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

https://doi.org/10.1016/j.jnutbio.2013.12.003Get rights and content

Abstract

The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10–100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3±1.11% and 68.8±1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5–10 μM) by up to 2.3-fold. On the molecular 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-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.

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 plant species, but only a few, such as Olea europaea, contain a high concentration in leaves and fruits [1], [2]. More importantly, appreciable amounts of OLA and MLA are also present in the non-glyceride fraction of pomace and virgin olive oils (244±28 mg/kg of OLA and 194±14 mg/kg of MLA) [3], [4], which are consumed in the Mediterranean diet [5]. Several important biological activities, e.g., antioxidant [6], anti-inflammatory [7] and vasodilatory effects [8], have been attributed to these triterpenes. In the last few years, OLA and MLA have also received increasing attention due to its antitumoral action. In this context, antiproliferative and apoptotic activities of both triterpenes have been demonstrated in a variety of human tumor cell lines, including astrocytoma [9], breast [10], [11] and colon cancer cells [4], [12]. Although the antitumoral effect of OLA and MLA has been mainly associated to their capacity to increase intracellular reactive oxygen species (ROS) [4], [9], [12], [13], other molecular mechanisms, such as activation of JNK/p53 pathway [14], activation of p38 MAPK through elevation of intracellular calcium [15] or suppression of NF-κB signaling [16], also seem to be related to its antitumoral activity in some epithelial cancers. In addition, in a monkey epithelial cell line 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 mainly involved in the development of multidrug resistance (MDR) in cancer cells [18].

Soft tissue sarcomas (STS) comprise a heterogeneous group of malignant tumors arising from soft tissues, including fat, smooth or skeletal muscle, fibrous tissue and vasculature, which embryologically derived from the mesoderm or neuroectoderm [19], [20]. Although surgical removal of the entire tumor remains the main therapeutic strategy for localized STS, doxorubicin (DXR)-based chemotherapy is widely used in the treatment of patients with locally advanced inoperable or metastatic disease [21], [22]. Nevertheless, therapies with DXR alone or in combination with ifosfamide have not achieved significant improvements in terms of overall survival [23]. Additionally, the effectiveness of DXR is limited because it provokes severe cardiotoxicity and the development of MDR, the latter mainly involving high cellular expression or activity of ABC transporters in the plasma membrane, such as P-glycoprotein (P-gp) and MRP-1 [24], [25]. As a result, many patients ultimately relapse, and the prognosis for patients with metastatic sarcoma remains poor. Therefore, the search for new therapeutic approaches is still a need and a combination therapy, which was able to sensitize cancer cells to DXR by counteracting such resistance mechanism without increasing general toxicity, would be a suitable strategy.

Although several studies have evaluated the efficacy of OLA and MLA in human carcinomas, only little is known regarding their effects in STS and whether a combination of these natural compounds with conventional chemotherapy is able to improve treatment outcome for this type of solid tumors. Our study focuses on the efficacy and molecular mechanisms involved in the antitumoral effects of OLA and MLA in STS cells, either as individual agents or in combination with DXR.

Section snippets

Cell culture and treatments

The human cell lines SW982 and SK-UT-1 were derived from synovial and leiomyosarcoma, respectively, which represent some of the most common STS in humans, accounting for approximately 5–10% and 24% of STS in adults. The cell lines were chosen because they show low sensitivity to DXR treatment [26] being a requisite for the aim of the study. SW982 and SK-UT-1 cells express the MDR protein MRP-1 at similar levels, but differ in the level of P-gp, which is barely expressed in SK-UT-1 cells. Both

Results

Growth inhibition by OLA and MLA (10–100 μM) was determined in synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells after 24 h of treatment. MLA inhibited STS cell proliferation in a concentration-dependent manner, and the average IC50 values were 45.3±0.04 μM in SW982 cells and 59.1±0.06 μM in SK-UT-1 cells. At 80 μM, MLA reduced cell growth by 70.3±1.1% and 68.8±1.5%, respectively (Fig. 1B). Flow cytometry analysis indicated that 80 μM MLA induced a significant cell death up to 37.5% after

Discussion

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

Acknowledgements

We thank Dra. A. Guinda for providing OLA.

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    Conflict of interest statement: The authors confirm that there are no conflicts of interest.

    ☆☆

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

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