Relevance of copper transporter 1 for cisplatin resistance in human ovarian carcinoma cells

Abstract

Defects in intracellular accumulation of the antitumour drug cisplatin are a commonly observed feature in the cells selected for cisplatin resistance. Copper transporter 1 (CTR1) has been suggested to play an important role in drug uptake and resistance. Here, we describe a detailed investigation of the involvement of CTR1 in cisplatin uptake and its relevance for cisplatin resistance using a well characterised sensitive/cisplatin-resistant cell line pair: A2780 human ovarian carcinoma cell line and its cisplatin-resistant variant A2780cis. A2780cis cells showed decreased cisplatin accumulation and lower CTR1 expression compared to A2780 cells. Co-incubation with copper sulphate affected neither cisplatin accumulation (determined by flameless atomic absorption spectrometry) nor its cytotoxicity (determined using an MTT-assay, MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). In both cell lines, CTR1 was localised near the nucleus as found using confocal fluorescence microscopy. The steady-state localisation of the protein in perinuclear region appears to involve its continuous endocytosis from cell surface. In contrast to copper, cisplatin exposure had no influence on the sub cellular localisation of CTR1. Co-localisation between CTR1 and a fluorescent cisplatin analogue labelled with carboxyfluorescein-diacetate could be observed in vesicular structures when continuous retrieval of the protein from cell membrane was inhibited. Our results strongly suggest that CTR1 mediates cisplatin uptake in the cell lines studied. Upon its transport across the plasma membrane by CTR1 the platinum drug is likely to be internalised along with the protein. Our findings imply that reduced CTR1 expression accounts for decreased cisplatin accumulation and represents one of the determinants of cisplatin resistance in A2780cis cell line.

Introduction

Over the past decades, cisplatin remains a widely used antitumour drug applied for treatment of various solid tumours including testicular, ovarian, head and neck, and small cell lung cancers [1]. Development of resistance in the course of the chemotherapy is, however, common and often leads to treatment failure. In order to understand mechanisms of cisplatin resistance, multiple studies have focused on biochemical and molecular alterations of resistant cells. Cancer cell resistance to cisplatin has been described to be multifactorial, with several mechanisms operating simultaneously in a single cell [1], [2]. Until now, reduced drug accumulation, increased drug inactivation within the cell, enhanced DNA repair and increased DNA damage tolerance have been found to contribute to cisplatin resistance [3]. Nevertheless, the most common feature of the cells with acquired resistance to cisplatin is impaired cellular accumulation of the drug [4].

Despite much effort, the mechanisms cisplatin employs to enter tumour cells remain largely obscure. Recent findings point out at the involvement of copper transporter 1 (CTR1) in the uptake of the platinum drug [5], [6]. The transporter appears to be clinically relevant, since high expression of CTR1 in patients with ovarian carcinoma was associated with good therapeutic response, while low levels of the protein lead to poor therapeutic outcome [7]. Deletion of CTR1 was reported to decrease cisplatin accumulation and to increase resistance in vitro and in vivo [8], [9]. Transfection of human ovarian carcinoma cells with CTR1 led to enhanced cellular accumulation of the drug but failed to increase DNA platination and cell sensitivity to cisplatin [10]. Some studies demonstrated that exposure of human tumour cells to clinically relevant concentrations of cisplatin triggers rapid degradation and loss of CTR1 [11], [12]. However, conflicting results showed that transfecting cervix carcinoma cells with the transporter did not lead to any changes in cellular accumulation and cytotoxicity of cisplatin [13]. Furthermore, expression of CTR1 in various cisplatin-resistant cell lines was reported to be similar to that in the respective sensitive counterparts [13], [14], [15].

Recent work from our laboratory presented characterisation of the cisplatin-sensitive/resistant human ovarian carcinoma cell line pair A2780/A2780cis regarding cellular platinum accumulation and efflux, cytotoxicity and expression of CTR1. Cellular platinum accumulation was significantly reduced in cisplatin-resistant cells as compared to their sensitive counterparts [16]. As no differences in cisplatin efflux between A2780 and A2780cis cells were observed, we concluded that primarily a decrease in cisplatin influx accounted for lower intracellular platinum concentrations in the resistant cell line. The A2780cis cell line exhibited 4.4-fold resistance to cisplatin, which correlated well with the decrease in the degree of DNA platination. Cisplatin-resistant cells expressed 1.5–1.8-fold lower levels of CTR1 suggesting a relationship between CTR1 expression, cisplatin uptake, DNA platination and cell sensitivity to the drug [16]. Interestingly, no reduction in CTR1 expression levels after cisplatin exposure was observed in both cell lines [16]. The present study aimed at further clarification of the role of CTR1 as a determinant of cisplatin uptake and resistance to the drug in the A2780/A2780cis cell line pair.

This paper describes a detailed investigation of sub cellular localisation of CTR1 under various conditions including exposure to copper and cisplatin, copper influence on cellular platinum accumulation and cytotoxicity, as well as studies of co-localisation between CTR1 and a cisplatin analogue labelled with fluorogenic carboxyfluorescein-diacetate. Based on the results of the study, relevance of CTR1 for cisplatin accumulation and drug resistance in the above-mentioned cell line pair is discussed.