Research article
Multidrug resistance mediated by ABC transporters in osteosarcoma cell lines: mRNA analysis and functional radiotracer studies

https://doi.org/10.1016/j.nucmedbio.2006.07.011Get rights and content

Abstract

Drug resistance remains a significant impediment to successful chemotherapy and constitutes a major prognostic factor in osteosarcoma (OS) patients. This study was designed to identify the role and prognostic significance of multidrug-resistance (MDR)-related transporters, such as multidrug resistance protein 1 (MDR1), multidrug-resistance-associated protein (MRP1) and breast-cancer-related protein (BCRP), in OS using cationic lipophilic radiotracers. We evaluated the chemosensitivity of four OS cell lines (Saos-2, 143B, MNNG/HOS and U-2OS) to doxorubicin (DOX), cisplatin (CIS) and methotrexate. The expression of MDR-related transporters was analyzed at mRNA level by quantitative polymerase chain reaction and at functional level by 99mTc sestamibi and 99mTc tetrofosmin. The effectiveness of MDR modulators [cyclosporin A (CsA) and imatinib] on transporter inhibition and on the reversal of resistance was also assessed.

MNNG/HOS and U-2OS cells expressing high levels of MDR1 were highly resistant to DOX and showed reduced accumulation and higher efflux for radiotracers. Although MRP1 was uniformly expressed in all cells, only U-2OS was resistant to CIS. CsA restored sensitivity to DOX and CIS, and enhanced the accumulation and efflux half-life of radiotracers in MDR1-expressing cell lines.

The chemosensitivity of OS cells to DOX was strongly dependent on mRNA MDR1 expression and could be circumvented by adding CsA. The kinetic parameters of radiotracers correlated with MDR1 expression levels, hence predicting DOX resistance. We concluded that sensitivity to chemotherapy is strongly dependent on the expression of MDR1 transporter and that radiotracer studies could prove clinically useful in predicting chemotherapy response and in evaluating the efficacy of MDR-reversing agents.

Introduction

Long-term disease-free survival in osteosarcoma (OS) patients has increased significantly with the introduction of adjuvant and neoadjuvant chemotherapy. To date, besides the detection of metastasis on diagnosis, histological response to preoperative chemotherapy based on the Huvos grading system is the sole reliable predictive parameter for clinical outcome [1], [2], [3], [4]. A relationship between histological subclassification and response to chemotherapy has been proposed [5] but is not yet used in clinical practice. In the meantime, systemic relapses still occur in 40% of cases [3], [4], mostly related to poor response to chemotherapy. Increasing drug dosage in histologically poor responders has not improved their outcome [2], [6], [7]. Nearly 50% of OS cases are either resistant to chemotherapy or acquire resistance during treatment [8]. One mechanism responsible for intrinsic or acquired drug resistance is the overexpression of transmembrane efflux proteins that have the ability to pump out certain substances from cells (reviewed in Borst and Elferink [9] and Gottesman [10]). The first transporter that was identified and characterized — multidrug resistance protein 1 (MDR1, also known as P-glycoprotein; encoded by the ABCB1 gene) [11], [12] — was a member of the ATP-binding cassette (ABC) superfamily. Subsequently, other members of this family, such as multidrug-resistance-associated protein (MRP1; encoded by the ABCC1 gene) and breast-cancer-related protein (BCRP; encoded by the ABCG2 gene), have proven to confer drug resistance to structurally and functionally unrelated drugs [13], [14], [15].

In vitro and clinical observations have shown that overexpression of MDR1 may be associated with drug resistance, tumor recurrence and poor outcome in OS patients, suggesting that MDR1 overexpression could be used as a prognostic factor [16], [17], [18]. However, others have failed to confirm this relationship, and the role of MDR1 as a marker of poor prognosis remains controversial [19], [20]. Different methodologies and the timing for MDR1 assessment (prechemotherapy or postchemotherapy) can account for these conflicting results. mRNA and protein expression studies do not provide information on the functional activity of drug efflux transporters. Furthermore, common chemotherapeutic regimens in OS include doxorubicin (DOX), a substrate of MDR1, but also methotrexate (MTX) and cisplatin (CIS), which are substrates for other multidrug resistance (MDR) transporters acting independently of MDR1.

MRP1 is overexpressed in several cell lines that display the MDR phenotype and has been reported in various tumors, including hematological malignancies [21], lung cancer [22] and Ewing's sarcoma [23]. The expression of MRP1 has not been extensively studied, and only a few studies correlate the expression of MRP1 with therapeutic response in OS [24], [25], [26]. BCRP is associated with resistance to mitoxantrone, camptothecins and MTX, and has been shown to be overexpressed in several resistant cell lines [15], [27], [28]. To our knowledge, there are no reports on BCRP expression in OS clinical samples.

A possible approach to circumvent MDR is the coadministration of inhibitors — compounds that inhibit the transport activity of MDR transporters. A large number of compounds, such as verapamil, cyclosporin A (CsA), PSC-833, VX710, tariquidar and imatinib mesylate, have been reported to reverse MDR in vitro and in vivo when combined with anticancer drugs [29], [30], [31], [32] but have yet to be proven useful in the clinical setting.

Molecular imaging provides a noninvasive way to assess in vivo the functional activity of MDR transporters using radiotracers that are substrates for pumps. Several 99mTc-labeled cationic lipophilic compounds, such as sestamibi (MIBI) and tetrofosmin (TFS), have been proposed as in vivo imaging probes for MDR functional activity [33].

In order to determine the role of MDR in current OS chemotherapy protocols at mRNA and functional levels, we studied the chemosensitivity of a panel of established human OS cell lines to drugs used in the recently started EURAMOS-1 protocol (DOX, CIS and MTX) (www.ctu.mrc.ac.uk/euramos) and correlated this with the gene expression levels of three MDR transporters (MDR1, MRP1 and BCRP). These cell lines have not been previously exposed to the drugs and are therefore not selected for drug resistance. The functional activity of MDR transporters was assessed via uptake and efflux kinetics using MIBI and TFS, and correlated with MDR expression pattern. Two MDR inhibitors (CsA and imatinib mesylate) were evaluated for their ability to reverse drug resistance, and radiotracers were used to monitor the effective inhibition of MDR transporters. CsA is a wide-spectrum MDR modulator that shows activity against MDR1 and MRP1 [32]. Imatinib mesylate, recently suggested as a potent inhibitor of BCRP transporters, has been shown to reverse BCRP-mediated resistance in BCRP-overexpressing cell lines [30].

Section snippets

Cell cultures

The human OS cell lines MNNG/HOS, 143B, U-2OS and Saos-2 were purchased from the American Type Culture Collection (Rockville, MD). All cell lines were cultured in RPMI 1640 (Gibco Invitrogen Life Technologies, Scotland, UK) and 10% heat-inactivated fetal calf serum (FCS; Gibco Invitrogen Life Technologies) at 37°C in a humidified incubator with 95% air and 5% CO2. None of the cell lines was previously selected for drug resistance. As a reference for gene expression, we used human

Chemosensitivity of OS cell lines and reversal of drug resistance

We used a panel of human established OS cell lines not selected for drug resistance in order to evaluate the cytotoxicity of DOX, CIS and MTX — the drugs used in the EURAMOS-1 protocol for the treatment of OS.

The chemosensitivity of OS cell lines to DOX and CIS was measured by FACS analysis. A dose-dependent increasing cell death was observed for Saos-2 and 143B cells after 24 h of exposure to DOX, whereas MNNG/HOS and U-2OS were highly resistant, with a low percentage of dead cells and with no

Discussion

This study was designed to evaluate the role and the prognostic significance of MDR-related transporters in the response of OS cell lines to chemotherapy.

We used a combination of drug response studies, qPCR and functional assays using radiotracers to determine whether there was a relationship between the drug resistance, gene expression and functional activity of MDR-associated transporters in OS cell lines.

In this study, we showed that different OS cell lines have different sensitivities to

Acknowledgments

We thank Novartis Pharmaceuticals for kindly providing us with imatinib mesylate (Gleevec), and Drs. Paul Eilers and Jan Oosting for their help with curve fitting and statistical analysis.

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    This research was supported by FARMA APS Portugal and a European Commission grant for a network of excellence (EuroBoNeT).

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