HNF4α Induced Chemosensitivity to Oxaliplatin and 5-FU Mediated by OCT1 and CNT3 in Renal Cell...

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics, Drug Transport and Metabolism

HNF4� Induced Chemosensitivity to Oxaliplatin and 5-FUMediated by OCT1 and CNT3 in Renal Cell Carcinoma

YOHANNES HAGOS,1,2 WAJA WEGNER,1 ANNETT KUEHNE,2 SASKIA FLOERL,2 VENKATA V. V. R. MARADA,1

GERHARD BURCKHARDT,1 MAJA HENJAKOVIC1

1Systemic Physiology and Pathophysiology, University Medical Center Goettingen, Goettingen 37073, Germany2PortaCellTec Biosciences GmbH, Goettingen 37073, Germany

Received 7 April 2014; revised 11 July 2014; accepted 31 July 2014

Published online 29 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24128

ABSTRACT: Increased expression of transporters-mediating uptake of antineoplastic drugs could render renal cell carcinoma (RCC) moresensitive to chemotherapy. Here, we studied the effect of hepatocyte nuclear factor 4� (HNF4�) on the expression of selected uptaketransporters in RCC lines. Organic cation transporters (OCTs) and organic anion transporters (OATs) mRNA levels in HNF4�-transfectedRCCs were measured by real-time PCR. Expression of HNF4�, �-catenin, N-cadherin, and E-cadherin was detected by immunofluorescence.OCT1, OAT2, and concentrative nucleoside transporter 3 (CNT3) were tested using tritium-labeled substrates and an apoptosis assay. MostRCC did not express uptake transporters in the absence or presence of HNF4�. In RCCNG1 cells, HNF4�-expression increased thechemosensitivity to oxaliplatin and enhanced the accumulation of methyl-4-phenylpyridinium acetate, a model substrate for OCT1.Furthermore, HNF4� enhanced OAT2 mRNA and increased caspase-3 activity upon incubation with a purported OAT2 substrate, 5-fluorouracil (5-FU). However, functional OAT2 protein was not upregulated. CNT3 mRNA was significantly elevated by HNF4�. Inhibitionof CNT3-mediated uridine uptake by 5-FU metabolite 5-fluoro-2′-deoxyuridine suggested the involvement of CNT3 in increased caspase-3activity. Our data suggest that HNF4� increases the expression of OCT1 and CNT3 in RCCNG1 cells, thereby increasing the chemosensitivityof tumor cells to oxaliplatin and 5-FU. C© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3326–3334, 2014Keywords: drug resistance; drug transport; renal transport; organic anion transporters (OAT); organic cation transporters (OCT)

INTRODUCTION

Renal cell carcinoma (RCC) is one of the 10 most common ma-lignant tumors, with highest prevalence in the United Statesand Europe.1 Nearly 70% of all cases are diagnosed as clearcell RCC of proximal tubules origin.2 Clear cell RCC is char-acterized by its chemoresistance, with limited response to con-ventional chemotherapeutic drugs.3 Heterogeneous mutationsof genes involved in angiogenesis, metabolism, and low accu-mulation of drugs are associated with the chemoresistance ofRCC cells.4 In chemoresistant cancer cells, low accumulationof anticancer drugs is associated with increased expressionof multidrug efflux transporters of the ATP-binding cassette(ABC) gene family.5 The role of multidrug resistance protein1 (MDR1, encoded by the ABCB1 gene) and other multidrugresistance-related transporters (MRPs) of the ABC family inchemoresistance of cancer cells was intensively studied in thelast three decades. The changes in expression of transportersbelonging to the solute carrier (SLC) family are possibly sim-ilarly important for chemoresistance, but have not been suffi-ciently investigated in cancer cells so far.

In epithelial cells of renal proximal tubules, members of theSLC22A family are involved in the secretion of organic cationsand anions.6,7 Organic cation transporter 2 (OCT2) is pre-dominantly localized in the basolateral membrane of proximaltubules and is involved in the transport of positively chargedendogenous substances and different drugs (e.g., metformin,

Correspondence to: Maja Henjakovic (Telephone: +49-551395901; Fax: +49-551395883; E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 103, 3326–3334 (2014)C© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

cisplatin, oxaliplatin).8 The expression of OCT1 and OCT3 isvery low or completely absent in human kidney tissue.6,9 Or-ganic anion transporter 1 (OAT1), OAT2, OAT3 in the basolat-eral membrane, and OAT4 in the luminal mediate the transportof endogenous [e.g., "-ketoglutarate, succinate, cyclic guanosinemonophosphate (cGMP)] and exogenous (e.g. methotrexate, 5-fluorouracil) substances in proximal tubules.7

In renal carcinoma cell lines, differential expression of OCTsand OATs was described.10 In our previous study, higher OCT3expression in the RCC cell line A498 in comparison with ACHNcells was identified to be responsible for enhanced chemosensi-tivity to melphalan, vincristin, and irinotecan.11 Furthermore,we showed a connection between high OCT1 expression and in-creased susceptibility to irinotecan and paclitaxel in lymphomacells.12

Several members of SLC28 (concentrative nucleoside trans-porters, CNTs) and SLC29 (equilibrative nucleoside trans-porters, ENTs) family are expressed in the kidneys and areknown to interact with drugs used in anticancer therapy.13

Only few regulators of SLC uptake transporter are so faridentified. The gene expressions of OCT1, OAT1, OAT2, andCNT1 have been shown to be regulated by hepatocyte nu-clear factor 4" (HNF4").14–17 The transcription factor HNF4"is expressed in the liver and in the proximal tubules of thekidneys.18 The influence of HNF4" on the protein expression ofOCTs, OATs, CNTs, and ENTs in renal cancer was so far notinvestigated.

The aim of this study was to elucidate the effect of HNF4"on the expression of selected renal drug transporters, espe-cially OCTs and OATs in RCC cell lines. For this purpose, weoverexpressed the transcription factor HNF4" in five different

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RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics, Drug Transport and Metabolism 3327

RCC cell lines and examined the changes of OCT1–3, OAT1–4, ENT1–3, and CNT1–3 gene expression, chemosensitivities,and transport activities. Our hypothesis was that the transcrip-tional regulator HNF4" may influence the protein expressionof OCTs and OATs, accordingly, increase the chemosensitivityof RCC cells to anticancer drugs using these transporters forentry into the cells.

MATERIAL AND METHODS

Cell Culture Conditions and Transfection

Renal cell carcinoma cell lines 786-O, A498, and ACHN wereobtained from ATCC (CRL-1932TM , CRL-7908TM , CRL-1611TM ;LGC Standards, Wesel, Germany). LN78 (human, female) andRCCNG1 (human, male) cells were derived from human clearRCC biopsies in Department of Nephrology and Rheumatology(University Medical Center Goettingen) after an appropriateethical approval. The used cell lines have been previously char-acterized and described by G. A. Muller and H. Dihazi, andobtained from the Department of Nephrology and Rheumatol-ogy, University Medical Center Goettingen, Germany.19,20

Renal cell carcinoma cell lines were cultured in Quantum263 (tumor cell medium with L-glutamine; PAA LaboratoriesGmbH, Coelbe, Germany) supplemented with 100 U/mL peni-cillin and 100 :g/mL streptomycin (PAA Laboratories GmbH)at 37◦C, 5% CO2, and 100% air humidity. For transient trans-fection, cells (1 × 105/well) were incubated up to 24 h in culturemedium without antibiotics. At a confluence of 70%–90%, cellswere transiently transfected with 0.5 :g HNF4" expressionvector (pBud-HNF4", kind gift from Holger Steinbrenner, Insti-tute for Biochemistry and Molecular Biology I, Heinrich-Heine-University, Dusseldorf, Germany, and described in Ref.21),control vector (pBudCE4.1; Life Technologies, Darmstadt, Ger-many), OAT2 expression vector (pEF5/FRT-OAT2, generatedby PortaCellTec biosciences GmbH, Goettingen) or controlvector (pEF5/FRT; Life Technologies), and CNT3 expressionvector (pCMV6-XL4-CNT3; OriGene Technologies, distribu-tor: AMS Biotechnology, Frankfurt am Main, Germany) usingLipofectamine R© 2000 (Life Technologies). After 5 h transfection,medium was changed to complete medium, and cells were incu-bated for 43 h under standard cell culture conditions followedby RNA isolation, immunofluorescence analysis or transportstudies. For determination of caspase-3 activities, transfectedcells were incubated up to 48 h with cytostatic drugs.

RNA Isolation, cDNA Synthesis, and TaqMan R© Real-Time PCR

Total RNA from transiently transfected RCCs (approximately1 × 106) was isolated using RNeasy R© Mini Kit (Qiagen, Hilden,Germany) according to manufacturer’s recommendations. Thequality and concentration of isolated RNA were determinedby Gene Quant II spectrophotometer (Amersham Biosciences,Freiburg im Breisgau, Germany). Superscript R© II ReverseTranscriptase (Life Technologies) and Oligo dT-Primer (Eu-rofin MWG Operon, Ebersberg, Germany) were used for reversetranscription of RNA.

The genes of interest were analyzed using TaqMan R©

Master-Mix (Life Technologies) and TaqMan R© Gene Expres-sion Assays (Life Technologies): HNF4", Hs00230853 m1;OAT1, Hs00537914 m1; OAT2, Hs00198527 m1; OAT3,Hs00188599 m1; OAT4, Hs00218486 m1; OCT1,Hs00427554 m1; OCT2, Hs00533907 m1; OCT3,

Hs00222691 m1; ENT1, Hs00191940 m1; ENT2,Hs00155426 m1; ENT3, Hs00983219 m1; CNT1,Hs00188418 m1; CNT2, Hs00188407 m1; CNT3,Hs0000223220 m1. In each experiment, mRNA levels ofGAPDH (Hs99999905 m1) and HPRT1 (Hs01003267 m1) wereused as housekeeping genes for sample normalization. For alltested genes, PCR conditions were as follows: 2 min at 50◦Cfollowed by 10 min at 95◦C and 40 amplification cycles (95◦Cfor 15 s and 60◦C for 60 s), analyzed by using Mx3000PTM real-time PCR cycler (Agilent Technologies, Boeblingen, Germany).Signals detected between 35 and 40 amplification cycles weredefined as low gene expression. The amplification efficienciesof all used TaqMan R© assays were 100% (±10%), in accordancewith the manufacturer’s information. The real-time PCR datawere analyzed using 2−��Ct method22 with �Ct = gene ofinterest − housekeeping gene (GAPDH or HPRT1) and ��Ct= �CtpBudCE4.1 – �CtpBud-HNF4".

Immunofluorescence Analysis of HNF4�, �-catenin, N-cadherin,and E-cadherin

Transfection efficiency of HNF4" in RCCNG1 cells was checkedby staining cells with goat anti-human HNF4" polyclonal an-tibody (Santa Cruz Biotechnology, Heidelberg, Germany; sc-6556) using immunofluorescence microscopy. Protein expres-sion of $-catenin, N-cadherin, and E-cadherin in RCC cells wasanalyzed after immunofluorescence staining with mouse anti-human $-catenin, mouse anti-human N-cadherin, and mouseanti-human E-cadherin monoclonal antibodies (Life Technolo-gies; 13–8400, 33–3900, 13–1700). RCCNG1, seeded and trans-fected on poly-D-lysine (Sigma-–Aldrich, Seelze, Germany)-coated glass cover slips (Carl Roth, Karlsruhe, Germany) werefixed in 3.8% paraformaldehyde/well (Carl Roth) for 8 minand permeabilized for 5 min in buffer consisting of 50 mMNa2HPO4/NaH2PO4 (pH 7.4), 0.5 mM NaCl, and 0.3% TritonX-100 (Carl Roth) at room temperature (RT). Afterwards, cellswere incubated for 2 h at RT with either 1 :g/mL goat anti-human HNF4", 3 :g/mL mouse anti-human $-catenin, 3 :g/mLmouse anti-human N-cadherin or 3 :g/mL mouse anti-humanE-cadherin antibody, blocked for 15 min with 1% BSA in PBS,and subsequently incubated for 1 h with corresponding sec-ondary antibodies (Life Technologies; Alexa Fluor R© 488 donkeyanti-goat IgG; Alexa Fluor R© 488 goat anti-mouse IgG). There-after, cells were incubated for 5 min with the nuclear stain, 4′

6-diamidino-2-phenylindole dihydrochloride (DAPI; Life Tech-nologies), and coverslips were prepared for immunofluores-cence microscopy. Transfection efficiency (ratio between HNF4"and DAPI positive cells) was analyzed using Image J (version1.44; National Institutes of Health). The specificity of immunos-taining was controlled by omitting the primary antibody andby using of isotype controls.

Apoptosis Assay

Apoptosis ofRCCNG1 cells was determined by using EnzChek R©

Caspase-3 Assay Kit #2 (Life Technologies). Transfectedcells were exposed with oxaliplatin (Santa Cruz Biotechnol-ogy) for 48 h, 5-fluorouracil (Sigma–Aldrich) and 5-fluoro-2′-deoxyuridine (5-FUDR; Sigma–Aldrich) for 16 h, or up to 0.01%DMSO, respectively. The incubations were stopped by wash-ing with PBS on ice. The cells were trypsinized and cen-trifuged for 3 min at 300g at room temperature. The pellet waswashed with PBS, resuspended in 50 :L lysis buffer, and lysed

DOI 10.1002/jps.24128 Hagos et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3326–3334, 2014

3328 RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics, Drug Transport and Metabolism

Table 1. Effect of HNF4" on mRNA Expression of OATs, and OCTs in RCC Cell Lines

RCC Cell Lines mRNA Level (2−��Ct ± SEM)

786-O A498 ACHN LN78 RCCNG1

HNF4" 8677.2 ± 3492.9*** 167.2 ± 75.7*** 16,377.83 ± 7423*** 7805.3 ± 3877.6*** 3367.4 ± 1993.1***

OCT1 0.8 ± 0.1 0.7 ± 0.1 2.5 ± 0.4 1.0 ± 0.3 2.9 ± 0.5*OCT2 BDL 1.3 ± 0.4 1.0 ± 0.0 BDL BDLOCT3 0.9 ± 0.1 1.2 ± 0.1 0.5 ± 0.2 0.4 ± 0.1 BDLOAT1 BDL BDL BDL BDL 18.6 ± 13.3*OAT2 BDL BDL BDL BDL 627.8 ± 400.2***OAT3 BDL BDL BDL BDL BDLOAT4 0.5 ± 0.1 1.7 ± 0.5 1.2 ± 0.2 3.5 ± 2.0 1.3 ± 0.3

*p < 0.5, statistical significance of the HNF4"-transfection compared with control transfected cells.***p < 0.001, statistical significance of the HNF4"-transfection compared with control transfected cells.HNF4", hepatocyte nuclear factor 4"; OAT, organic anion transporter; OCT, organic cation transporter; BDL, below detection limit.

completely by three freeze-thaw cycles consisting of 1 min freez-ing in liquid nitrogen. Caspase-3 activity was measured accord-ing to manufacturer’s recommendation and calculated as theratio of fluorescence to mg of total protein.

Transport Studies

RCCNG1, transiently transfected with HNF4" expression vec-tor or control vector, were investigated for their ability to takeup different [3H] labeled substances. First, cells in each wellwere washed with 1 mL of prewarmed (37◦C) mammalianRinger’s solution containing: 130 mM NaCl, 4 mM KCl, 1 mMCaCl2, 1 mM Mg2SO4, 1 mM NaH2PO4, 20 mM HEPES, and20 mM D-glucose, pH adjusted with 1 M NaOH to 7.4. Subse-quently, cells were incubated with 200 :L of substrate solu-tions following our established protocols for OCT1 dependentmethyl-4-phenylpyridinium acetate (MPP), OAT1-dependentp-aminohippuric acid (PAH), and OAT2-dependent cGMP up-take.

The intracellular accumulation of [3H]MPP (PerkinElmer,Hamburg, Germany) was tested in cells incubated for 5 min at37◦C in a solution containing 20 nM [3H]MPP and 980 nM un-labeled MPP (Sigma–Aldrich). The [3H]MPP uptake was sup-pressed by addition of 100 :M unlabeled MPP.

For testing of [3H]PAH (PerkinElmer) uptake, cells were in-cubated for 10 min with 10 :M PAH (1 :M [3H]PAH and 9 :Munlabeled PAH) at 37 ◦C. The inhibition of [3H]PAH intracellu-lar accumulation was characterized by incubation with 500 :Munlabeled PAH.

For investigation of [3H]cGMP (PerkinElmer) influx, cellswere incubated 5 min with 10 :M (100 nM [3H]cGMP and9.9 :M unlabeled cGMP (BioLog, Bremen, Germany), with orwithout 200 :M indomethacin (Sigma–Aldrich), at 37 ◦C.

The uptake of [3H]5-FU (BIOTREND Chemikalien, Cologne,Germany) was tested after incubation of the cells for 5 minat 37◦C with 100 nM [3H]5-FU, with or without 200 :M in-domethacin, 200 :M adenosine (Sigma–Aldrich), or 200 :Muridine (Sigma–Aldrich), respectively. Supplementary, the up-take of [3H]5-FU was measured in HEK293, stably transfectedwith human OAT2 or control vector, respectively.

CNT3 dependent [3H]uridine (PerkinElmer) accumulationwas tested in HEK293 cells, transiently transfected with CNT3expression vectors or control vector, and incubated for 5 min at37◦C with 100 nM [3H]uridine, with or without 200 :M adeno-sine, 1–500 :M 5-FU, and 1–500 :M 5-FUDR. For testing of

sodium-dependent CNT3-mediated [3H]uridine uptake, sodiumwas replaced by tetraethylammonium (Sigma–Aldrich).

After incubation with radioactively labeled substances andpotential inhibitors, cells were washed three times with PBSat 4◦C, and cell lysis was induced by incubation for 120 minwith 500 :L of 1 M NaOH. Thereafter, cell lysates were neu-tralized with an equivalent volume of 1 M HCl, and 900 :L ofthese solutions were transferred to scintillations vials. 2.5 mLLumasafe scintillation solution was added to each vial, andradioactivity was counted by a liquid scintillation counter (Tri-Carb 1500; PerkinElmer). The remaining 100 :L of each probewas used for the determination of total protein concentrationby the Bradford protein assay, and radioactive counts were nor-malized per milligram of total protein.

Statistical Analysis and Calculation of IC50 Value

Data in the figures and Table 1 are presented as mean ± SEM.Statistical analysis was performed with two-sided unpaired t-test (GraphPad Prism 4, version 4.03). Differences were consid-ered as significant at the level of p < 0.05.

The inhibitory effect [I(%)] of 5-FUDR on CNT3-mediated[3H]uridine uptake was calculated according to the formula:

I(%) = 100 − vtransporterinhibited × 100

vtransporterw/oinhibitor

Vtransporter: transporter-mediated uptake = net uptake(pmol/min); I (%): inhibitory effect.

For IC50 calculation, the inhibitory effect I (%) was plot-ted against inhibitor concentrations and fitted using Hillequation.

I = Io + Imaxcn

cn + ICn50

Io: inhibitory baseline effect (%); Imax: maximal inhibitory effect(%); n: Hill exponent; c: inhibitor concentration (:mol/L); IC50:inhibitor concentration causing half-maximal inhibitory effect(:mol/L).

Hagos et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3326–3334, 2014 DOI 10.1002/jps.24128


Figure 1. HNF4" protein in RCCNG1 cells. The cells were transiently transfected with control vector pBudCE4.1 (a–c) and HNF4" expressionvector pBud-HNF4" (d–f). HNF4" expression and cellular localization were analyzed using immunofluorescence staining (green color HNF4",blue color DAPI; excitation wavelength 488 and 365 nm, respectively). Data are representative for ≥ three independent experiments.

Figure 2. Immunofluorescence analysis of $-catenin and N-cadherin in RCCNG1 cells. The protein expression and localization of $-catenin(a–c) and N-cadherin (d–f) in RCCNG1 cells were investigated using immunofluorescence analysis (green color $-catenin or N-cadherin, bluecolor DAPI; excitation wavelength 488 and 365 nm, respectively). Data are representative for ≥ three independent experiments.

RESULTS

Expression of HNF4� and Cell–Cell Contact Proteins �-catenin,N-cadherin, and E-Cadherin in RCCNG1 Cells

The expression and localization of HNF4" protein were charac-terized in RCCNG1 cells using immunofluorescence. Endoge-nous HNF4" protein expression was not detected in controlcells (Figs. 1a–1c). HNF4" transfected cells expressed HNF4"protein exclusively in cell nuclei with a transfection efficiencyof up to 30% (Figs. 1d–1e).

The cell structure and cell–cell contacts of RCCNG1 cellswere investigated using immunofluorescence staining of renalepithelial cell markers $-catenin, N-cadherin, and E-cadherin,in combination with the cell nucleus stain DAPI. Protein ex-pression of $-catenin, N-cadherin, and E-cadherin was not af-fected by HNF4" transfection in this cell line (data not shown).High expression of $-catenin was detected in RCCNG1 cells(Figs. 2a–2c). The $-catenin positive cells displayed heteroge-

neous cell morphology (Figs. 2a–2c). Beta-catenin was localizedin cell membranes, cell nuclei, and cytoplasm. Only few prolif-erating cells showed N-cadherin membrane expression, othercells expressed low levels of endogenous N-cadherin protein(Figs. 2d–2f). E-cadherin protein was not expressed in RCCNG1(data not shown).

Effect of HNF4� on Gene Expression of Organic Cation andAnion Transporters in RCC Cells

Renal cell carcinoma cell lines were transfected with HNF4"or control vector and changes in the expression of target geneswere investigated using real-time PCR. Housekeeping genes(GAPDH and HPRT1) were not significantly altered by HNF4"transfection (data not shown).

Only in RCCNG1, HNF4" over-expression caused a sig-nificant increase of OCT1, OAT1, and OAT2 compared withvector transfected cells (Table 1). Thereby, OAT1 remainedat a low mRNA level (detected after approx. 36 cycles). Our



Figure 3. Effect of HNF4" on oxaliplatin chemosensitivity and[3H]MPP uptake in RCCNG1 cells. (a) Activity of caspase-3 was an-alyzed in RCCNG1 cells transiently transfected with HNF4" or controlvector, and incubated for 48 h with 1 :M oxaliplatin, 10 :M oxaliplatin,and 0.008% DMSO. (b) [3H]MPP uptake and inhibition of the uptakewith 100 :M unlabeled MPP were detected 48 h after transfectionwith pBud-HNF4" or control vector pBudCE4.1. Data are presented asmean ± SEM. n = 5. *p < 0.05; ***p < 0.001, compared with HNF4"overexpressing cells. ##p < 0.01 and ###p < 0.001, for the inhibitoryeffect of unlabeled MPP.

experiments showed no significant effect of HNF4" overexpres-sion on [3H]PAH uptake in RCCNG1. The exposure of the cellswith an excess of 500 :M unlabeled PAH did not decrease theintracellular concentration of [3H]PAH, suggesting that RC-CNG1 cells do not express enough functional OAT1, even af-ter HNF4" transfection. Therefore, the following experimentswere performed in RCCNG1 cells, focusing on the functionalexpression of OCT1 and OAT2.

HNF4� Increased Oxaliplatin Chemosensitivity

A high expression of OCT1 is known to correlate with increasedanticancer activity of oxaliplatin.23 Therefore, we tested thecytostatic effect of oxaliplatin on HNF4" transfected and con-trol cells. The activity of caspase-3 was elevated significantlyin HNF4" transfected cells compared with vector transfectedcells treated with 1 and 10 :M oxaliplatin (Fig. 3a).

HNF4" transfection significantly enhanced the uptake of[3H]MPP, a known OCT1 substrate, in RCCNG1 cells, com-pared with the control cells (Fig. 3b). Coincubation with unla-beled MPP (100 :M) resulted in a significant inhibition of the[3H]MPP accumulation in pBud-HNF4" and pBudCE4.1 trans-fected cells. [3H]MPP uptake in vector transfected cells demon-strated that RCCNG1 cells endogenously express a MPP trans-porting protein (Fig. 3b). The involvement of OCT2 and OCT3

Figure 4. Effect of HNF4" on 5-FU chemosensitivity and [3H]cGMPuptake in RCCNG1 cells. (a) Expression vector for HNF4" or emptyvectors were transiently transfected in RCCNG1 cells. Caspase-3 ac-tivity was measured after 16 h incubation with 1 :M 5-FU, 10 :M5-FU, and 0.008% DMSO. (b) [3H]cGMP uptake and the inhibition of[3H]cGMP uptake by 200 :M indomethacin were analyzed in HNF4",OAT2, and vector transfected cells. Data are represented as mean ±SEM. n = 4. n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001,compared with HNF4" or OAT2 overexpressing cells. #p < 0.05 and###p < 0.001, for the inhibitory effect of unlabeled indomethacin.

can be excluded because RCCNG1 cells do not express OCT2 orOCT3 (Table 1).

HNF4� Increased 5-FU Chemosensitivity

OAT2 was described to transport 5-FU.24 Therefore, thecaspase-3 activity was determined in RCCNG1 cells incubatedwith increasing concentrations of 5-FU, after transfection withHNF4" or control vector (Fig. 4a). The activity of caspase-3was not affected in vector transfected cells. However, in HNF4"transfected cells incubated with 1 :M and 10 :M 5-FU, signif-icantly higher caspase-3 activity was detected compared withcontrol cells (Fig. 4a).

Since the increased caspase-3 activity in HNF4" express-ing cells suggested a role of OAT2, the functional expressionof OAT2 was tested in RCCNG1 by the uptake of radiola-beled cGMP and inhibition with indomethacin. The absenceof a significant indomethacin inhibition of cGMP uptake indi-cated that RCCNG1 with or without HNF4" did not expressenough functional OAT2 protein (Fig. 4b). In contrast, signifi-cantly higher [3H]cGMP accumulation and a marked inhibitionwith indomethacin were detected in RCCNG1 transfected withhuman OAT2 compared with control cells (Fig. 4b).

The involvement of OAT2 in 5-FU chemosensitivity of RC-CNG1 cells was excluded by further uptake experiments inHEK293 cells expressing OAT2. OAT2 expressing HEK293cells showed 70-fold higher cGMP uptake compared with con-trol cells. OAT2 mediated cGMP accumulation was inhibitedby indomethacin by around 80% (data not shown). The accu-mulation of [3H]5-FU was only 1.7-fold higher in HEK293 cells



Figure 5. HNF4" induced changes in mRNA expression of ENTs andCNTs in RCCNG1 cells. The expressions of selected drug transporterswere analyzed in RCCNG1 cells transiently transfected with HNF4"expression vector or empty vector, using Taqman real-time PCR. The�Ct values were calculated as �Ct = gene of interest − GAPDH and��Ct as ��Ct = �CtpBudCE4.1 – �CtpBud-HNF4". Data are presented asmean ± SEM. n = 4; n.s.: not significant and *p < 0.05, compared withHNF4" overexpressing cells.

expressing OAT2 compared with control cells, and the [3H]5-FUuptake was not significantly inhibited by indomethacin (datanot shown). Therefore, OAT2 is not responsible for 5-FU up-take in OAT2 expressing HEK293 cells. It is not clear whichtransporter accounts for the 1.7-fold increase in 5-FU uptake.

The expression of the nucleoside and nucleobase trans-porters, ENTs and CNTs,13 was investigated to test for fur-ther candidate genes responsible for the HNF4"-increased 5-FU chemosensitivity in RCCNG1. mRNAs of ENT1, ENT2, andENT3 were detected after 28–29 PCR cycles, but were not af-fected by HNF4" (Fig. 5). The expression of CNT1 was not sig-nificantly elevated, and CNT2 was detected after ≥ 35 amplifi-cation cycles in real-time PCR. In contrast, CNT3 mRNA levelwas significantly increased by HNF4" transfection in RCCNG1(Fig. 5). Therefore, in subsequent experiments, the function ofCNT3 protein was investigated.

The uptake of radioactively labeled 5-FU was measuredin RCCNG1, transiently transfected with HNF4" expressionvector or control vector, respectively. The intracellular accu-mulation of [3H]5-FU in RCCNG1 was slightly, but signifi-cantly enhanced by HNF4" transfection (Fig. 6). Coincubation

Figure 6. [3H]5-FU uptake in RCCNG1 cells. RCCNG1 cells weretransiently transfected with either expression vector pBud-HNF4" orcontrol vector pBudCE4.1 and incubated for 48 h. The accumulationof 5-FU was determined after 5 min incubation with 100 nM [3H]5-FU. The [3H]5-FU uptake was inhibited by coincubation with 200 :Madenosine or 200 :M uridine. Data are presented as mean ± SEM. n =4. n.s.: not significant; **p < 0.01, compared with HNF4" overexpress-ing cells. ##p < 0.01 and ###p < 0.001, inhibitory effect of adenosin anduridine compared with control cells.

Figure 7. Effect of 5-FU on [3H]uridine uptake in HEK293 cells.HEK293, cells transiently transfected with expression vector pCMV6-XL4-CNT3 or control HEK293 cells were incubated for 48 h, and trans-port activity of CNT3 was tested in presence of 10 :M of NBTI, anENT1–3 inhibitor. The accumulation of uridine was determined after5 min incubation with 100 nM [3H]uridine in sodium-free and sodium-containing test solution, and after coincubation with 200 :M adenosine.In addition, CNT3 mediated [3H]uridine uptake was tested after co-incubation with 1–500 :M 5-FU. Data are presented as mean ± SEM.n(NBTI without Na+; NBTI; NBTI+Adenosin) = 4, n(1–500 :M 5-FU) = 2. n.s.: notsignificant; ***p < 0.001, compared with CNT3 overexpressing cells.###p < 0.001, inhibitory effect of sodium-free test conditions or coincu-bation with adenosine on CNT3-mediated uridine accumulation.

with CNT3 inhibitors, adenosine and uridine, resulted ina significant inhibition of CNT3-mediated [3H]5-FU uptakeinto HNF4" transfected cells (Fig. 6). In addition, 5-FUchemosensitivity of HNF4" transfected RCCNG1 was signifi-cantly inhibited in presence of adenosine or uridine (data notshown).

Furthermore, the involvement of CNT3 in 5-FU inducedcaspase-3 activity was investigated in CNT3-transfectedHEK293 cells. The accumulation of radioactively labeled uri-dine, a known substrate for CNT3,25 was tested in the absenceand presence of sodium and inhibited with increasing concen-trations of 5-FU in CNT3 transfected HEK293 and control cells.For inhibition of [3H]uridine equilibration mediated by ENTproteins, experiments were performed in the presence of S-(4-Nitrobenzyl)-6-thioinosine (NBTI). CNT3 in transiently trans-fected HEK293 cells caused a 6-fold higher accumulation of[3H]uridine compared with control cells (Fig. 7). In the absenceof sodium or in the presence of sodium and adenosine, CNT3mediated uridine uptake was significantly inhibited (Fig. 7).Unexpectedly, [3H]uridine uptake was not significantly inhib-ited by increasing concentrations of 5-FU (Fig. 7).

Since CNT3 does not interact with 5-FU, we reasoned thatthe active metabolite, 5-FUDR, may be a substrate. In presenceof ENTs inhibitor NBTI, [3H]uridine uptake in CNT3 trans-fected HEK293 cells was significantly inhibited by 5-FUDR ina concentration dependent manner (Fig. 8a). The calculatedIC50 value of CNT3 for 5-FUDR was 5.7 ± 0.6 :M (Fig. 8a).In addition, in HNF4" transfected RCCNG1 cells incubatedwith 1 :M 5-FUDR in presence of 10 :M NBTI, a significantlyhigher caspase-3 activity was detected as compared with con-trol cells (Fig. 8b). Interestingly, the activity of caspase-3 wasnot affected in HNF4" transfected RCCNG1 cells, exposed with1 :M 5-FU in presence of NBTI (Fig. 8b).



Figure 8. Effect of 5-FUDR on [3H]uridine uptake in HEK293 cellsand on the caspase-3 activity in RCCNG1 cells. (a) HEK293 cells,transiently transfected with expression vector pCMV6-XL4-CNT3 orcontrol HEK293 cells were incubated for 48 h, and CNT3-mediated[3H]uridine uptake was determined after coincubation with 1–500 :M5-FUDR, in the presence of 10 :M of ENT1–3 inhibitor NBTI. The5-FUDR (inhibitor) concentration causing half-maximal inhibitory ef-fect (IC50) on [3H]uridine accumulation in CNT3 expressing cells wascalculated. Means ± standard average deviation (av. dev., for n = 2)were calculated from experiments at two different days. (b) Expres-sion vector for HNF4" or empty vectors were transiently transfectedin RCCNG1 cells. Caspase-3 activity was measured after 16 h incuba-tion with 1 :M 5-FU, 1 :M 5-FUDR and 0.01% DMSO, in presence of10 :M NBTI. Data are presented as mean ± SEM. n = 4. n.s.: not signif-icant; *p < 0.05, compared with HNF4" overexpressing cells. 5-FUDR:5-fluoro-2′-deoxyuridine.

DISCUSSION

The resistance of clear cell RCC to conventional anticancerdrugs is responsible for the high number of cancer relateddeaths. The loss or decrease of the expression of uptake trans-porters may be partially involved in the chemoresistance ofRCC. The aim of this work was to enhance, by the transcrip-tional factor HNF4", the expression of renal uptake trans-porters and to increase chemosensitivity of RCC cell lines toselected anticancer drugs.

In our renal carcinoma cell lines, mRNA levels of OCTs andOATs were below detection limit and expressed very lowly inRCCNG1. The changes of gene expression in tested RCC cell

lines are not surprising because it has been shown that can-cer cells can modify numerous epigenetic and genetic mecha-nisms, resulting in altered gene expression, cell survival, andchemoresistance.26,27

Interestingly, only in RCCNG1 cells, OCT1 and OAT2 mRNAwere elevated by HNF4" over-expression. The low, but de-tectable protein expression of N-cadherin and the absence ofE-cadherin protein suggested the origin of RCCNG1 in a prox-imal tubule cell. N-cadherin is the predominant cadherin inproximal tubule cells and absent in other nephron segments.28

E-cadherin is present only at low levels in the membranes ofthe proximal tubule cells.28 The high expression of $-cateninwas detected in cell cytoplasm and nuclei of RCCNG1 cells.The $-catenin over-activation was considered to be a hallmarkof Wnt-driven RCC.29 From all these data, it appears that RC-CNG1 cells are more closely related to proximal tubule cellsthan other tested cell lines.

The effect of HNF4" on OCT1 expression in renal carcinomacells is very interesting for anti-cancer therapy, because theexpression of OCT1 is very low or absent in normal humankidney tissue. A higher uptake of cytostatic drugs by OCT1would be toxic for renal cancer cells. Independent studies es-tablished the involvement of human OCT1 and OCT2 in oxali-platin cytotoxicity.23,30 In line with the real-time data, HNF4"significantly enhanced the oxaliplatin induced apoptosis in RC-CNG1 cells. Furthermore, the uptake of typical model substratefor OCTs, [3H]MPP, was significantly enhanced by HNF4" intransfected RCCNG1, and the uptake of [3H]MPP was signifi-cantly reduced by unlabeled 100 :M MPP. Thus, if tumor cellsexpress OCT1 they may be treated with oxaliplatin.

In the present work, HNF4" considerably elevated OAT2mRNA in RCCNG1 cells. Accordingly, we found that HNF4" en-hanced chemosensitivity of RCCNG1 cells to 5-FU, a reportedOAT2 substrate.24 In the last two decades, continuous intra-venous infusion of 5-FU in combination with other therapiesor a pro-drug of 5-FU (capecitabine) was used for treatment ofrenal cell cancer.31,32 However, the uptake of OAT2 substratecGMP33 was not enhanced by HNF4" transfection in RCCNG1.Our experiments in HEK293 stably transfected with humanOAT2 confirmed that 5-FU was not a transported substrate ofOAT2. The reason for the disagreement between our resultsand published data is unclear, but can be due to the usage ofdifferent expression systems. Kobayashi et al.24 performed thefunctional tests of OAT2 function in Xenopus laevis oocytes.

Since, in our hands, OAT2 was not involved in 5-FU up-take, real-time PCR analysis of HNF4" transfected and con-trol RCCNG1 was performed to identify additional transportergenes responsible for the enhanced 5-FU chemosensitivity. Outof tested genes, the mRNA of CNT3 was significantly increasedby HNF4" in RCCNG1 cells. The CNT1 promoter activation isknown to be regulated by HNF4",17 but nothing was knownabout the promoter of CNT3. CNT3 mediates a 10-fold higherintracellular accumulation of selective nucleosides than CNT1and CNT2.34 All three CNTs accept uridine as substrate.35

In our experiments, the uptake of [3H]5-FU was enhanced byHNF4"and inhibited by adenosine and uridine, suggesting thatthe observed [3H]5-FU accumulation in RCCNG1 after HNF4"stimulation might be CNT3 mediated. The uptake of the nu-cleobase 5-FU by CNTs was not described to our knowledge,but it was reported that 5-FU did not inhibit CNT3 dependenturidine uptake in HeLa cells.36 In agreement with literaturedata, CNT3 mediated uridine uptake was not suppressed by



increasing 5-FU concentrations in HEK293 transiently trans-fected with CNT3, in presence of the ENTs inhibitor, NBTI.

The possible explanation for HNF4"-enhanced chemosensi-tivity in RCCNG1 cells with 5-FU and absence of 5-FU-inducedinhibition of uridine uptake in CNT3 overexpressing HEK293cells may be due to the existence of high level of ENTs proteinsin RCCNG1. ENT1 and ENT2 have been shown to transport5-FU, and high levels of ENT1 in colorectal cancer tissue cor-related with low clinical response to 5-FU.30,37 Furthermore,ENT1 and ENT3 are known to be involved in the transloca-tion of 5-FU metabolite 5-fluoro-2′-deoxyuridine (5-FUDR).38,39

Human CNT1 and CNT3 efficiently transported 5-FUDR ex-pressed in oocytes.40,41 CNTs have been described as uptaketransporter and ENTs are known to mediate bi-directional fa-cilitated diffusion of nucleosides.34 Our hypothesis was thatENTs are responsible for the equilibration of 5-FU and 5-FUDRlevels in RCCNG1 cells, and HNF4"-increased CNT3 expres-sion may induce a higher accumulation of 5-FUDR in HNF4"-transfected RCCNG1 cells. Accordingly, CNT3 mediated re-absorption of metabolite 5-FUDR probably induces a higherapoptosis in HNF4" transfected RCCNG1 cells. Therefore, theinfluence of 5-FUDR on CNT3-mediated uridine uptake inHEK293 cells was tested. In agreement with published studyperformed in oocytes,40 uridine accumulation was significantlyinhibited by increasing 5-FUDR concentrations in CNT3 ex-pressing HEK293 cells. Additional experiments showed HNF4"increased 5-FUDR chemosensitivity in RCCNG1 cells in pres-ence of ENTs inhibitor NBTI. Without protein activity of ENTs,no significant difference was detected between HNF4" trans-fected and control RCCNG1 cells after incubation with 5-FU.These data proved our hypothesis that ENTs transport 5-FUand 5-FUDR in a bi-directional manner and CNT3 accumulatedthe cytostatic metabolite 5-FUDR, but not 5-FU.

CONCLUSIONS

This study demonstrates for the first time that HNF4"-stimulated protein expression of influx transporter OCT1 andCNT3 leads to a higher chemosensitivity toward oxaliplatin,5-FU, and 5-FU metabolite 5-FUDR, respectively, in RCCNG1,but not in other tested RCC cell lines. Therefore, the SLC-transporter mediated accumulation of anticancer drugs couldpotentially help to overcome the chemoresistance at least in asubset of tumor cells.

ACKNOWLEDGMENTS

This work was supported by Deutsche Forschungsgemeinschaft(DFG; German Research Foundation) and University MedicalCenter Goettingen. The funders had no role in study design,data collection and analysis, decision to publish, or preparationof the manuscript.

The authors have declared that no competing interests exist.

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HNF4α Induced Chemosensitivity to Oxaliplatin and 5-FU Mediated by OCT1 and CNT3 in Renal Cell...

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