Induction of proteins involved in multidrug resistance (P-glycoprotein, MRP1, MRP2, LRP) and of CYP 3A4 by rifampicin in LLC-PK₁ cells

Abstract

P-glycoprotein, multidrug resistance-related proteins (MRPs) and lung resistance-related protein (LRP) are involved in multidrug resistance in tumor cells but are also expressed in normal tissues. In the LLC-PK₁ tubular renal cell line, a 15-day treatment with 25 μM rifampicin significantly increased the mRNA levels of P-glycoprotein, MRP1, MRP2, LRP and cytochrome P450 3A4 (CYP 3A4). Western blot analysis confirmed a moderate increase in the expression of P-glycoprotein and MRP2, but not MRP1 also at the protein level. The intracellular uptake of doxorubicin was significantly lower in rifampicin pretreated cells. A pretreatment with 6-[82S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]cyclosporin D, valspodar (PSC 833), a specific inhibitor of P-glycoprotein, with (3-(3-(2-(7-chloro-2-quinidinyl)ethenyl-phenyl)((3-diimethyl amino-3oxo propyl)thio)methyl)thio)propanoic acid, sodium salt (MK-571), a specific inhibitor of MRP1, and with verapamil, that inhibits both proteins, significantly increased doxorubicin cell accumulation in rifampicin pretread cells. In rifampicin treated cells cultured on porous membranes, doxorubicin showed a polarized transport, that was reduced by a pretreatment with PSC 833. A chronic treatment with rifampicin induces the expression of transport proteins and of CYP 3A4 and could therefore alter the renal elimination kinetics of drugs that are their substrates.

Introduction

The most important mechanism responsible for resistance against certain anticancer agents is linked to decreased intracellular accumulation through enhanced cellular efflux of the antitumor compounds and usually involves the expression of P-glycoprotein, multidrug resistance-related proteins (MRPs) or lung resistance-related protein (LRP) (Loo and Clarke, 1999). P-glycoprotein is a 170 kDa ATP-dependent pump expressed constitutively in various normal cells and has been identified on apical membranes of cells with excretory or protective functions such as the enterocytes, the proximal renal tubular cells and the endothelial cells of brain capillaries (Thiebaut et al., 1987). MRPs are a family of drug transporters of 190 kDa involved in multidrug resistance but also expressed in normal tissues; MRP1 and MRP2 are expressed on the basolateral and apical membranes of cells, respectively (Borst et al., 1999). LRP is a 110 kDa major vault transporter protein, originally isolated from P-glycoprotein-negative multidrug resistant cells (Scheffer et al., 2000b) that appears to function as a bidirectional nucleo-cytoplasmic transporter of molecules, is widely expressed in normal human tissues and is important in intracellular drug sequestration (Sugawara et al., 1997). All these proteins seem to act as a defense mechanism against potential toxic substances, and are now recognised as the major determinants of the pharmacokinetics of various drugs. P-glycoprotein shows some similarities with cytochrome P450 3A4 (CYP 3A4), since both act to eliminate xenobiotics, are often expressed in the same cells, have common substrates and can be inhibited by the same molecules (Schuetz et al., 1996).

Many environmental factors can affect the expression of P-glycoprotein and MRPs. In vitro data have revealed that P-glycoprotein, the product of multidrug resistance (MDR1) gene in humans and of mdr1a and mdr1b genes in rodents, can be induced by a variety of drugs and hormones Greiner et al., 1999, Seree et al., 1998, but the molecular mechanism of induction has not been completely elucidated (Geick et al., 2001). Recently it has been shown that P-glycoprotein is inducible in human gut by the prototypic CYP 3A4 inducer rifampicin; in this study Greiner et al. (1999) have suggested that P-glycoprotein induction may be restricted to some cell types, however, few data exist about the induction of P-glycoprotein in other tissues. An organ specific mdr1 inducibility has been previously shown in rodents: in fact, in mice, the P-glycoprotein inducer dexamethasone increased mdr1b expression in colon but not in kidney (Seree et al., 1998) and mdr1a and mdr1b were not inducible in rat liver in the presence of rifampicin or dexamethasone (Salphati and Benet, 1998). In humans, rifampicin does not induce lymphocyte P-glycoprotein expression (Becquemont et al., 2000). In addition, it has been recently shown that MRP2 can be induced by rifampicin in vivo in human duodenum (Fromm, 2000) and in monkey liver (Kauffmann et al., 1998).

In the normal kidney P-glycoprotein is constitutively expressed and plays a major role in the renal excretion of many substrates (Ernest and Bello-Reus, 1998); MRP1, MRP2 and LRP are also expressed in the normal kidney Borst et al., 1999, Sugawara et al., 1997. An induction of renal P-glycoprotein has been demonstrated using substrates such as cyclosporin A and 6-[82S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]cyclosporin D, valspodar (PSC 833) (Jette et al., 1996) and cisplatin (Demeule et al., 1999). Furthermore the expression of renal MRP2 can be also induced by cisplatin (Demeule et al., 1999).

The LLC-PK₁ cell line is a proximal tubular renal cell line derived from pig kidney (Hull et al., 1976) which has structure and function similar to those of renal proximal tubular cells (Handler et al., 1980). These cells form an oriented monolayer with microvilli and tight junctions (Horio et al., 1990) and express low amounts of P-glycoprotein Crivellato et al., 1999, Decorti et al., 1998 as well as of MRP1 and MRP2 Decorti et al., 2001, Evers et al., 1996, and thus represent an useful in vitro model for studying drug transport pathways and the effect of drug induction in the proximal renal tubule.

The aim of this study was therefore to evaluate the effect of a treatment with rifampicin on the expression of P-glycoprotein, MRP1, MRP2, LRP and CYP 3A4 in LLC-PK₁ cells.