Cisplatin-Induced Downregulation of OCTN2 Affects Carnitine Wasting

Published OnlineFirst September 21, 2010; DOI: 10.1158/1078-0432.CCR-10-1239 Published OnlineFirst on September 21, 2010 as 10.1158/1078-0432.CCR-10-1239

Cancer Therapy: Preclinical Clinical Cancer Research Cisplatin-Induced Downregulation of OCTN2 Affects Carnitine Wasting

Cynthia S. Lancaster1, Chaoxin Hu1, Ryan M. Franke1, Kelly K. Filipski1, Shelley J. Orwick1, Zhaoyuan Chen1, Zhili Zuo2, Walter J. Loos3, and Alex Sparreboom1

Abstract Purpose: Carnitine is an essential cofactor for mitochondrial fatty acid oxidation that is actively reab- sorbed by the luminal transporter Octn2 (Slc22a5). Because the nephrotoxic agent cisplatin causes urinary loss of carnitine in humans, we hypothesized that cisplatin may affect Octn2 function. Experimental Design: Excretion of carnitine and acetylcarnitine was measured in urine collected from mice with or without cisplatin administration. The transport of carnitine was assessed in cells that were transfected with OCT1 or OCT2. The effect of cisplatin treatment on gene expression was analyzed using a mouse GeneChip array and validated using quantitative reverse transcriptase-PCR. Results: In wild-type mice, urinary carnitine excretion at baseline was ∼3-fold higher than in mice lacking the basolateral cisplatin transporters Oct1 and Oct2 [Oct1/2(−/−) mice], indicating that carnitine itself undergoes basolateral uptake into the kidney. Transport of carnitine by OCT2, but not OCT1, was confirmed in transfected cells. We also found that cisplatin caused an increase in the urinary excretion of carnitine and acetylcarnitine in wild-type mice but not in Oct1/2(−/−) mice, suggesting that tubular transport of cisplatin is a prerequisite for this phenomenon. Cisplatin did not directly inhibit the transport of carnitine by Octn2 but downregulated multiple target genes of the transcription factor peroxisome proliferator activated receptor α, including Slc22a5, in the kidney of wild-type mice that were absent in Oct1/2(−/−) mice. Conclusion: Our study shows a pivotal role of Oct1 and Oct2 in cisplatin-related disturbances in carnitine homeostasis. We postulate that this phenomenon is triggered by deactivation of peroxisome proliferator activated receptor α and leads to deregulation of carnitine-shuttle genes. Clin Cancer Res; 16(19); 4789–99. ©2010 AACR.

Cisplatin is among the most widely used anticancer circulating blood level, and this disproportionate accu- agents; however, its clinical use is limited by its poten- mulation of cisplatin in kidney tissue contributes to tial to induce severe nephrotoxicity. Cisplatin concentra- nephrotoxicity (1). Recent studies have indicated that tion in proximal tubular cells is about five times the the initial uptake of cisplatin into renal tubular cells is mediated by the organic cation transporter OCT2 (2), and that mice lacking the orthologue transporters Authors' Affiliations: 1Department of Pharmaceutical Sciences, St. Jude Oct1 and Oct2 [Oct1/2(−/−) mice] are protected from Children's Research Hospital, Memphis, Tennessee; 2Curtin Health Innovation Research Institute, Western Australian Biomedical Research experiencing severe cisplatin nephrotoxicity (3, 4). Fur- Institute, School of Biomedical Sciences, Curtin University of Technology, thermore, various physiologic hallmarks of cisplatin Perth, Australia; and 3Department of Medical Oncology, Erasmus MC-Daniel den Hoed Cancer Center, Rotterdam, the Netherlands nephrotoxicity in humans, such as changes in blood urea nitrogen, serum creatinine, and glucose (5), were Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). also specifically altered in wild-type mice undergoing cis- Current affiliation for K.K. Filipski: Cancer Prevention Fellowship platin treatment (3, 4). Program, The National Cancer Institute, Bethesda, Maryland. Decreased glucose levels (hypoglycemia) can occur when Current affiliation for C. Hu: Department of Pathology, Johns Hopkins fatty acid oxidation is defective, leading to glucose con- University School of Medicine, Baltimore, Maryland. sumption without regeneration through gluconeogenesis.

Current affiliation for Z. Chen: TDM Pharmaceutical Research, Newark, Carnitine (also known as vitamin BT; β-hydroxy-γ- Delaware. trimethylaminobutyric acid) is a hydrophilic zwitterion C.S. Lancaster and C. Hu contributed equally to this work. that plays an essential role in this process by the trans- Corresponding Author: Alex Sparreboom, St. Jude Children's Research fer of long-chain fatty acids inside mitochondria for β- Hospital, 262 Danny Thomas Place, CCC, Room I5308, Memphis, TN 38105. Phone: 901-595-5346; Fax: 901-595-3125; E-mail: alex. oxidation, and as such is critical in the production of [email protected]. cellular energy (6). This pathway of carnitine-dependent doi: 10.1158/1078-0432.CCR-10-1239 transport of fatty acids to mitochondria consists of carni- ©2010 American Association for Cancer Research. tine palmitoyltransferase I (Cpt1), carnitine-acylcarnitine

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Materials and Methods Translational Relevance Chemicals and reagents Carnitine (vitamin BT), an essential cofactor for mi- [3H]Carnitine (80 Ci/mmol) and [14C]tetraethylammo- tochondrial fatty acid oxidation, is maintained at nium (55 mCi/mmol; TEA) were purchased from Ameri- steady levels in the blood by a rescue mechanism that can Radiolabeled Chemicals, Inc. Unlabeled carnitine, involves renal tubular resorption of filtered carnitine acetylcarnitine, TEA bromide, and cisplatin were purchased by OCTN2, a luminal organic cation transporter. The from Sigma. Deuterated carnitines were obtained from physiologic significance of OCTN2 has been confirmed Cambridge Isotope Laboratories. Cisplatin-glutathione by the identification of mutations that cause a poten- conjugates were produced by incubating glutathione tially lethal autosomal recessive disease known as pri- (3.33 mmol/L) with cisplatin (1.67 mmol/L) in a buffer mary systemic carnitine deficiency. Furthermore, containing 154 mmol/L sodium chloride and 10 mmol/L heterozygosity for OCTN2 mutations is associated with sodium phosphate (pH 7.4) at 37°C as previously de- an intermediate carnitine-deficiency phenotype, sug- scribed (12). All other compounds used in this study were gesting that even partial loss of transporter function of reagent grade or better. may be detrimental. We found that the widely used anticancer drug cisplatin causes a dramatic urinary loss of Animals carnitine in mice in a manner that is dependent on Adult (8–12-week old) male FVB wild-type mice (n = transporter-mediated uptake of cisplatin in renal tubu- 11) and Oct1/2(−/−) mice (n = 12) were obtained from lar cells. These cisplatin-related disturbances in carni- Taconic. The experimental protocols were reviewed and tine homeostasis are proposed to be triggered by approved by St. Jude Children's Research Hospital Animal deactivation of the transcription factor peroxisome Care Committee in accordance with the “Guide for the α proliferator-activated receptor- , leading to deregula- Care and Use of Laboratory Animals.” The animals were tion of OCTN2 and a number of other carnitine-shuttle acclimatized for at least 1 week before experimentation, genes. Our study is of direct relevance to humans be- fed with a standard National Institute of Nutrition diet, cause interference with OCTN2 may cause reduced car- and allowed access to tap water ad libitum. For collection nitine levels in patients treated with cisplatin and may of urine, animals were housed in metabolic cages in a contribute to treatment-related complications. temperature-controlled environment with a 12-hour reverse light cycle. After collection of a 24-hour baseline urine sample, mice were given a single i.p. injection of cisplatin at a dose of 10 mg/kg. Urine was subsequently translocase (Cact), and carnitine palmitoyltransferase II collected at 24, 48, and 72 hours after administration. (Cpt2; ref. 7; Supplementary Fig. S1). Carnitine is normal- ly maintained at a steady level in the blood through a res- Carnitine analysis cue mechanism that involves reabsorption in the kidney of Quantitative determination of carnitine and acetylcarni- filtered carnitine by the solute carrier OCTN2 (SLC22A5; tine in mouse urine was done using liquid chromatography CT1; ref. 8). The existence of this reabsorption mechanism with tandem mass spectrometric detection. In brief, 40 μL for carnitine and the occurrence of mutations in OCTN2 samples were diluted 5-fold with water containing d3- that leads to primary systemic carnitine deficiency strongly carnitine hydrochloride (10 μmol/L) and d3-acetylcarnitine support the critical physiologic importance of OCTN2 (9). hydrochloride (2 μmol/L) as internal standards, and 25 μL Indeed, metabolic abnormalities similar to human prima- aliquots were injected onto a Waters 2695 Separation ry systemic carnitine deficiency have been described in ju- Module. Chromatographic separation was achieved on a venile visceral steatosis mice (10) and shown to be caused reversed-phase column (150 × 2.1 mm, i.d.) packed with by a null mutation (1114T>G; L352R) in the mouse Octn2 a3-μm ODS-3 stationary phase (GL Sciences) using a mo- gene, Slc22a5 (11). Furthermore, heterozygosity for bile phase [an 85:15 (v/v) mixture of 0.2% of the ion- OCTN2 mutations in humans is associated with an inter- pairing agent IPCC-MS3 in water and 0.1% formic acid mediate carnitine-deficiency phenotype, suggesting that in acetonitrile] delivered at a flow rate of 0.3 mL/min. even partial loss of transporter function may be detrimen- The column effluent was monitored using a Micromass tal. In this context, it is particularly noteworthy that cis- Quattro LC triple-quadrupole mass spectrometric detector platin was previously reported to cause excessive urinary equipped with an electrospray interface and controlled by loss of carnitine in cancer patients (5), although the mech- Masslynx v4.1 software. The observed mass transitions (m/z) anism by which this occurs remains unknown. Against this in positive ion mode included 162.2→84.8 (carnitine), background, we investigated the impact of cisplatin treat- 165.2→102.7 (d3-carnitine), 204.2→84.8 (acetylcarnitine), ment on carnitine homeostasis in wild-type mice and and 207.2→84.7 (d3-acetylcarnitine). Samples were ana- Oct1/2(−/−) mice to (a) elucidate the molecular basis of lyzed along with an eight-point calibration curve ranging the putative interaction of cisplatin with carnitine-shuttle from 1 to 200 μmol/L (carnitine) or 0.25 to 50 μmol/L genes and to (b) further our understanding of treatment- (acetylcarnitine); three quality control samples were ana- related side effects associated with hypocarnitinemia. lyzed in quadruplicate; and results were interpolated on a

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quadratic regression curve with 1/x2 weighting. The percent Cellular transport studies deviation from nominal values, within-run precision, and HEK293 cells transfected with human OCTN1, human between-run precision for each analyte were always within OCTN2, mouse Octn2, or an empty pcDNA3 vector were ±3%, <10.7%, and <2.9%, respectively. kindly provided by Dr. Akira Tsuji (Kanazawa, Japan). These cells were cultivated in DMEM containing 10% fetal Gene expression analysis calf serum, 1 mg/mL G418, 100 units/mL penicillin, and RNA was extracted using the RNEasy mini kit (Qiagen). 100 μg/mL streptomycin. Stably transfected 293Flp-In RNA samples were amplified from four animals per group cells expressing OCT2 or an empty pcDNA5 vector have [wild-type mice or Oct1/2(−/−) mice], both before and at been described previously (2). These cell lines were collect- 72 hours after administration of cisplatin (10 mg/kg, i.p.), ed and reseeded in complete DMEM containing 10% fetal and then analyzed using the Mouse 430v2 GeneChip array bovine serum and hygromycin B (100 μg/mL). All uptake (Affymetrix). Additional samples were obtained from wild- and inhibition assays were done on monolayer cultures in type mice at 24, 48, and 72 hours after cisplatin adminis- six-well plates in a humidified incubator at 37°C under tration. For real-time reverse transcriptase-PCR (RT-PCR) 5% CO2 and 95% humidity, as described (2). For evaluat- analyses, RNA was reverse transcribed in samples from ing OCT1 function, Xenopus laevis oocytes (BD Biosciences) three animals per group using SuperScript III first-strand injected with transporter cRNA and water-injected controls synthesis supermix for quantitative RT-PCR (Invitrogen) were used as previously described (13). according to the manufacturer's recommendations. The relative expression levels for mRNA were measured using Computational OCTN2 docking analysis Taqman probes, Universal Master Mix, and Assay-on- An OCTN2 homology model was generated similar to Demand products from Applied Biosystems. Each reaction that described previously for OCT1 (14). The sequence was carried out in triplicate, and transcripts of each sample of human OCTN2 was taken from the SwissProt database were normalized to the housekeeping gene Gapdh. (entry O76082; http://us.expasy.org/sprot/) and submitted to the meta-server 3D-Jury (15) to identify a protein tem- Nephrotoxicity analysis plate. The template of GlpT (PDB entry: 1PW4; ref. 16) After collection of kidneys, tissues from all animals were was identified from this search. The homology module of fixed overnight in 10% neutral-buffered formalin, embed- InsightII (MSI, Inc.) and the ClustalW algorithm (17) ded in paraffin, sectioned (4 μm), and stained with hema- were then applied in sequence alignment, and the Blosum toxylin and eosin (Lab Vision). Microscopic evaluation scoring matrix (18) was used to obtain the best-fit align- was done independently by two experienced veterinary ment, which was selected according to the value of the align- pathologists blinded to the composition of the groups. ment score and to the reciprocal positions of residues that Nephrotoxicity was assessed from the percentage of ob- are important for the transport function of OCTN2 (19, served damaged tubules and graded on a five-point scale 20). The resultant structure of OCTN2 was optimized using as 0 (<10%; absent), 1 (11–25%; minimal), 2 (26–50%; a molecular mechanics method with the following para- mild), 3 (51–75%; moderate), and 4 (>75%; severe). meters: a distance-dependent dielectric constant of 1.0, non- bonded cutoff of 8 Å, Amber force field (20) and Kollman- Assessment of tubular apoptosis all-atom charges, and conjugate gradient minimization until Caspase-3 analysis was done on formalin-fixed, paraffin- 0.05 kcal/(mol Å). The minimized structure was validated embedded sections of kidney samples of four animals per using the PROCHECK as previously described (21). group after deparaffinization in xylene. Heat-induced epi- TheputativeactivesitesofOCTN2wereindicated tope retrieval was done by heating slides in a pressure based on our experimental data obtained in OCTN2- cooker at 98°C for 30 minutes in Target Retrieval buffer overexpressed HEK293 cells. The transmembrane domains (pH 9.0; DAKO). Endogenous peroxidases were blocked (TMD) 1–7 were found to be responsible for organic cation by incubating slides for 5 minutes with 3% aqueous hydro- transport and for sodium dependence in carnitine trans- gen peroxide, and nonspecific protein binding was blocked port. Carnitine transport by OCTN2 requires the linkage by incubating for 30 minutes with Superblock (Pierce). between TMD1–7 and TMD11 (19). Furthermore, the resi- The primary antibody, rabbit anti-human cleaved caspase- dues of Q180, Q207, S467, and P478 are critical for the 3 (BioCare Medical), was used at 1:100 for 60 minutes, function of OCTN2 (20). Collectively, these prior studies followed by incubation for 30 minutes with the polymer have indicated that the putative active site of OCTN2 for Rabbit on Rodent (BioCare Medical). Next, slides were carnitine recognition is identified as the cave between incubated with the chromagen 3,3′-diaminobenzidine TMD1–7 and TMD11, which is near the residues men- (DAKO) for 5 minutes and counterstained with dilute he- tioned above. Next, automated docking studies were matoxylin. In each slide, 10 random high-power fields done with GOLD 3.0 (Genetic Optimisation for Ligand (40×) were selected for immunohistochemistry scoring. Docking). A radius of 10 Å from the residue of S467, The number of cells with strong cytoplasmic or nuclear which is critical to the activity of the OCTN2, was used staining for caspase-3 was counted for each tissue to ob- to design the binding site. Thirty genetic algorithm runs tain the average number of positive cells per high-power were performed in each docking calculation. For each of field for each kidney. the genetic algorithm runs, a maximum number of

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Fig. 1. Involvement of the organic cation transporter Oct2 in carnitine homeostasis. A, urinary excretion of carnitine and acetylcarnitine at baseline in wild-type mice (n = 11) and Oct1/2(−/−)mice(n = 12). B, expression of carnitine-related transporter genes [Slc6a14 (Atb0+), Slc22a1 (Oct1, Slc22a2 (Oct2), Slc22a4 (Octn1), Slc22a5 (Octn5), Slc22a8 (Oat3), Slc22a16 (Oct6), Slc22a21 (Octn3), Slc25a20 (Cact), and Slc25a29 (Cacl; Ornt3)] and enzymes [Cdv3 (Tpp36), Cpt1a, Cpt1b, Cpt1c, Cpt2, Crat, and Crot] at baseline in wild-type mice (n = 4) and Oct1/2(−/−) mice (n =4).*,P < 0.05 versus Oct1/2(−/−)mice. C, transport of TEA (60 μmol/L; 60-min incubation), a positive control, and carnitine (2 μmol/L) in water-injected Xenopus laevis oocytes and oocytes injected with organic cation transporter 1 (OCT1) cRNA. D, transport of TEA (60 μmol/L; 30-min incubation), a positive control, and carnitine (2 μmol/L) in 293Flp-In cells transfected with an empty pcDNA5 vector or organic cation transporter 2 (OCT2). E, inhibition of OCT2-mediated transport of TEA (2 μmol/L) by cimetidine (1 mmol/L), a positive control, and carnitine (1 mmol/L). *, P < 0.05 versus vehicle control. All data represent mean (columns) and SEM (error bars).

150,000 operations were performed on a population of total number for additional caspase-3 and microarray anal- 100 individuals, with a selection pressure of 1.1. Opera- yses, and real-time RT-PCR analyses were done in three of tor weights for crossover, mutation, and migration were these animals. All data are presented as mean and SEM, un- set to 95, 95, and 10, respectively. The GoldScore fitness less otherwise stated. Statistical analyses were done using a function was used to identify the best-fitting binding two-tailed t test (two groups) or a one-way ANOVA (multi- mode. An in silico screen was done on the DrugBank ple groups), after confirming normality using the Shapiro- database (http://www.drugbank.ca/), which includes Wilk W test. P < 0.05 was considered statistically significant. >1,480 Food and Drug Administration–approved small- All statistical calculations were done using the software pack- molecule drugs and >3,200 investigational agents. age NCSS v.2004 (Number Cruncher Statistical System).

Statistical analysis Results All in vitro experiments were done three times at least in triplicate. The animal experiments were done three times Carnitine undergoes OCT2-mediated basolateral at least triplicate. Urinary excretion and histology data were uptake in renal cells available in 11 wild-type mice and 12 Oct1/2(−/−)mice. We recently reported that Oct1 and Oct2 in mice are Four mice in each group were randomly selected from the key proteins involved in the transfer of cisplatin from

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the circulation into renal tubular cells (3). Based on this that carnitine was not transported by OCT1 (Fig. 1C), finding, we hypothesized that the ability of cisplatin to af- but that the intracellular uptake of carnitine was signifi- fect carnitine homeostasis is possibly indirectly influenced cantly increased in cells overexpressing OCT2 (Fig. 1D). by Oct1/Oct2 deficiency. While addressing this question, Furthermore, carnitine itself caused ∼40% inhibition of we made the serendipitous observation that the baseline OCT2 function as assessed by changes in the transport of urinary excretion of carnitine and its acetylated form, acet- TEA (Fig. 1E), a known OCT2 substrate. ylcarnitine, was about ∼3-fold lower in Oct1/2(−/−) mice than in wild-type mice (Fig. 1A), although the urine flow Cisplatin-induced carnitine wasting is dependent rate was the same (0.859 ± 0.152 versus 1.11 ± 0.093 mL/ on Oct1/Oct2 24 hours; P = 0.19). Analysis of kidneys from Oct1/2(−/−) Experiments done in wild-type mice indicated that even a mice revealed that the expression of genes previously im- single administration of cisplatin (10 mg/kg) resulted in plicated in renal carnitine handling, including Slc22a4 significant increases in the urinary excretion of carnitine (Octn1), Slc22a5 (Octn2), Slc22a21 (Octn3), and Slc22a8 (Fig. 2A) and acetylcarnitine (Fig. 2B) within the first days (Oat3; ref. 22), was not changed compared with wild-type after dosing. Within this time frame, cisplatin treatment in mice (Fig. 1B). This suggests that carnitine itself may un- wild-type mice was not associated with significant changes dergo carrier-mediated basolateral uptake in renal tubular in urinary creatinine (baseline, 0.47 ± 0.21 mg/24 hours; cells of wild-type mice by Oct1 and/or Oct2. To confirm day 1, 0.46 ± 0.02 mg/24 hours; day 2, 0.50 ± 0.15 mg/24 this possibility, we assessed transport of carnitine in model hours; P = 0.93), suggesting that membrane damage was not systems overexpressing these proteins in vitro.Wefound the cause for the concurrently observed urinary carnitine

Fig. 2. Influence of cisplatin on carnitine homeostasis and nephrotoxicity. Effect of cisplatin (10 mg/kg, i.p.) on urinary carnitine (A) and acetylcarnitine (B) loss in wild-type mice (n = 11) and Oct1/2(−/−) mice (n = 12) within the first 3 d after treatment. Data represent mean (columns) and SEM (error bars). *, P < 0.05 versus baseline (time zero). C, comparative cisplatin-related nephrotoxicity in wild-type mice and Oct1/2(−/−) mice 72 h after the administration of cisplatin (10 mg/kg, i.p.) from representative animals. Severe renal tubular necrosis (>75% of the cortex affected per section), characterized by dilated tubules filled with necrotic tubular epithelial cells, was observed in kidneys of all wild-type mice n = 11) but in none of the Oct1/2(−/−) mice (n = 12). D, immunohistochemistry for caspase-3, a marker of apoptosis. Quantification of the number of positively labeled cells within 10 high-power fields (40×) from the outer cortex indicated an average value of 11.7 ± 0.638 for wild-type mice (n =4) and 4.95 ± 0.330 for Oct1/2(−/−) mice (n =4; P = 0.000082). The circles indicate representative positively labeled cells and squares represent areas of artifact.

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Fig. 3. Influence of cisplatin on mouse Octn2 and human OCTN2 function. A, transport of carnitine (10 nmol/L) and TEA (60 μmol/L) in HEK293 cells transfected with an empty pcDNA3 vector, human OCTN1 (hOCTN1), human OCTN2 (hOCTN2), and mouse Octn2 (mOctn2). B, concentration dependence of carnitine transport in HEK293 cells transfected with an empty pcDNA3 vector (triangles) and human OCTN2 (circles). The solid line represents OCTN2-mediated carnitine transport, with an observed Michaelis-Menten

(Km) constant of 3.43 ± 0.38 μmol/L. C, inhibition of carnitine (10 nmol/L; 30 min) transport by mouse Octn2 (mOctn2) and human Octn2 (hOCTN2) in the presence of vinorelbine (60 μmol/L), a positive control, cisplatin (100 μmol/L), cisplatin-glutathione conjugates (Pt-GSH; 100 μmol/L equivalent), carboplatin (100 μmol/L), or oxaliplatin (100 μmol/L). *, P < 0.05 versus control. D, lack of cisplatin (30-min incubation) transport in HEK293 cells transfected with mouse Octn2 (mOctn2) and human OCTN2 (hOCTN2) compared with cells transfected with an empty pcDNA3 vector. All data represent mean (columns or symbols) and SEM (error bars).

changes. Cisplatin-induced changes in urinary carnitines pared with human OCTN2 (Fig. 3A) and that transport were absent in Oct1/2(−/−) mice (Fig. 2A and B), suggesting by human OCTN2 was saturable with a Michaelis-Menten that renal tubular transport of cisplatin by Oct1 and Oct2 is a constant of 3.43 μmol/L (Fig. 3B). Subsequent studies fo- prerequisite for treatment-related disturbances in carnitine cused specifically on the influence of cisplatin on the func- excretion. All wild-type mice developed severe nephrotoxici- tion of mouse Octn2 and human OCTN2. Although ty (>75% damaged tubules) by day 3, as exemplified by in- cisplatin slightly inhibited carnitine transport by both creases in the number of dilated tubules filled with necrotic transporters in vitro (Fig. 3C), the degree to which OCTN2 epithelial cells (Fig. 2C), and enhanced apoptosis (caspase-3 function was affected was less substantial than that ob- activity) compared with Oct1/2(−/−)mice(Fig.2D). served in the presence of vinorelbine used as a positive The cumulative, baseline-corrected urinary loss of total control.4 A similar lack of OCTN2-inhibitory ability was carnitines within the first 3 days of cisplatin administration noted for carboplatin and oxaliplatin, consistent with was dramatically increased in mice experiencing severe the notion that neither cisplatin (Fig. 3D) nor these two nephrotoxicity by day 3 compared with those that did not platinum analogues are themselves transported substrates (4.61 μmol versus 0.424 μmol; P = 0.014). (23). An in silico docking analysis on the carnitine-binding site within the OCTN2 protein confirmed a relatively low Cisplatin does not directly inhibit OCTN2 activity predicted affinity for cisplatin (affinity score 39.83) com- We next tested the possibility that cisplatin inhibits re- pared with the top-ranking agent in the DrugBank database, nal tubular transporters involved in the reabsorption of desloratadine (affinity score 70.58), a known highly potent carnitine and acetylcarnitine. Functional characterization inhibitor of OCTN2 (ref. 24; Supplementary Table S1). of transfected HEK293 cells confirmed that carnitine was In vivo, cisplatin is known to undergo extensive intracel- more efficiently transported by OCTN2 than by the related lular metabolic transformation following dissociation of solute carrier, OCTN1 (Fig. 3A), although both transporters have a substrate preference for carnitine relative to the prototypical cation TEA. In addition, we found that 4 C. Hu, et al. Inhibition of OCTN2-mediated carnitine transport by anti- carnitine transport by mouse Octn2 was reduced com- cancer drugs, in preparation.

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the chlorides from the molecule and can form glutathione and Slc22a21 (Octn3) was not affected by cisplatin treat- adducts (12), which subsequently accumulate in urine (25). ment (data not shown). A subsequent time-course anal- However, at equimolar concentrations (100 μmol/L), ysis in samples obtained from wild-type mice revealed the presence of cisplatin-glutathione adducts did not result a significant downregulation of Octn2 mRNA as early in dramatically altered carnitine transport by OCTN2 com- as 24 hours after cisplatin administration (Fig. 4B). In pared with unconjugated cisplatin. To further rule out the contrast to Octn2, we noted a temporal induction of effect of in vivo cisplatin metabolites in OCTN2 function, some carnitine-palmitoyltransferases involved in the for- carnitine transport was assessed in the presence of urine ex- mation of acetylcarnitine, in particular Cpt1c mRNA tracts from patients treated with cisplatin (2). The extent of (∼60-fold by day 2), although expression was normal- inhibition of OCTN2-mediated carnitine transport by these ized or even reduced by day 3 compared with baseline extracts was ∼58%, but this inhibition was also noted in levels (Fig. 4B). samples taken before cisplatin administration (data not shown). In line with a similar recent observation (26), this Discussion result suggests that the observed inhibition by human urine can be explained by carnitine(s) in the samples regardless This study shows that the nephrotoxic agent cisplatin of the presence of cisplatin and/or cisplatin metabolites. can dramatically affect urine levels of carnitine, an essential cofactor for mitochondrial fatty acid oxidation, Cisplatin-induced expression changes of through a process that involves downregulation of the ex- carnitine genes pression of Octn2, a transporter involved in tubular reab- As a next step toward understanding the mechanism sorption of filtered carnitine. In addition, we found that underlying the Oct1/Oct2–dependent changes in urinary cisplatin altered the expression of Cpt1 isoforms, the carnitine and acetylcarnitine by cisplatin, we performed rate-limiting carnitine-palmitoyltransferases required for transcriptional profiling of kidney biopsies following cis- mitochondrial fatty acid oxidation and energy production. platin administration in vivo. Several well-characterized The cisplatin-induced changes in urinary carnitine and carnitine genes, such as Slc22a5 (Octn2) and carnitine- gene expression were completely absent in Oct1/2(−/−) palmitoyltransferases (Cpt1a, Cpt1b,andCpt2), showed mice, suggesting that transport of cisplatin itself from strong repression in kidneys of treated wild-type mice the circulation into renal tubular cells is a prerequisite by day 3, whereas their alteration was weaker or not for treatment-related disturbances in carnitine homeosta- detected in treated Oct1/2(−/−) mice (Fig. 4A). Expres- sis. The current study complements previous knowledge sion of the carnitine transporter genes Slc22a4 (Octn1) on the interaction of cisplatin with renal organic cation

Fig. 4. Influence of cisplatin on renal expression of carnitine-shuttle genes. A, real-time RT-PCR analyses for Slc22a5 (Octn2); Cpt1a, Cpt1b, Cpt1c, and Cpt2 done on kidney biopsies taken before (untreated) and 72-h after cisplatin (10 mg/kg, i.p.; treated) administration to wild-type mice (n = 3) and Oct1/2(−/−) mice (n = 3). Note the difference in scale for the Y axes. *, P < 0.05 versus untreated. B, additional analyses were done on samples obtained from wild-type mice at baseline and at 24, 48, and 72 h after cisplatin (10 mg/kg, i.p.) administration (n = 4 per time point). Note the difference in scale for the Y axes. *, P < 0.05 versus untreated. All data were normalized to the housekeeping gene Gapdh and expressed as a percentage relative to untreated wild-type, which was set at 100%. Data represent mean (columns) and SEM (error bars).

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Fig. 5. Proposed pathway of urinary carnitine loss by cisplatin in wild-type mice (A) but not in Oct1/2(−/−) mice (B). See Discussion for details. Acox1, acyl-CoA oxidase; Cpt, carnitine palmitoyltransferase; Ctr1, copper transporter 1 (Slc31a1); Cyp2e1, cytochrome P450 isoform 2e1; IL-1β, interleukin-1β; P38, P38 mitogen-activated protein kinase (P38 MAPK, Mapk14); Oct, organic cation transporter; Octn2, novel organic cation transporter 2; Ppar-α, peroxisome proliferator activated receptor-α; Pgc1, peroxisome proliferator activated receptor-γ-coactivator-1 (Ppargc1α); PPRE, peroxisome proliferator response element; ROS, reactive oxygen species; Rxr-α, retinoid X receptor; Tnf-α, tumor necrosis factor-α; Tnfr2, tumor necrosis factor receptor 2 (Tnfrsf1a).

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transporters and provides further mechanistic insight into observed phenomena. For example, we found that the ad- the role these proteins play in drug-related side effect ministration of cisplatin was not associated with substan- profiles. tial concurrent changes in urinary excretion of creatinine. Over the last few decades, a number of studies have es- The observed changes in urinary carnitine excretion in- tablished that drug-induced alteration of the function of duced by cisplatin in wild-type mice are congruent with a proteins involved in carnitine homeostasis can lead to del- previous observation that fatty acid oxidation is inhibited eterious phenotypic changes. An example of this has been in a mouse model of cisplatin-induced renal failure (33). recently reported for the nephrotoxic antibiotic cephalori- Because both carnitine and acetylcarnitine are transported dine (27). In particular, these studies have shown that substrates of Octn2 (34), it is likely that the observed cephaloridine increases the fractional renal excretion of cisplatin-induced downregulation of this transporter in carnitine, presumably due to interference with a reabsorp- wild-type mice, but not in Oct1/2(−/−) mice, contributes tion process in the kidney. Similar phenomena have been to the wasting of carnitines. In addition, the Oct1/Oct2- described with usage of the antiepileptic valproate (28) genotype–dependent changes in the expression of carnitine- and the antibiotic pivampicillin (29). Several of these palmitoyltransferases following cisplatin administration agents share a significant structural similarity with carnitine are in line with the finding that cisplatin initially increases in that they also contain quaternary nitrogens and exist as but then subsequently decreases the activity of both mito- zwitterions under physiologic conditions. Although not chondrial Cpt1 and peroxisomal acyl-CoA oxidase (acyl- definitively established, it is plausible that in many in- CoA; ref. 33). Because Cpt1 is the rate-limiting enzyme stances, including cephaloridine (30), the underlying in fatty acid oxidation, the observed temporal expression mechanism involved in the effects of these agents on carni- changes induced by cisplatin can explain why the initially tine homeostasis is related to direct inhibition of OCTN2 occurring phenotypic alterations are noted with acetylcar- through a competitive mechanism (24). nitine, and not with carnitine. Paradoxically, of the three Interestingly, in a previous study involving a small cohort known Cpt1 enzymes, we found that renal expression of of adult patients, treatment with cisplatin was also associ- Cpt1c, previously believed to be localized exclusively in ated with a significant increase in the urinary excretion of the central nervous system (35), was exceptionally suscep- carnitine, which eventually normalized about 7 days after tible to cisplatin treatment. However, unlike Cpt1a and discontinuing therapy (5). Similar observations have been Cpt1b, Cpt1c is unable to catalyze the acyl transfer from fatty made in patients undergoing combination chemotherapy acyl-CoAs to carnitine (36), suggesting that it may lack a di- with ifosfamide-doxorubicin-cisplatin (31), paclitaxel- rect role in the observed cisplatin-induced carnitine changes. carboplatin, or vinorelbine-carboplatin (32). In the case It is unclear at present whether the urinary carnitine of cisplatin treatment, the increases in urinary carnitine changes induced by cisplatin and the subsequent inhibi- were hypothesized to be due to inhibition of active tubu- tion of fatty acid oxidation constitutes an adaptive lar reabsorption of carnitine and acylcarnitines, with aver- metabolic response to renal tubular injury or, alternatively, age losses amounting to as much as 1 mmol of total whether cisplatin nephrotoxicity is ultimately the result of carnitine per day (5). The reported extent of urinary car- reduced expression of Octn2 (and Cpt1), thereby affecting nitine loss in patients undergoing cisplatin chemotherapy local carnitine stores and altering intracellular levels of tox- and the occurrence of peak changes on day 2 after drug ic long-chain fatty acid intermediates. The possibility that administration closely match our current observations in carnitine loss and the associated gene expression changes wild-type mice. This finding supports the contention that are coincidental and not causatively linked with nephro- the mouse is an appropriate animal model to further toxicity is suggested by the finding that these phenomena study the mechanisms and therapeutic implications of are delayed relative to changes in urinary activity of N-acetyl- this phenomenon. β-D-glucosaminidase, an early marker of renal tubular Although cisplatin could theoretically increase renal ex- injury (42). We are planning additional studies involving cretion of carnitine as a result of transport inhibition, this carnitine-deficient rodent models, such as juvenile visceral possibility is less likely considering the fact that urinary steatosis mice that lack functional Octn2, to resolve this issue. changes for carnitine itself were not observed before day 2 It is noteworthy in this context that a link between cisplatin after drug administration, by which time the major fraction toxicity and carnitine wasting has recently been suggested in of the cisplatin dose (>95%) has already been excreted (3). a number of investigations demonstrating that exogenous Furthermore, using cellular models, we found that cisplatin carnitine supplementation alleviates cisplatin-induced did not substantially inhibit the direct transport of carnitine injury of the kidney in mice (37) and rats (38–41). These by mouse Octn2 or human OCTN2, and that cisplatin was prior studies have generally found that carnitine strongly not itself a transported substrate under the applied condi- inhibits cisplatin-induced injury to DNA, mitochondrial tions. This lack of interaction of cisplatin with OCTN2 was dysfunction, lipid peroxidation, and apoptosis of epithelial further confirmed with a computational modeling analysis. cells in the kidney, and these effects have been typically In consideration of the specific time profile and extent of ascribed to intrinsic antioxidative, antiapoptotic, and anti- total carnitine loss associated with cisplatin treatment, it inflammatory properties of carnitine. Our current studies, is also unlikely that gross cellular damage to renal tubular however, provide support for an alternate mechanism by cells would be the principal contributing mechanism to the which exogenous carnitine supplementation can reduce

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Lancaster et al.

cisplatin nephrotoxicity. In particular, we found that carni- retinoid X receptor-α heterodimer (33), decreases the ex- tine is both a substrate and an inhibitor of OCT2, which pression of Ppar-α targets such as Octn2 and ultimately suggests that carnitine can inhibit transporter-mediated up- leads to reduced reabsorptive capacity and loss of carni- take of cisplatin in renal proximal tubular cells, and subse- tines in urine. In support of this proposed pathway, we quently ameliorate cisplatin nephrotoxicity. Support for found in a preliminary analysis that the expression of this possibility comes from our recent studies indicating key tumor necrosis factor-α–related genes such as Cxcl2 that chemical inhibitors of OCT2, such as cimetidine, can (MIP-2) and known Ppar-α target genes such as Pargc-1α be used as a strategy to prevent severe nephrotoxicity in and Acox1 (acyl-CoA oxidase) are specifically affected in mice receiving cisplatin (42). The previous observation the kidney of wild-type mice (Supplementary Table S2). that carnitine does not have an appreciable effect on the In conclusion, we report that cisplatin treatment is asso- in vivo tumoricidal action of cisplatin (37) is in line with ciated with excessive urinary loss of carnitines, and that this the notion that OCT2 is either absent or detectable at very process is dependent on renal tubular uptake of cisplatin by low levels in most tumors (3, 4), and that OCT2 inhibitors the basolateral organic cation transporters Oct1 and Oct2. do not seem to affect the accumulation of cisplatin in Unlike other agents previously linked with hypocarnitine- tumor cells (42). mia, cisplatin-induced carnitine wasting is associated with The mechanism by which cisplatin causes deregulation an unusual regulatory mechanism likely involving deacti- of Octn2 expression in the kidney of wild-type mice is not vation of the transcription factor Ppar-α and subsequent entirely clear. Recent studies have provided evidence that downregulation of the luminal carnitine transporter Octn2. the expression of mouse Octn2 is directly governed by the Additional studies are in progress using Ppar-α–deficient transcription factor peroxisome proliferator activated mice to further understand the significance of this tran- receptor α (Ppar-α), and that this process is mediated scription factor in cisplatin-related carnitine wasting and through a functional peroxisome proliferator response its pharmacodynamic implications. element located in the first intron (43). Our proposed mechanism of action of cisplatin-induced downregulation Disclosure of Potential Conflicts of Interest of Octn2 and other genes regulating fatty acid catabolism is shown in Fig. 5. Following cisplatin administration, the No potential conflicts of interest were disclosed. drug is taken up into renal tubular cells by Oct1 and Oct2, Acknowledgments with perhaps a minor role of the copper transporter Ctr1 (44), where the interaction between cisplatin and Cyp2e1 We thank David Finkelstein, John Killmar, Kelli Boyd, Peter Vogel, and results in the generation of reactive oxygen species (45). Laura Janke for expert assistance with data analysis; Sharyn Baker for critical The accumulation of reactive oxygen species then triggers, review of the manuscript; and Akira Tsuji for providing the OCTN- overexpressing cell lines. directly or indirectly, the activation of Mapk14 (p38 mitogen-activated protein kinase; ref. 46), leading to Grant Support increased production of tumor necrosis factor-α (47). As a result, the tubular expression of multiple cytokines, American Lebanese Syrian Associated Charities and USPHS Cancer including IL-1β, is increased (48), and this subsequently Center Support Grant (3P30CA021765). α The costs of publication of this article were defrayed in part by the causes a decrease in the expression of Pargc-1 , a tissue- payment of page charges. This article must therefore be hereby marked specific transcriptional coactivator of Ppar-α and its advertisement in accordance with 18 U.S.C. Section 1734 solely to obligate partner retinoid X receptor (49). The reduced indicate this fact. availability of Pargc-1α, along with the ability of cisplatin α Received 05/10/2010; revised 06/17/2010; accepted 07/04/2010; to directly impair DNA-binding activity to the Ppar- / published OnlineFirst 09/21/2010.

References 1. Pabla N, Dong Z. Cisplatin nephrotoxicity: mechanisms and renopro- 7. Nalecz KA, Miecz D, Berezowski V, Cecchelli R. Carnitine: transport tective strategies. Kidney Int 2008;73:994–1007. and physiological functions in the brain. Mol Aspects Med 2004;25: 2. Filipski KK, Loos WJ, Verweij J, Sparreboom A. Interaction of cisplatin 551–67. with the human organic cation transporter 2. Clin Cancer Res 2008; 8. Ohtani Y, Nishiyama S, Matsuda I. Renal handling of free and 14:3875–80. acylcarnitine in secondary carnitine deficiency. Neurology 1984;34: 3. Filipski KK, Mathijssen RH, Mikkelsen TS, Schinkel AH, Sparreboom A. 977–9. Contribution of organic cation transporter 2 (OCT2) to cisplatin- 9. Scaglia F, Wang Y, Singh RH, et al. Defective urinary carnitine induced nephrotoxicity. Clin Pharmacol Ther 2009;86:396–402. transport in heterozygotes for primary carnitine deficiency. Genet 4. Ciarimboli G, Deuster D, Knief A, et al. Organic cation transporter 2 Med 1998;1:34–9. mediates cisplatin-induced oto- and nephrotoxicity and is a target for 10. Koizumi T, Nikaido H, Hayakawa J, Nonomura A, Yoneda T. Infantile protective interventions. Am J Pathol 2010;176:1169–80. disease with microvesicular fatty infiltration of viscera spontaneously 5. Heuberger W, Berardi S, Jacky E, Pey P, Krahenbuhl S. Increased occurring in the C3H-H-2(0) strain of mouse with similarities to urinary excretion of carnitine in patients treated with cisplatin. Eur Reye's syndrome. Lab Anim 1988;22:83–7. J Clin Pharmacol 1998;54:503–8. 11. Lu K, Nishimori H, Nakamura Y, Shima K, Kuwajima M. A missense 6. Longo N, Amat di San Filippo C, Pasquali M. Disorders of carnitine mutation of mouse OCTN2, a sodium-dependent carnitine cotran- transport and the carnitine cycle. Am J Med Genet C Semin Med sporter, in the juvenile visceral steatosis mouse. Biochem Biophys Genet 2006;142:77–85. Res Commun 1998;252:590–4.

4798 Clin Cancer Res; 16(19) October 1, 2010 Clinical Cancer Research

Cisplatin Downregulation of OCTN2 and Carnitine Wasting

12. Goto S, Yoshida K, Morikawa T, Urata Y, Suzuki K, Kondo T. Aug- 31. Hockenberry MJ, Hooke MC, Gregurich M, McCarthy K. Carnitine mentation of transport for cisplatin-glutathione adduct in cisplatin- plasma levels and fatigue in children/adolescents receiving cisplatin, resistant cancer cells. Cancer Res 1995;55:4297–301. ifosfamide, or doxorubicin. J Pediatr Hematol Oncol 2009;31:664–9. 13. Hu S, Franke RM, Filipski KK, et al. Interaction of imatinib with human 32. Mancinelli A, D'Iddio S, Bisonni R, Graziano F, Lippe P, Calvani M. organic ion carriers. Clin Cancer Res 2008;14:3141–8. Urinary excretion of L-carnitine and its short-chain acetyl-L-carnitine 14. Perry JL, Dembla-Rajpal N, Hall LA, Pritchard JB. A three-dimensional in patients undergoing carboplatin treatment. Cancer Chemother model of human organic anion transporter 1: aromatic amino acids Pharmacol 2007;60:19–26. required for substrate transport. J Biol Chem 2006;281:38071–9. 33. Portilla D, Dai G, McClure T, et al. Alterations of PPARα and its coac- 15. Ginalski K, Elofsson A, Fischer D, Rychlewski L. 3D-Jury: a simple tivator PGC-1 in cisplatin-induced acute renal failure. Kidney Int approach to improve protein structure predictions. Bioinformatics 2002;62:1208–18. 2003;19:1015–8. 34. Wu X, Huang W, Prasad PD, et al. Functional characteristics and tis- 16. Huang Y, Lemieux MJ, Song J, Auer M, Wang DN. Structure and sue distribution pattern of organic cation transporter 2 (OCTN2), an mechanism of the glycerol-3-phosphate transporter from Escherichia organic cation/carnitine transporter. J Pharmacol Exp Ther 1999;290: coli. Science 2003;301:616–20. 1482–92. 17. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the 35. Obici S, Feng Z, Arduini A, Conti R, Rossetti L. Inhibition of hypotha- sensitivity of progressive multiple sequence alignment through se- lamic carnitine palmitoyltransferase-1 decreases food intake and quence weighting, position-specific gap penalties and weight matrix glucose production. Nat Med 2003;9:756–61. choice. Nucleic Acids Res 1994;22:4673–80. 36. Wolfgang MJ, Kurama T, Dai Y, et al. The brain-specific carnitine 18. Henikoff S, Henikoff JG. Amino acid substitution matrices from pro- palmitoyltransferase-1c regulates energy homeostasis. Proc Natl tein blocks. Proc Natl Acad Sci U S A 1992;89:10915–9. Acad Sci U S A 2006;103:7282–7. 19. Inano A, Sai Y, Kato Y, Tamai I, Ishiguro M, Tsuji A. Functional regions 37. Chang B, Nishikawa M, Sato E, Utsumi K, Inoue M. L-Carnitine inhi- of organic cation/carnitine transporter OCTN2 (SLC22A5): roles in bits cisplatin-induced injury of the kidney and small intestine. Arch carnitine recognition. Drug Metab Pharmacokinet 2004;19:180–9. Biochem Biophys 2002;405:55–64. 20. Seth P, Wu X, Huang W, Leibach FH, Ganapathy V. Mutations in nov- 38. Aleisa AM, Al-Majed AA, Al-Yahya AA, et al. Reversal of cisplatin- el organic cation transporter (OCTN2), an organic cation/carnitine induced carnitine deficiency and energy starvation by propionyl-L- transporter, with differential effects on the organic cation transport carnitine in rat kidney tissues. Clin Exp Pharmacol Physiol 2007;34: function and the carnitine transport function. J Biol Chem 1999; 1252–9. 274:33388–92. 39. Martinez G, Costantino G, Clementi A, et al. Cisplatin-induced kidney 21. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM. injury in the rat: L-carnitine modulates the relationship between AQUA and PROCHECK-NMR: programs for checking the quality of MMP-9 and TIMP-3. Exp Toxicol Pathol 2009;61:183–8. protein structures solved by NMR. J Biomol NMR 1996;8:477–86. 40. Sayed-Ahmed MM, Eissa MA, Kenawy SA, Mostafa N, Calvani M, 22. Kobayashi Y, Ohshiro N, Tsuchiya A, Kohyama N, Ohbayashi M, Osman AM. Progression of cisplatin-induced nephrotoxicity in a Yamamoto T. Renal transport of organic compounds mediated by carnitine-depleted rat model. Chemother 2004;50:162–70. mouse organic anion transporter 3 (mOat3): further substrate spec- 41. Tufekci O, Gunes D, Ozogul C, et al. Evaluation of the effect of acetyl ificity of mOat3. Drug Metab Dispos 2004;32:479–83. L-carnitine on experimental cisplatin nephrotoxicity. Chemotherapy 23. Yonezawa A, Masuda S, Yokoo S, Katsura T, Inui K. Cisplatin and 2009;55:451–9. oxaliplatin, but not carboplatin and nedaplatin, are substrates for 42. Franke RM, Kosloske AM, Lancaster CS, et al. Influence of Oct1/ human organic cation transporters (SLC22A1-3 and multidrug and Oct2-deficiency on cisplatin-induced changes in urinary N-acetyl- toxin extrusion family). J Pharmacol Exp Ther 2006;319:879–86. β-D-glucosaminidase. Clin Cancer Res 2010 [Epub ahead of print]. 24. Diao L, Ekins S, Polli JE. Novel inhibitors of human organic cation/ 43. Wen G, Ringseis R, Eder K. Mouse OCTN2 is directly regulated by carnitine transporter (hOCTN2) via computational modeling and peroxisome proliferator-activated receptor α (PPARα) via a PPRE in vitro testing. Pharm Res 2009;26:1890–900. located in the first intron. Biochem Pharmacol 2010;79:768–76. 25. Leone R, Fracasso ME, Soresi E, et al. Influence of glutathione ad- 44. Pabla N, Murphy RF, Liu K, Dong Z. The copper transporter Ctr1 ministration on the disposition of free and total platinum in patients contributes to cisplatin uptake by renal tubular cells during cisplatin after administration of cisplatin. Cancer Chemother Pharmacol 1992; nephrotoxicity. Am J Physiol 2009;296:F505–11. 29:385–90. 45. Liu H, Baliga R. Cytochrome P450 2E1 null mice provide novel 26. Haschke M, Vitins T, Lude S, et al. Urinary excretion of carnitine as a protection against cisplatin-induced nephrotoxicity and apoptosis. marker of proximal tubular damage associated with platin-based Kidney Int 2003;63:1687–96. antineoplastic drugs. Nephrol Dial Transplant ;25:426–33. 46. Bragado P, Armesilla A, Silva A, Porras A. Apoptosis by cisplatin re- 27. Ganapathy ME, Huang W, Rajan DP, et al. β-Lactam antibiotics as quires p53 mediated p38α MAPK activation through ROS generation. substrates for OCTN2, an organic cation/carnitine transporter. J Biol Apoptosis 2007;12:1733–42. Chem 2000;275:1699–707. 47. Ramesh G, Reeves WB. p38 MAP kinase inhibition ameliorates cis- 28. Ohtani Y, Matsuda I. Valproate treatment and carnitine deficiency. platin nephrotoxicity in mice. Am J Physiol 2005;289:F166–74. Neurology 1984;34:1128–9. 48. Ramesh G, Reeves WB. TNF-α mediates chemokine and cytokine 29. Holme E, Greter J, Jacobson CE, et al. Carnitine deficiency in- expression and renal injury in cisplatin nephrotoxicity. J Clin Invest duced by pivampicillin and pivmecillinam therapy. Lancet 1989; 2002;110:835–42. 2:469–73. 49. Kim MS, Sweeney TR, Shigenaga JK, et al. Tumor necrosis factor 30. Kano T, Kato Y, Ito K, Ogihara T, Kubo Y, Tsuji A. Carnitine/organic and interleukin 1 decrease RXRα, PPARα, PPARγ,LXRα,andthe cation transporter OCTN2 (Slc22a5) is responsible for renal secretion coactivators SRC-1, PGC-1α, and PGC-1β in liver cells. Metabolism of cephaloridine in mice. Drug Metab Dispos 2009;37:1009–16. 2007;56:267–79.

www.aacrjournals.org Clin Cancer Res; 16(19) October 1, 2010 4799

Cisplatin-Induced Downregulation of OCTN2 Affects Carnitine Wasting

Cynthia S. Lancaster, Chaoxin Hu, Ryan M. Franke, et al.

Clin Cancer Res Published OnlineFirst September 21, 2010.

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