Cellular pharmacology of cisplatin in relation to the expression of human copper transporter CTR1 in different pairs of cisplatin-sensitive and -resistant cells

Abstract

The molecular mechanism of cisplatin uptake remains poorly defined and impaired drug accumulation may be implicated in the acquisition of resistance to cisplatin. Thus, we used cell lines of different tumor types (ovarian carcinoma A2780 and IGROV-1, osteosarcoma U2-OS, cervix squamous cell carcinoma A431) and stable cisplatin-resistant sublines, exhibiting variable levels of resistance (between 2.5 and 18.4), to investigate the mechanisms of cellular accumulation of cisplatin. Among the resistant lines we found that reduced cisplatin uptake was a common feature and ranged between 23 and 76%. In an attempt to examine the role of human copper transporter 1 (CTR1) in cisplatin accumulation by human cells, we selected the well characterized A431 cell line and the resistant variant A431/Pt. As compared with A431/Pt cells, A431/Pt transfectants overexpressing CTR1 (3.4-fold) exhibited increased uptake of copper, thereby supporting the expression of a functional transporter. However, no changes in cisplatin uptake and cellular sensitivity to drug were observed. Also overexpression of CTR1 in A431 cells did not produce modulation of cisplatin accumulation. An analysis of the expression of other factors that could affect drug accumulation indicated that A431/Pt cells displayed increased expression of ATPase, Cu²⁺ transporting, alfa polypeptide. In conclusion, our results indicate that the overexpression of a functional CTR1 in a human cell line characterized by impaired cisplatin uptake fails (a) to restore cellular drug accumulation to the level of the parental cell line and (b) to modulate cisplatin sensitivity. Our data are consistent with the interpretation that the defects in cellular accumulation by resistant cells are not mediated by expression of CTR1, that plays a marginal role, if any, in cisplatin transport.

Introduction

Cisplatin (DDP) is a chemotherapeutic agent used for the treatment of a variety of solid tumors including squamous cell carcinomas [1]. The development of resistance to DDP during treatment is common and constitutes a major obstacle to the cure of sensitive tumors. Resistance to DDP is thought to be due to the selection of drug-resistant cells that arise through spontaneous somatic mutation [2]. Determination of in vitro sensitivity to DDP of tumor cell lines or samples from patients before and after treatment with DDP indicated that only modest level of resistance may be sufficient for treatment failure [3], [4], [5]. This is consistent with the rapid emergence of low levels of DDP resistance documented in human tumor xenografts [6].

To understand the clinically relevant mechanisms of resistance, preclinical studies have been aimed at clarifying the biochemical/molecular alterations of resistant cells. These studies did not conclusively identify the bases of cellular resistance to DDP, but they contributed to define the multifactorial alterations involved, such as reduced drug accumulation [7], [8], increased detoxification through thiol-mediated mechanisms [9], [10], reduced DNA platination and enhanced DNA–platinum adduct removal [11]. Moreover, a frequently identified alteration of cells with acquired DDP resistance both in vitro and in vivo involves impaired drug uptake [5], [7], [12], [13], [14], [15], [16], [17], [18].

The mechanism of cellular DDP accumulation is poorly defined. Several evidences suggest that drug uptake is mediated by a transport mechanism or channel [19], [20], [21], [22]. The copper transporter 1 (CTR1) has been recently implicated in mediating the DDP uptake and acquisition of DDP resistance in yeast, mouse, and mammals [23], [24]. Conflicting results were obtained by Chauhan et al. [25] in the human epidermoid carcinoma cell line KB-3-1 and in the DDP-resistant variant KB-CP20, as the overexpression of human CTR1 gene in KB-CP20 failed to change DDP accumulation.

In an attempt to examine the role of human CTR1 as a determinant of DDP resistance/sensitivity and accumulation in human cells, we used the human cervix squamous cell carcinoma cell line A431 and the DDP-resistant variant A431/Pt. In particular, we cloned and overexpressed the human CTR1 cDNA in A431 and A431/Pt cells. We found that the overexpression of CTR1 failed to increase DDP accumulation and to change DDP sensitivity in both cell lines. Our results support that the defects in cellular accumulation by resistant cells are not mediated by expression of CTR1, that plays a marginal role, if any, in cisplatin transport.