Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells

Abstract

Drug resistance, one of the major obstacle in the successful anticancer therapy, can be observed at the outset of therapy (intrinsic resistance) or after exposure to the antitumor agent (acquired resistance). To gain a better insight into the mechanisms of intrinsic resistance we have analyzed two human cell types derived from untreated tumors: MCF-7 breast cancer and A549 non small cell lung cancer (NSCLC). We have examined: the cytotoxic effect induced by doxorubicin (DOX); the time course of drug accumulation by flow cytometry and intracellular drug distribution by confocal microscopy; the expression and distribution of proteins related to anthracycline resistance, such as P-gp (P-glycoprotein), MRP1 (multidrug resistance-associated protein) and LRP (lung resistance-related protein). The cytotoxicity assays showed that A549 cells were less sensitive than MCF-7 cells to the DOX treatment in agreement with the different DOX uptake. Moreover, while in A549 cells DOX was mostly located in well defined intracytoplasmic vesicles, in MCF-7 cells it was mainly revealed inside the nuclei. The analysis of P-gp and MRP expression did not show significant differences between the two cell lines while a high expression of LRP was detected at the nuclear envelope and cytoplasmic levels in A549 cells. These findings suggest that the lower sensitivity to DOX treatment showed by lung carcinoma cells could be ascribed to drug sequestration by LRP inside the cytoplasmic compartments.

Introduction

Successful chemotherapeutic treatment of tumors is often seriously hampered by their poor response to anticancer drugs. In fact, tumor cells can be insensitive to drug treatment at the outset of therapy, thus exhibiting an intrinsic resistant phenotype (Childs and Ling, 1994).

The mechanisms responsible for intrinsic drug resistance are still poorly understood. It is well known that tumor cells exposed to antineoplastic agents may develop, both in vivo and in vitro, a multidrug resistant (MDR) phenotype, which is generally associated with altered expression of drug transporter proteins, such as P-glycoprotein (P-gp) (Gottesman & Pastan, 1993, Shapiro & Ling, 1995) and the family of multidrug resistance-associated proteins (MRPs) (Cole et al., 1992, Berger & Elbling, 1997). These proteins act as ATP-dependent molecular pumps, which extrude a number of antitumor drugs from the cell, thus decreasing their concentrations at intracellular targets. It is generally believed that an intrinsic MDR phenotype is caused by multiple mechanisms; recent evidence suggests that changes in transport proteins may also contribute to this more elusive form of drug resistance (Berger et al., 2000, Meschini et al., 2000). Recently, overexpression of a 110 kDa vesicular protein, termed lung resistance-related protein (LRP), has been demonstrated in a number of tumor cell lines intrinsically resistant to chemotherapeutic drugs (Laurencot et al., 1997). LRP has been found to be identical with the human major vault protein (MVP), which is the major component of vaults (Scheffer et al., 1995, Izquierdo et al., 1996). Vaults are multisubunit complexes of ribonucleoprotein particles exhibiting an octagonal barrel-shaped structure, with protruding caps and an invaginated waist. These structures are highly conserved among eukaryotes, suggesting that they are essential for cell function (Kedersha et al., 1990); however, their physiological role in normal tissues and in intrinsic or acquired drug resistance is yet to be defined. Vaults are mainly located within the cytoplasm, where they exhibit a punctate distribution, while a minor fraction concurs to the formation of nuclear pore complexes. Therefore, based on their subcellular localization, vaults have been implicated in vesicular and nucleocytoplasmic drug transport (Scheffer et al., 1995). In fact, the entrapment of drugs into vesicular compartments and decreased nuclear/cytoplasmic ratios have been observed in some MDR cell lines overexpressing LRP (Dietel et al., 1990, Schuurhuis & Broxterman, 1991).

While one or more drug transport proteins may contribute to the intrinsic MDR phenotype, a more general mechanism for drug resistance has recently emerged and has gained increasing popularity with tumor cell biologists, namely the inability of cells to undergo apoptosis, which is now generally acknowledged as leading to cell death upon exposure to anticancer agents. Intrinsic or acquired defects in the apoptotic machinery could therefore profoundly alter the therapeutic outcome and have become an area of active research.

To investigate the mechanisms responsible for intrinsic drug resistance, we have analyzed two different human cell lines, MCF-7 (breast carcinoma) and A549 (non small cell lung carcinoma, NSCLC). Both cell lines were derived from untreated tumors and their response to chemotherapeutic agents is a fair reproduction of the clinical behaviour of the tumor types from which they originated. Breast adenocarcinomas, which represent the most common form of cancer in women, usually respond to pharmacological and endocrine treatments at the outset of therapy, even though they frequently progress from hormone-dependent to hormone-independent growth, and very easily acquire MDR and metastatic properties (Clarke et al., 1990; Leonessa et al., 1992). Lung cancers are classified in 2 main histological groups, small cell lung cancers (SCLCs) and non small cell lung cancers (NSCLCs). The latter are especially characterized by a poor prognosis (Cole et al., 1992, Doyle, 1993); NSCLCs are frequently chemoresistant from diagnosis and in vitro NSCLC cells often exhibit an intrinsic multidrug resistant phenotype (Scheper et al., 1993). Only low amounts of the MDR1 gene product have been detected in unselected NSCLC cell lines, suggesting that other mechanisms could be involved in the intrinsically resistant phenotype (Doyle, 1993). Overexpression of LRP, originally observed in a NSCLC cell line selected for resistance against doxorubicin (DOX) and subsequently detected in other cell lines of different histogenetic origin (Scheper et al., 1993, Scheffer et al., 1995), has been proposed to play a major role in this phenomenon.

In the present study, we have examined: (1) the cytotoxic effect induced by DOX treatment in MCF-7 and A549 cells and the ability of the cells to undergo apoptosis in response to drug treatment; (2) the time course of drug accumulation by flow cytometric analysis and the intracellular distribution of the drug by laser confocal microscopy; and (3) the intracellular levels and distribution of transport proteins related to anthracycline resistance, including P-gp, MRP1 and LRP.

Our data seem to indicate that the lower sensitivity to doxorubicin observed in A549 non small lung cancer cells is mainly due to the high intracellular expression of LRP. Moreover, the association of LRP with intrinsic drug resistance in vitro has been pointed out and the potential value of this protein as marker of clinical drug resistance has been argued.