Modulation of MRP1 protein transport by plant, and synthetically modified flavonoids

Abstract

The influence of novel synthetic and plant origin flavonoids on activity of multidrug resistance-associated protein (MRP1) was investigated in human erythrocytes used as a cell model expressing MRP1 in plasma membrane. The fluorescent probe, BCPCF (2′, 7′-bis-(3-carboxy-propyl)-5-(and-6)-carboxyfluorescein), was applied as a substrate for MRP1 multidrug resistance transporter. The effect of compounds belonging to different classes of natural flavonoids: flavone, flavonol, isoflavones and flavanolignan was compared with action of new synthetic derivatives of genistein. Most of the flavonoids showed strong or moderate ability to inhibit transport carried out by MRP1. Inhibitory properties of flavonoids were compared to the effects of indomethacin, probenecid and MK-571 known as MRP1 inhibitors. Studying the influence of new synthetic genistein derivatives on BCPCF transport we have found that the presence of hydrophobic groups substituting hydrogen of hydroxyl group at the position 4′ in ring B of isoflavone is more important for inhibitory properties than hydrophobic substitution at the position 7 in ring A. In case of naturally occurring isoflavones the replacement of hydrogen at position 4′ by hydrophobic ring structure seems also to be favourable for inhibition potency.

Introduction

Multidrug resistance (MDR) is the main obstacle in successful chemotherapy of diverse cancer diseases. Several cellular mechanisms are responsible for drug resistance of tumour cells (Gottesman, 2002). One of the most important mechanism is an overexpression of multidrug resistance transporters in plasma membranes of resistant tumour cells. These proteins are members of ABC protein superfamily (ATP-binding cassette proteins) and are represented by P-gp (P-glycoprotein, ABCB1), MRP1 (multidrug resistance-associated protein, ABCC1) and several other MDR transporters. The outward transport carried out by MDR transporters overexpressed in plasma membrane prevents cancer cells against effective intracellular accumulation of cytotoxic drugs. MRP1 like other MDR transporters confers resistance to multiple anticancer drugs. Two main drug binding sites were recognized in transmembrane domains 10–11 and 16–17 of the protein (Daoud et al., 2001).

Besides resistant tumour cells P-gp and MRP1 are also commonly expressed in different kinds of normal tissues, where ABC multidrug transporters are a part of the defence system against cytotoxic compounds. They play a role in the absorption, disposition and elimination of xenobiotics (Borst and Elferink, 2002). MRP proteins protect central nervous system, kidney, liver and other tissues against various xenobiotics and they take part in maintaining intracellular homeostasis (Borst and Elferink, 2002, Hooijberg et al., 2003, Sun et al., 2003).

MRP1 ubiquitously expressed in normal tissues is also detected in erythrocyte membrane. MRP1 is a universal organic anion transporter that can transport glutathione conjugates, compounds conjugated with sulphate or glucuronic acid, but also hydrophobic and amphipathic drugs and dyes (Borst and Elferink, 2002, Konig et al., 1999, Schinkel and Jonker, 2003). The human MRP family of proteins contains several members. In human erythrocytes three ABCC transporters have been found: MRP1 (ABCC1), MRP4 (ABCC4) and MRP5 (ABCC5) (Klokouzas et al., 2003, Boadu and Sager, 2004). In red blood cells expression of MRP1 is especially well established (Abraham et al., 2001, Dekkers et al., 2000, Klokouzas et al., 2001, PuƗaski et al., 1996, Rychlik et al., 2000). The expression of MRP1 in human erythrocytes accounts for transport of oxidized glutathione (GSSG) and dinitrophenyl-S-glutathione conjugates (DNP-SG) (PuƗaski et al., 1996, Akerboom et al., 1992). Dekkers et al., 1998, Dekkers et al., 2000 have demonstrated that MRP1 plays a role in the maintaining of transbilayer lipid asymmetry of erythrocyte membrane. Inhibition of MRP1 influences not only the distribution of NBD-labelled phospholipid analogues but also that of the endogenous phospholipids. In human red blood cells multidrug resistance-associated protein may act as a floppase that translocates phosphatidylserine and phosphatidylcholine from inner to outer membrane leaflet (Dekkers et al., 2000). It was also shown that MRP1 reconstituted into proteoliposomes facilitates a flop of fluorescent labelled phosphatidylcholine (NBD-PC) from outer to inner leaflet of vesicles (Huang et al., 2004, Kamp and Haest, 1998). MK-571, a potent MRP1 inhibitor that reverses MRP1-mediated drug resistance (Gekeler et al., 1995) almost completely inhibits the NBD-PC translocation (Huang et al., 2004).

For many years after discovery of MRP1 its effective inhibitors have not been known. P-gp modulators inhibit activity of MRP1 only when used in a very high concentration or they do not affect its transport properties at all. The compounds such as probenecid, benzbromarone and indomethacin that can reverse the resistance in cells expressing MRP1 are generally inhibitors of organic anion transport. Klokouzas et al. (2001) have shown that clotrimazole, an anti-mycotic drug, inhibits MRP1-mediated transport of dinitrophenyl S-glutathione both in erythrocytes and in MRP1-expressing lung tumour cells. This transport was inhibited by MRP1-specific antibody QCRL-3. Genistein was the first flavonoid reported as inhibitor of MRP1 (Hooijberg et al., 1997, Teodori et al., 2002). It has also been shown that this isoflavone binds strongly to the NBD2 cytosolic domain of mouse and Leishmania P-glycoprotein (Di Pietro et al., 2002). Recently other plant-derived flavonoids were discovered as inhibitors of MRP1 both in tumour cells (Leslie et al., 2001, Nguyen et al., 2003) and in erythrocytes (Bobrowska-Hägerstrand et al., 2001, Bobrowska-Hägerstrand et al., 2003).

The aim of this paper is to compare the inhibition potency of different plant derived flavonoids (flavones, isoflavones, flavanolignan) and four new synthetic genistein derivatives. Different substitutions were introduced to genistein molecule to obtain new synthetic isoflavones that are much more hydrophobic than genistein itself and that may interact more easily with membrane interior than precursor compound.

The inhibition of erythrocyte MRP1 by both synthetic and natural flavonoids was examined by measuring the efflux of fluorescent MRP1 substrate, BCPCF from erythrocytes. Another similar compound — BCECF, a structural analogue of BCPCF was recognized as MRP1 substrate (Draper et al., 1997a, Draper et al., 1997b, Rychlik et al., 2003). However, in MRP5 — overexpressing cells reduced of BCECF accumulation was also observed (McAleer et al., 1999) and therefore some contribution of this transporter to BCECF transport in intact erythrocyte could not be excluded. In contrast, Rychlik et al. (2003), in experiments performed on inside-out vesicles proved that transport of BCECF was blocked by antibody QCRL3, specific against MRP1 (Rychlik et al., 2003). BCPCF, a structural analogue of BCECF was used as fluorescent substrate for MRP1 in our previous studies (Bobrowska-Hägerstrand et al., 2001, Łania-Pietrzak et al., 2005). In current studies we show that MK-571, the most potent inhibitor of MRP1-mediated transport effectively blocks BCPCF efflux from erythrocytes.