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istry xx (2014) xxx–xxx

Journal of Nutritional Biochem

Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin byinhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein☆,☆☆

Victor Hugo Villara, Oliver Vöglera, Francisca Barcelóa, Manuel Gómez-Floritb, Jordi Martínez-Serraa,c,Antònia Obrador-Heviaa,d, Javier Martín-Brotoa,d, Valentina Ruiz-Gutiérreze, f, Regina Alemanya, f,⁎

aGroup of Clinical and Translational Research, Department of Biology, Institut Universitari d'Investigacions en Ciències de la Salut (IUNICS), University of the Balearic Islands, Palma deMallorca, Spain

bGroup of Cell Therapy and Tissue Engineering, Institut Universitari d'Investigacions en Ciències de la Salut (IUNICS), University of the Balearic Islands, Palma de Mallorca, SpaincDepartment of Hematology, University Hospital Son Espases, Palma de Mallorca, SpaindDepartment of Oncology, University Hospital Son Espases, Palma de Mallorca, Spain

eInstituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Seville, SpainfCIBER:CB06/03 Fisiopatología de la Obesidad y la Nutrición, CIBERobn, Instituto de Salud, Carlos III (ISCIII), Spain

Received 18 June 2013; received in revised form 27 November 2013; accepted 16 December 2013

Abstract

The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in theMediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have beenextensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecularmechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 andleiomyosarcoma SK-UT-1 cells. As single compound, MLA (10–100 μM)wasmore potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3±1.11% and68.8±1.52% at 80 μM, respectively. Importantly, OLA (80 μM)orMLA (30 μM)enhanced the antitumoral effect of DXR (0.5–10 μM)byup to 2.3-fold. On themolecular level,efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells.Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediatedMRP-1 transport, whereasMLA acted as a non-competitive one.Moreover, noneof both triterpenes induceda compensatory increase inMRP-1expression. In summary,OLAorMLAsensitized cellularmodels of STS toDXRandselectively inhibitedMRP-1activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.© 2013 Elsevier Inc. All rights reserved.

Keywords: Triterpenes; Doxorubicin; MRP-1; Drug resistance; Soft tissue sarcomas

1. Introduction

The pentacyclic triterpenes oleanolic acid (OLA) (3β-hydroxy-12-oleanen-28-oic acid) and maslinic acid (MLA) (2α,3β-dihydroxy-12-oleanen-28-oic acid) (Fig. 1) are natural compounds found in many

☆ Conflict of interest statement: The authors confirm that there are noconflicts of interest.

☆☆ Research was supported by the “Asociación Española Contra el Cáncer(Junta de Baleares)” and the Institute of Health Carlos III through thesubprogram “CIBERobn (Centro de Investigación Biomédica en Red de laFisiopatología de la Obesidad y Nutrición)”. V.H.V. was supported by apredoctoral fellowship from “Govern de les Illes Balears, Conselleriad'Educació, Cultura i Universitats” under a program of joint financing withthe European Social Fund.

⁎ Corresponding author. Department of Biology, Institut Universitarid'Investigacions en Ciències de la Salut (IUNICS), University of the BalearicIslands, Carretera de Valldemossa, km 7.5, E-07122 Palma de Mallorca, Spain.Tel.: +34-971-259619; fax: +34-971-173184.

E-mail address: regina.alemany@uib.es (R. Alemany).

0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.jnutbio.2013.12.003

plant species, but only a few, such as Olea europaea, contain a highconcentration in leaves and fruits [1,2]. More importantly, appreciableamounts of OLA and MLA are also present in the non-glyceridefraction of pomace and virgin olive oils (244±28 mg/kg of OLA and194±14 mg/kg of MLA) [3,4], which are consumed in the Mediter-ranean diet [5]. Several important biological activities, e.g., antioxi-dant [6], anti-inflammatory [7] and vasodilatory effects [8], have beenattributed to these triterpenes. In the last few years, OLA and MLAhave also received increasing attention due to its antitumoral action.In this context, antiproliferative and apoptotic activities of bothtriterpenes have been demonstrated in a variety of human tumor celllines, including astrocytoma [9], breast [10,11] and colon cancer cells[4,12]. Although the antitumoral effect of OLA and MLA has beenmainly associated to their capacity to increase intracellular reactiveoxygen species (ROS) [4,9,12,13], other molecular mechanisms, suchas activation of JNK/p53 pathway [14], activation of p38 MAPKthrough elevation of intracellular calcium [15] or suppression ofNF-κB signaling [16], also seem to be related to its antitumoral activityin some epithelial cancers. In addition, in a monkey epithelial cell line

Fig. 1. MLA, but not OLA, inhibited cell growth in human STS cells. (A) Chemical structures of OLA and MLA. (B) Synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells weretreated with vehicle (V), OLA and MLA (10–100 μM) for 24 h. Cell viability was assessed using the MTT assay as described in Materials and methods. Each value represents mean±S.E.M. of three individual experiments performed in triplicate. ⁎Pb.05 and ⁎⁎⁎Pb.001 vs. vehicle-treated cells.

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OLA also inhibited the activity of the multidrug resistance-related protein 1 (MRP-1) [17], an ATP-binding cassette (ABC)transporter located in the plasma membrane, which is mainlyinvolved in the development of multidrug resistance (MDR) in cancercells [18].

Soft tissue sarcomas (STS) comprise a heterogeneous group ofmalignant tumors arising from soft tissues, including fat, smooth orskeletal muscle, fibrous tissue and vasculature, which embryologi-cally derived from themesoderm or neuroectoderm [19,20]. Althoughsurgical removal of the entire tumor remains the main therapeuticstrategy for localized STS, doxorubicin (DXR)-based chemotherapy iswidely used in the treatment of patients with locally advancedinoperable or metastatic disease [21,22]. Nevertheless, therapies withDXR alone or in combination with ifosfamide have not achievedsignificant improvements in terms of overall survival [23]. Addition-ally, the effectiveness of DXR is limited because it provokes severecardiotoxicity and the development of MDR, the latter mainlyinvolving high cellular expression or activity of ABC transporters inthe plasma membrane, such as P-glycoprotein (P-gp) and MRP-1[24,25]. As a result, many patients ultimately relapse, and theprognosis for patients with metastatic sarcoma remains poor.Therefore, the search for new therapeutic approaches is still a needand a combination therapy, which was able to sensitize cancer cells toDXR by counteracting such resistance mechanism without increasinggeneral toxicity, would be a suitable strategy.

Although several studies have evaluated the efficacy of OLA andMLA in human carcinomas, only little is known regarding their effectsin STS and whether a combination of these natural compounds withconventional chemotherapy is able to improve treatment outcome forthis type of solid tumors. Our study focuses on the efficacy andmolecular mechanisms involved in the antitumoral effects of OLA and

MLA in STS cells, either as individual agents or in combinationwith DXR.

2. Materials and methods

2.1. Cell culture and treatments

The human cell lines SW982 and SK-UT-1 were derived from synovial andleiomyosarcoma, respectively, which represent some of the most common STS inhumans, accounting for approximately 5–10% and 24% of STS in adults. The cell lineswere chosen because they show low sensitivity to DXR treatment [26] being a requisitefor the aim of the study. SW982 and SK-UT-1 cells express the MDR protein MRP-1 atsimilar levels, but differ in the level of P-gp, which is barely expressed in SK-UT-1 cells.Both cell lines were obtained from the American Type Culture Collection (Manassas,VA, USA). Synovial sarcoma cells were grown in Leibovitz's L-15 medium (InvitrogenS.A, Barcelona, Spain), whereas leiomyosarcoma SK-UT-1 cells were cultured in DMEMmedium (LabClinics S.A, Barcelona, Spain) supplemented with MEM–nonessentialamino acids (dilution, 1:100) and 1 mM sodium pyruvate. Both media contained 2 mML-glutamine and were supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. Tissue culture supplements were allpurchased from LabClinics. When the cells reached 60–70% confluence, vehicle[ethanol or dimethyl sulfoxide (DMSO)], OLA (3β-hydroxy-12-oleanen-28-oic acid),MLA (2α,3β-dihydroxy-12-oleanen-28-oic acid) or DXR as single agents or theircombinations were added to the medium for 24 h. OLA from olive oil was kindlyprovided by Dra. A Guinda (Instituto de la Grasa, Consejo Superior de InvestigacionesCientíficas, Seville, Spain), and MLA was purchased from Cayman Europe. DXR waspurchased from Sigma-Aldrich (Madrid, Spain). Stock solution of OLA and MLA wasprepared at 10 mM in ethanol or DMSO, respectively, or in water in the case of DXR.

2.2. Cell viability

Synovial sarcoma and leiomyosarcoma cells were plated in 96-well plates at adensity of 11×103 or 8×103 cells per well in 200 μl of their respective growth mediumand grown for 24 h. To test the effectiveness of compounds on cell growth, cells wereexposed to increasing concentrations of OLA or MLA (10–100 μM) for 24 h. Thecompound concentration resulting in 50% inhibition of cell viability (IC50) wasdetermined using GraphPad Software. To determine whether OLA and MLA would

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increase sensitivity of these cell lines to DXR, cells were treated simultaneously withvarying concentrations of DXR (range, 0.5–10 μM) and OLA (80 μM) or MLA (30 μM) orthe respective vehicle. After 24 h, the viability of the cells was measured using themethylthiazoletetrazolium (MTT) method. Absorbance was measured at 570/650 nm(for SW982 cells) and 590/650 nm (for SK-UT-1 cells) on an enzyme-linkedimmunosorbent assay plate reader (Asys Hitech GmbH, Austria). The slight adjustmentof wavelength for each cell line was necessary to avoid saturation of the signal, whichoccurred when analyzing a similar cell number of both lines. The mean percentage ofcell survival relative to that of vehicle-treated cells was estimated from data of threeindividual experiments performed by triplicate.

2.3. Cell apoptosis

Analysis of the cell cycle by flow cytometry was performed on SW982 and SK-UT-1cells as described previously [26]. Cell populations in the different phases of cell cycle(sub-G1, G0/G1, S/G2/M) were determined based on their DNA content in a BeckmanCoulter Epics XL flow cytometer. Cellular apoptosis was also determined by assessmentof the cleavage of caspase 3 and by measuring the expression of the anti-apoptoticprotein p21 (CIP1/WAF1) by immunoblot analysis.

2.4. Quantification of intracellular DXR

SW982 and SK-UT-1 cells were treated with vehicle or DXR (1 μM for SW982 or 0.1μM for SK-UT-1 cells) in the absence or presence of OLA (10–80 μM) and MLA (10–50μM) for 24 h. After this time, cells were harvested, washed and resuspended inphosphate-buffered saline (PBS) at 37°C. Fluorescence attributable to intracellular DXRwas immediately measured by flow cytometry (Beckman Coulter Epics XL flowcytometer).

2.5. Immunoblot analysis

Preparation of cell extracts and protein assays were performed as describedpreviously [26]. Primary polyclonal antibodies anti-p21 (CIP1/WAF1) (21 kDa), anti-caspase 3 (35 kDa) and anti-cleaved caspase 3 (Asp175) (17–19 kDa) and themonoclonal anti-β-actin (45 kDa) were obtained from Cell Signaling Technology(Beverly, MA, USA). The monoclonal anti-MDR1 (P-gp) (170 kDa) and anti-MRP-1(190 kDa) antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz,CA, USA). All antibodies were diluted 1:1000. IRDye 800CW-conjugated anti-rabbitand anti-mouse secondary antibodies were purchased from Bonsai Technologies(Spain). The intensity of band immunoreactivity was analyzed with the Odysseyimaging system from LI-COR. To assess the changes of protein levels, theintensity of each protein band was normalized against the intensity of thecorresponding β-actin band.

2.6. Activity of P-gp and MDR-associated protein 1

To examine the effect of both triterpenes on P-gp and MDR-associated protein 1(MRP-1) function, the fluorescent dyes rhodamine 123 (Rho-123) and 5(6)-carboxy-fluorescein diacetate (CFDA) were used. Synovial sarcoma cells were incubated with0.5 μM of Rho-123 dissolved in complete medium or CFDA dissolved in FBS-freemedium for 30 min at 37°C, whereas leiomyosarcoma cells were only incubated withCFDA. After removing the extracellular free dye, the cells were incubated in dye-freemedia containing vehicle, verapamil (10 μM), probenecid (250 μM), OLA (10–80 μM) orMLA (10–80 μM). The efflux of Rho-123 and CFDA was analyzed after 2 h by flowcytometry (Beckman Coulter Epics XL flow cytometer).

2.7. Measurement of intracellular ROS production

The intracellular generation of ROS was measured using the cell-permeablefluorescence probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Synovialsarcoma SW982 and leiomyosarcoma SK-UT-1 cells were incubated for 30 minwith 10 μM DCFH-DA dissolved in FBS-free medium. After removing theextracellular free dye, the cells were incubated in dye-free media containingvehicle (ethanol or DMSO), OLA (10–80 μM) or MLA (10–80 μM). Theintracellular fluorescence due to oxidation of DCFH was detected after 30 minby flow cytometry (Beckman Coulter Epics XL flow cytometer) with excitation at488 nm and emission at 530 nm.

2.8. Glutathione measurement

The 5,5′-dithiobis-2-nitrobenzoic acid (DNTB) colorimetric method was used tomeasure glutathione (GSH) content as described previously [27]. Leiomyosarcoma cellswere treated with vehicle, OLA (10–80 μM), MLA (10–80 μM) or H2O2 (60 mM, as apositive control) for 2 h. After this time, cells were washed with cold PBS and scrappedoff with a rubber policeman in 300 μl of 1.67% (w/v) freshly preparedmeta-phosphoricacid. This procedure provoked the lysis of cells and the precipitation of proteins. Underthese conditions, GSH is the only measurable soluble thiol within the cells. Sampleswere centrifuged and aliquots of 40 μl of the supernatant were mixed with 140 μl of 0.3M sodium phosphate buffer, pH 8.7, and 20 μl of 0.04% DNTB in 1% sodium citrate

buffer, pH 6.8. After 5 min, the absorbance at 413 nm was measured and the level ofsoluble thiol was determined by comparison with GSH standards. Values werenormalized to the corresponding protein contents.

2.9. Kinetics

Kinetic parameters for MRP-1 inhibition by triterpenes were analyzed byincubating leiomyosarcoma SK-UT-1 cells with different concentrations of CFDA inthe absence or presence of OLA and MLA. Cells were incubated with variousconcentrations of CFDA (0, 0.1, 1 and 10 μM) dissolved in FBS-free medium for 30min at 37°C. After removing the extracellular free dye, each group of cells wasincubated in dye-free media containing vehicle, OLA (10–80 μM) or MLA (10–80 μM)for 1 h to allow the export of CFDA. Cells were then harvested and washed, andintracellular CFDA fluorescence was analyzed by flow cytometry. T0 was theintracellular CFDA fluorescence measured at t=0. The export rate of CFDA in thepresence of each concentration of triterpene was calculated as the difference betweenthe intracellular CFDA fluorescence value found at T0 and after 1 h. This value wasdivided by the export time of CFDA (1 h) in order to calculate first response units perhour (FRU/h). The kinetic parameters Km and Vmax were calculated with the Michaelis–Menten equation using GraphPad Software. Each value is the mean of threeindependent experiments.

2.10. Data analysis

The results are expressed as the mean±S.E.M. One-way analysis of variancefollowed by Bonferroni's multiple comparison test was performed using GraphPadSoftware. Differences were considered statistically significant at Pb.05.

3. Results

Growth inhibition by OLA and MLA (10–100 μM) was determinedin synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells after24 h of treatment. MLA inhibited STS cell proliferation in aconcentration-dependent manner, and the average IC50 values were45.3±0.04 μM in SW982 cells and 59.1±0.06 μM in SK-UT-1 cells. At80 μM, MLA reduced cell growth by 70.3±1.1% and 68.8±1.5%,respectively (Fig. 1B). Flow cytometry analysis indicated that 80 μMMLA induced a significant cell death up to 37.5% after 24 h, whencompared to vehicle-treated leiomyosarcoma cells (data not shown).In contrast, OLA showed only a moderate effect on cell viability at aconcentration of 80 μM (inhibition of 14.5% and 18.1%) (Fig. 1B) anddid not induce significant cell death. As a whole, these data indicatethat MLA, but not OLA, inhibits STS cell growth with a predominantlypro-apoptotic effect.

According to the dose–response effects on cell viability (Fig. 1B),the efficacy between OLA and MLA differed greatly. Whereas MLAalone was able to reduce cell viability by up to 85% in SK-UT-1 cells atthe maximum dose (100 μM), OLA alone only showed a minor effectat all doses tested. To be able to investigate whether OLA and, inparticular, MLA may improve cell susceptibility to DXR, the dose foreach triterpene was adjusted individually in the combinationexperiments to 30 μM for MLA and 80 μM for OLA. MLA alone atthis concentration did not induce significant apoptosis after 24 h oftreatment (data not shown), and its effects on cell viability weresimilar with those of OLA alone, especially in SK-UT-1 cells. As shownin Fig. 2, higher antitumoral effect (inhibition of cell viability andinduction of cell death) was observed for the combined treatmentswith DXR and OLA or MLA than for each compound alone (Fig. 2).Combination of DXR with OLA or MLA consistently increased theantiproliferative effect of DXR by 1.3- to 2.3-fold in both cell lines.Maximal inhibition of combined treatments was achieved using DXRat 5 and 10 μM, which reduced cell viability in leiomyosarcoma cellsup to 60% (Fig. 2A and B). In line with this observation, combination ofDXR (1–10 μM) and OLA (80 μM) or MLA (30 μM) increased theapoptotic effect of DXR in leiomyosarcoma cells. In this context, OLAand MLA increased DXR-induced cleavage of caspase 3 by 1.8±0.01-fold and 2.7±0.10-fold, respectively (Fig. 2C). Likewise, the decreaseof the anti-apoptotic protein p21CIP expression triggered by DXRalone at 1 or 10 μM was enhanced by 34.9±1.3% and 19.8±5.3%,

Fig. 2. OLA and MLA enhanced the antitumoral effects of DXR in human STS cells. Synovial sarcoma SW982 (A) and leiomyosarcoma SK-UT-1 cells (B) were treated with vehicle (V) orDXR (0.5, 1, 5 and 10 μM) alone or combined simultaneously with OLA (80 μM) or MLA (30 μM) for 24 h. Cell viability was assessed using the MTT assay as described in Materials andmethods. Each value represents mean±S.E.M. of three individual experiments performed in triplicate. ⁎Pb.05, ⁎⁎Pb.01 and ⁎⁎⁎Pb.001 vs. DXR-treated cells. (C) Apoptotic effect of DXR(1 and 10 μM) as single compound or combined with OLA (80 μM) (left panel) orMLA (30 μM) (right panel) after 24 h. Panels show the immunoreactive bands of procaspase 3, cleavedcaspase 3 and anti-apoptotic protein p21CIP in representative immunoblots.

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respectively, in the presence of OLA, and by 48.8±5.0% and 57.1±1.3%, respectively, in the presence of MLA (Fig. 2C).

To address the question whether the improved sensitivity ofsarcoma cells to DXR induced by these triterpenes could be due to anincrease in the accumulation of DXR, intracellular DXR levels wereevaluated in the presence and absence of OLA or MLA. OLA (10–80μM) applied simultaneously with DXR augmented intracellular DXRaccumulation after 24 h, raising its levels to 1.6±0.26-fold and 2.0±0.15-fold in SW982 cells and to 1.5±0.14-fold and 1.9±0.09-fold inSK-UT-1 cells at a dose of 50 and 80 μM, respectively (Fig. 3).Similarly, MLA at 50 μM also increased DXR accumulation by 1.4±0.02-fold in SW982 cells and 1.3±0.05-fold in SK-UT-1 cells (Fig. 3).Intracellular fluorescence intensity in OLA- or MLA-treated cells was

not different from vehicle-treated cells (data not shown). Theseresults indicate that both triterpenes are able to enhance intracellularDXR accumulation.

The effect of OLA and MLA on the activity of the MDR-relevantefflux pumps P-gp and MRP-1 were then evaluated using thefluorescent dyes Rho-123 and 5-CFDA, which are widely used P-gpand MRP-1 substrates, respectively. Because of the lower expressionof P-gp protein in leiomyosarcoma SK-UT-1 cells (Fig. 4A), we onlymeasured the efflux activity of P-gp in synovial sarcoma SW982 cells.Neither OLA nor MLA (10–80 μM) increased significantly theintracellular retention of Rho-123 (Fig. 4B). However, the P-gpinhibitor verapamil (10 μM), used as positive control, augmentedthe intracellular Rho-123 accumulation by 1.5±0.11-fold in these

Fig. 3. OLA and MLA increased intracellular DXR in human STS cells. Synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells were incubated with vehicle (V) or DXR (1 μM forSW982 or 0.1 μM for SK-UT-1 cells) in the absence or presence of OLA (10–80 μM) orMLA (10–50 μM) as described inMaterials andmethods. After 24 h of incubation, intracellular DXRwas measured by its fluorescence intensity with flow cytometry. (Upper panels) Median of intracellular DXR fluorescence in the absence (V) or presence of triterpenes normalized tovehicle-treated cells (taken as 100%). Each column represents mean±S.E.M. of three independent experiments. (Lower panels) Representative flow cytometry analysis of theintracellular DXR fluorescence detected with excitation at 488 nm and emission at 580 nm in synovial sarcoma (left panel) and leiomyosarcoma cells (right panel). ###Pb.001 vs.vehicle-treated cells and ⁎⁎Pb.01 and ⁎⁎⁎Pb.001 vs. DXR-treated cells.

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cells. In contrast, OLA at 10 and 50 μM significantly increasedintracellular 5-CFDA accumulation by 4.4±0.73-fold and 5.5±0.92-fold in synovial sarcoma cells (Fig. 5A) and by 3.5±0.29-fold and 6.2±0.16-fold in leiomyosarcoma SK-UT-1 cells, being also in leiomyo-sarcoma cells more effective than the MRP-1 inhibitor probenecid(250 μM), used as positive control (Fig. 5B). In a similar way,intracellular 5-CFDA levels were also increased in the presence of MLAat 10 and 50 μMby 8.0±0.29-fold and 15.8±1.29-fold, respectively, inSW982 cells, and by 2.9±0.36-fold and 8.5±0.47-fold, respectively, inleiomyosarcoma cells (Fig. 6A). However, neither OLA (80 μM) norDXR (1 or 10 μM) alone or in combination for 24 h alteredsignificantly the expression of MRP-1 in these STS cells (MRP-1 levelsrose 2.8±12.1% in OLA-treated cells, 12.6±3.8% and 1.4±3.9% in DXR-

treated cells at 1 or 10 μM and 18.9±12.2% and 19.9±9.6% in cellstreated with OLA plus DXR 1 or 10 μM, when compared with vehicle-treated cells) (Fig. 5B). Similar results were obtained with thetreatment of SK-UT-1 cells with MLA (30 μM) alone (MRP-1 levelsrose 13.1±8.6% when compared with vehicle-treated cells) or incombination with DXR (MRP-1 levels rose 24.8±9.4% and 19.4±4.0%in cells treated with MLA plus DXR 1 and 10 μM, respectively)(Fig. 6B).

Similar to results from other groups, OLA and MLA at 80 μMincreased ROS production by 2.1±0.16-fold and 3.9±0.20-fold,respectively, in SW982 cells, and by 2.5±0.33-fold and 5.5±1.10-fold, respectively, in leiomyosarcoma cells. Due to this fact, we nextevaluatedwhether these triterpenes inhibit MRP activity indirectly by

Fig. 4. OLA and MLA did not inhibit P-gp activity in human synovial sarcoma SW982 cells. (A) P-gp and MRP-1 protein expression in human STS cells. Panels show immunoreactivebands of P-gp (170 kDa) and MRP-1 proteins (190 kDa) in synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells in representative immunoblots. The amount of total proteinloaded was 45 or 30 μg for the detection of P-gp or MRP-1 proteins, respectively. (B) Intracellular Rho-123 fluorescence was estimated by flow cytometry in SW982 cells after 2 h oftreatment with vehicle, OLA (10–80 μM), MLA (10–80 μM) or verapamil (10 μM). (Right) Median of intracellular Rho-123 fluorescence in the absence (vehicle, V) or presence oftriterpenes normalized to vehicle-treated cells (taken as 100%). Each column represents mean±S.E.M. of three independent experiments. (Left) Representative flow cytometryanalysis of the intracellular Rho-123 fluorescence in the absence (vehicle) or presence of OLA (10 and 80 μM) or MLA (10 and 80 μM) detected with excitation at 488 nm and emissionat 530 nm. T0 was the intracellular Rho-123 fluorescence measured at t=0. ⁎⁎⁎Pb.001 vs. vehicle-treated cells.

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reducing intracellular GSH — an important antioxidant factor thatmodulates the activity of these transporters. The incubation ofleiomyosarcoma cells with different concentrations of OLA or MLA(10–80 μM) for 2 h did not modify the intracellular GSH level(Fig. 7A). This indicates that inhibition of MRP activity by thesetriterpenes is not caused by a decrease of intracellular GSHconcentration due to ROS generation. In order to get a deeperinsight in the mechanism of OLA and MLA on MRP activity, wemonitored the variation of intracellular retention of CFDA inleiomyosarcoma cells, which mainly express this transporter, byflow cytometry. Different concentrations of the fluorescencesubstrate CFDA were used while OLA or MLA was added at fixedconcentrations of 10, 50 and 80 μM. The export rates of CFDA aredisplayed as a direct function of CFDA concentration in Fig. 7B. Thekinetic parameters Vmax and Km (obtained by using the Michaelis–Menten equation) indicated that OLA (10–80 μM) did not changesignificantly the Vmax values of CFDA export but dose-dependentlyaugmented its Km values from 0.8±0.1 to 1.2±0.1 and 1.5±0.1 μMat 50 μM and 80 μM, respectively, when compared to vehicle-treatedcells (see table in Fig. 7B). In contrast, increasing concentrations of

MLA did not affect the Km values of CFDA but dose-dependentlydecreased its Vmax values from 50.2±3.5 to 36.8±2.2 and 30.8±3.2FRU/h at 50 and 80 μM, respectively, when compared to vehicle-treated cells (see table in Fig. 7B). Together, these results suggestthat OLA behaved as a competitive inhibitor of the MRP transporter,while MLA acted in a non-competitive manner. In short-timeincubation experiments (Figs. 4, 6 and 7), MLA was used at 80 μMto permit a direct comparison with OLA and to test whether someeffects would be detectable at higher concentrations.

4. Discussion

OLA and MLA are natural compounds present as minor non-nutritional components in pomace and virgin olive oil [3,4], which areregularly consumed in the traditional Mediterranean diet [5] andassociated with low incidence of cancer [28]. Special interest has beengiven to the antitumoral effects of these pentacyclic acids [29,30], butstill, almost nothing is known about their efficacy in STS cells andtheir ability to modulate the antitumoral effects of the standardchemotherapeutic DXR.

Fig. 5. OLA inhibited MRP-1 activity without altering its expression in human STS cells. Intracellular 5(6)-CFDA fluorescence was estimated by flow cytometry in synovial sarcomaSW982 (A) and leiomyosarcoma SK-UT-1 cells (B) after 2 h of treatment with vehicle, OLA (10–80 μM) or probenecid (250 μM). (Left) Median of intracellular CFDA fluorescence in theabsence or presence of OLA normalized to vehicle-treated cells (taken as 100%). Each column represents mean±S.E.M. of three independent experiments. (Right) Representative flowcytometry analysis of the intracellular CFDA fluorescence in the absence (vehicle) or presence of OLA (10 and 80 μM) detected with excitation at 488 nm and emission at 530 nm. T0was the intracellular CFDA fluorescence measured at t=0. ⁎Pb.05, ⁎⁎Pb.01 and ⁎⁎⁎Pb.001 vs. vehicle-treated cells. (B, lower panel) Levels of MRP-1 were evaluated after treatment withOLA (80 μM) alone or in combination with DXR (1 and 10 μM) for 24 h in leiomyosarcoma cells. The intensity of MRP-1 protein band was normalized against the intensity of thecorresponding β-actin band. Immunoreactive bands are representative of three independent experiments.

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Our study demonstrates that MLA exhibited stronger antipro-liferative and pro-apoptotic effects than OLA in two human STS celllines (Figs. 1B and 2). Intracellular production of ROS is able totrigger apoptosis, and twice as high efficacy with which MLAprovoked intracellular ROS generation could be an explanation forthe observed higher antitumoral effect of MLA compared to OLA.Likewise, in HT-29 colon cancer cells OLA also differed from MLAregarding its capacity to generate significant levels of superoxideanions and had only a moderate antiproliferative activity (−16%cell growth) [4]. The low potential of OLA to induce cell death hasalso been confirmed in other human colon carcinoma cell lines[4,31]. Independently of this difference, both triterpenes sensitizedSTS cells to DXR and were able to selectively reduce MRP-1 effluxpump activity (Figs. 5 and 6) at concentrations that may beachieved following a typical Mediterranean diet based on theregular consumption of virgin olive oil and olives [4]. Mostprobably as a consequence, DXR accumulated in these cells,which was accompanied by a stronger antiproliferative and

apoptotic effect. Because MRP-1 and P-gp are able to extrudeDXR out of the cell, synovial sarcoma cells constitutively expressingboth proteins (Fig. 4A) may be able to partially overcome thespecific inhibition of MRP-1. This would explain why these cellswere less sensitive to the combination treatment of OLA or MLAplus DXR than leiomyosarcoma cells, which mainly express MRP-1(Figs. 2 and 4A).

An increase of ROS production, such as the one mentioned for OLAand MLA, could be related to the observed inhibition of MRP-1. GSHnot only provides endogenous antioxidant protection against ROS butalso forms glutathione-S conjugates with xenobiotics, and theseconjugates are then removed from cells by MRP-1, but not P-gp. Theincrement of intracellular ROS through OLA and MLA could lead to areduction of intracellular GSH levels and, in this way, indirectly inhibitthe export of DXR byMRP-1 transporters because anthracyclines (e.g.,DXR) are predominantly cotransported with GSH [32,33]. However,this proposed molecular mechanism of action for OLA and MLA doesnot seem to be relevant for their inhibitory effect on MRP-1 activity in

Fig. 6. MLA inhibited MRP-1 activity without altering its expression in human STS cells. Intracellular 5(6)-CFDA fluorescence was estimated by flow cytometry in synovial sarcomaSW982 (A) and leiomyosarcoma SK-UT-1 cells (B) after 2 h of treatment with vehicle, MLA (10–80 μM) or probenecid (250 μM). (Left) Median of intracellular CFDA fluorescence in theabsence or presence of MLA normalized to vehicle-treated cells (taken as 100%). Each column represents mean±S.E.M. of three independent experiments. (Right) Representative flowcytometry analysis of the intracellular CFDA fluorescence in the absence (vehicle) or presence of MLA (10 and 80 μM) detected with excitation at 488 nm and emission at 530 nm. T0was the intracellular CFDA fluorescence measured at t=0. ⁎Pb.05 and ⁎⁎⁎Pb.001 vs. vehicle-treated cells. (B, lower panel) Levels of MRP-1 were evaluated after treatment with MLA(30 μM) alone or in combination with DXR (1 and 10 μM) for 24 h in leiomyosarcoma cells. The intensity of MRP-1 protein band was normalized against the intensity of thecorresponding β-actin band. Immunoreactive bands are representative of three independent experiments.

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STS cells as the increment in ROS production provoked by OLA andMLA did not alter intracellular GSH levels (Fig. 7), although this wasonly tested in the absence of DXR.

The efflux of the MRP-1-specific substrate CFDA in leiomyosar-coma cells was inhibited by OLA (Fig. 7). The kinetic parametersindicated a competitive inhibition, meaning that CFDA and OLA haveaffinity to the same binding site and are able to displace each otherdepending on the respective concentration. This suggests that OLAitself is probably a substrate for MRP transporters, which would againexplain its low efficacy in our study or as single antitumoral drug inthose tumors overexpressingMRP-1 [17]. In contrast, MLA behaved asa non-competitive inhibitor of CFDA export, which is normallyachieved by binding to a site on the protein other than thesubstrate-binding site (allosteric inhibition). Therefore, the maxi-mum export rate is reduced because even higher substrate concen-tration cannot displace the inhibitor from its binding site and revertthe inhibitory effect. Interestingly, neither MLA nor OLA altered theefflux kinetics of P-gp in our study (Fig. 4), which is consistent with

previous results in non-tumoral monkey epithelial cells obtained byBraga and colleagues who reported that OLA inhibited MRP-1 activitywithout affecting that of P-gp [17]. Although structure–activityrelationship studies have identified some features common to theinhibitors of both, P-gp and MRP-1 transporters, differences in theinhibitor requirements and substrate preferences of these trans-porters have also been described [34]. MRP-1 has a preference fornegatively charged substrates, whereas compounds with cationiccharge are only transported by P-gp [35]. Indeed, OLA possesses sucha negatively charged type I recognition unit as its molecular structurepresents a carboxylic acid group, thus fulfilling an important requisitefor MRP-1 substrates. In spite of that, the current knowledge aboutmolecular requirements of MRP-1 inhibitors cannot explain theobserved differences between OLA and MLA.

If the inhibition of MRP-1 activity by OLA and MLA actuallyaffected DXR accumulation in tumor cells of patients, the therapeuticapplicability of such a combination therapy in STS would depend onthe expression of MRP-1 in the particular tumor of each patient. We

Fig. 7. OLA inhibited MRP-1 transport function through a competitive inhibition mechanism, whereas MLA acted through a non-competitive one, without altering intracellular GSHlevels in human leiomyosarcoma SK-UT-1 cells. (A) SK-UT-1 cells were treated with vehicle, OLA (10–80 μM), MLA (10–80 μM) and H2O2 (60 mM, as positive control) for 2 h, andintracellular GSH levels were determined. (B) Kinetic inhibition parameters of triterpenes were evaluated using the fluorescence dye CFDA (0, 0.1, 1 and 10 μM) as transporter drug inthe absence (vehicle) or presence of OLA or MLA (10–80 μM). CFDA export rate (FRU/h) was plotted against CFDA concentration (μM). The kinetic parameters Km and Vmax werecalculated with the Michaelis–Menten equation using GraphPad Software. Each value is the mean of three independent experiments.

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have recently demonstrated that, in a subset of 52 high-risk locallyadvanced STS tumor patients derived from a large international studywith 328 participants, 15%were positive forMRP-1 expression [26,36]and, even more important, that its expression correlated with worseprognosis in these patients [37]. In this context, OLA and MLA may bevaluable adjuvants combined with DXR in the treatment of STStumors. Furthermore, none of both triterpenes induced a compensa-tory increase in MRP-1 expression (Figs. 5 and 6), which is inagreement with results from other groups [17,38] but is usually acommon problem for compounds blocking P-gp activity. In addition,

MRP-1 does not appear to be as important as P-gp in protecting non-tumoral cells from chemotherapeutic drugs and other toxicants[39,40], meaning that inhibition of MRP-1 should not augmentgeneral toxic side effects of DXR. Accordingly, OLA, MLA andalso other novel olenane-type pentacyclic triterpenoids presentedlow toxicity in normal cells and animal models, when administeredalone [41–43].

In summary, our study reveals that OLA and MLA increase DXR-induced antitumoral effects in STS cells and inhibit the activity of theMDR proteinMRP-1. Although the data do not present direct evidence

10 V.H. Villar et al. / Journal of Nutritional Biochemistry xx (2014) xxx–xxx

that the observed intracellular accumulation of DXR was caused byMRP-1 inhibition, this anthracycline is a known substrate of MRP-1and it can be deduced that such an inhibition would also affectthe transport of DXR. Therefore, OLA and MLA are promisingadjuvants for the treatment of STS tumors expressing MRP-1 withDXR and perhaps also with other chemotherapeutics that aresubstrates of this efflux pump.

Acknowledgements

We thank Dra. A. Guinda for providing OLA.

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