Menten constant, and Vmax denotes the maximum velocity. Data represent the mean of triplicate observations from experiments performed on at least three separate occasions, and are expressed as average percentage of uptake values in cells transfected with an empty vector (VC). Error bars represent SE. The asterisk denotes significant differences from VC (P < 0.05).
Fig. 2.
Fig. 2.
Identification of OCT2 as a transporter in dorsal root ganglia (DRG). (A) Detection of Oct2 by immunofluorescence in DRG of wild-type and Oct1/2(−/−) mice. Oct2 is depicted by green fluorescence, whereas DNA is depicted in blue (DAPI). (B) OCT2 expression detected in human DRG (Human 1 and Human 2) by RT-PCR (depicted by the 128-bp product). Oct2 expression was also detected in DRG of wild-type mice (lane 3; 306bp product) but not in DRG isolated from Oct1/2(−/−) mice.
Fig. 3.
Fig. 3.
Oxaliplatin disposition and toxicity in Oct1/2(−/−) mice. (A and B) Time course of urinary excretion (A) and total plasma concentrations (Ct) and unbound plasma concentrations (Cu) (B) in male wild-type (urinary excretion: n = 29; plasma concentrations: n = 4) and Oct1/2(−/−) mice (urinary excretion: n = 25; plasma concentrations: n = 4) following a single i.p. administration of oxaliplatin (40 mg/kg). Data are presented as the mean (symbols or bars) and SE (error bars). The asterisk denotes a significant difference from wild-type mice (P < 0.05). (C and D) Cumulative overall survival (C) and overall toxicity (D) as determined from weight changes from baseline in male wild-type and Oct1/2(−/−) mice following a single i.p. administration of oxaliplatin (40 mg/kg). Data are presented as the mean (symbols) and SE (error bars) of six observations per group. (E) Representative histopathology in wild-type and Oct1/2(−/−) mice before (“untreated”) and 72-h after administration of oxaliplatin (40 mg/kg) (“treated”). Both in wild-type and Oct1/2(−/−) mice, the main lesions were damage to the small-intestinal epithelium (40× magnification) and bone marrow hypocellularity (10× magnification). The former was associated with crypt loss, implying the prior occurrence of crypt necrosis, the latter with severe myelosuppression.
Fig. 4.
Fig. 4.
Sensitivity to cold in response to oxaliplatin. (A) Baseline values for the number of paw lifts or licks performed by wild-type or Oct1/2(−/−) mice exposed to −4 °C for 5 min. (B) The number of paw lifts or licks performed by wild-type FVB mice exposed to various temperatures (−4 °C, 4 °C, and 30 °C) for 5 min both before and after treatment with oxaliplatin (40 mg/kg). Data are presented as the mean (symbols or bars) and SE (error bars) where n represents the number of events observed. P values above the bars denote statistical comparison between baseline and after treatment.
Fig. 5.
Fig. 5.
OCT2 regulation of oxaliplatin-induced neuropathy. (A) Sensitivity to cold associated with a single dose of oxaliplatin (40 mg/kg) in wild-type and Oct1/2(−/−) mice, as determined by a cold-plate test. Data are presented as the number of paw lifts or licks at baseline and following exposure to a temperature of −4 °C for 5 min at 24 h [wild type: n = 25; Oct1/2(−/−): n = 17] or 48 h [wild type: n = 25; Oct1/2(−/−): n = 16] after drug administration. (B) Mechanical allodynia associated with a single dose of oxaliplatin (40 mg/kg) in wild-type and Oct1/2(−/−) mice, as determined by a Von Frey Hairs test. Data are presented as the force required to promote paw withdrawal in grams (g) at baseline and following 24 h [wild type: n = 11; Oct1/2(−/−): n = 11] or 48 h [wild type: n = 11, Oct1/2(−/−): n = 10] after drug administration. (C) Change in sensitivity to cold in wild-type (n = 7) and Oct1/2(−/−) (n = 7) mice 24 h after receiving oxaliplatin (5 mg/kg) alone or in combination with cimetidine (30 mg/kg, i.v. bolus). (D) Change in mechanical allodynia in wild-type (n = 8) and Oct1/2(−/−) (n = 7) mice 48 h after receiving oxaliplatin (5 mg/kg) alone or in combination with cimetidine (30 mg/kg, i.v. bolus). (E) Change in sensitivity to cold in wild-type mice (n = 7) 24 h after receiving oxaliplatin (5 mg/kg) alone (n = 7) or in combination with cimetidine (i.v. bolus) at a concentration of 5 mg/kg (n = 7) or 30 mg/kg (n = 9). Bars represent the mean and error bars are the SE. The asterisk denotes significant difference from baseline and between strains (P < 0.05).
Fig. 6.
Fig. 6.
Transporter genes in dorsal root ganglia of Oct1/2(−/−) mice. Comparative expression of 84 transporter genes at baseline in dorsal root ganglia of wild-type mice and Oct1/2(−/−) mice (n = 3 each). Each symbol represents an average reading for a single gene, the solid line is the line of identity, and the dotted lines are the 95% CIs. The colored symbols represent transporter genes previously implicated in oxaliplatin toxicity.
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