Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2',7'-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles

The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants K(m) and V(max) of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry.     

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

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.     

Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles

The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants K_m and V_max of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry.     

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.     

Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)

Ivermectin is a potent antiparasitic drug from macrocyclic lactone (ML) family, which interacts with the ABC multidrug transporter P-glycoprotein (Pgp). We studied the interactions of ivermectin with the multidrug resistance proteins (MRPs) by combining cellular and subcellular approaches. The inhibition by ivermectin of substrate transport was measured in A549 cells (calcein or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, BCECF) and in HL60-MRP1 (calcein). Ivermectin induced calcein and BCECF retention in A549 cells (IC(50) at 1 and 2.5microM, respectively) and inhibited calcein efflux in HL60-MRP1 (IC(50)=3.8microM). The action of ivermectin on the transporters ATPase activity was followed on membranes from Sf9 cells overexpressing human Pgp, MRP1, 2 or 3. Ivermectin inhibited the Pgp, MRP1, 2 and 3 ATPase activities after stimulation by their respective activators. Ivermectin showed a rather good affinity for MRPs, mainly MRP1, in the micromolar range, although it was lower than that for Pgp. The transport of BODIPY-ivermectin was followed in cells overexpressing selectively Pgp or MRP1. In both cell lines, inhibition of the transporter activity induced intracellular retention of BODIPY-ivermectin. Our data revealed the specific interaction of ivermectin with MRP proteins, and its transport by MRP1. Although Pgp has been considered until now as the sole active transporter for this drug, the MRPs should be taken into account for the transport of ivermectin across cell membrane, modulating its disposition in addition to Pgp. This could be of importance for optimizing clinical efficacy of ML-based antiparasitic treatments. This offers fair perspectives for the use of ivermectin or non-toxic derivatives as multidrug resistance-reversing agents.     

Neither lipophilicity nor membrane-perturbing potency of phenothiazine maleates correlate with the ability to inhibit P-glycoprotein transport activity

Although phenothiazines are known as multidrug resistance modifiers, the molecular mechanism of their activity remains unclear. Since phenothiazine molecules are amphiphilic, the interactions with membrane lipids may be related, at least partially, to their biological effects. Using the set of phenothiazine maleates differing in the type of phenothiazine ring substitution at position 2 and/or in the length of the alkyl bridge-connecting ring system and side chain group, we investigated if their ability to modulate the multidrug resistance of cancer cells correlated with model membrane perturbing potency. The influence exerted on lipid bilayers was determined by liposome/buffer partition coefficient measurements (using the absorption spectra second-derivative method), fluorescence spectroscopy and calorimetry. Biological effects were assessed by a flow cytometric functional test based on differential accumulation of fluorescent probe DiOC(2)(3) by parental and drug-resistant cells. We found that all phenothiazine maleates were incorporated into lipid bilayers and altered their biophysical properties. With only few exceptions, the extent of membrane perturbation induced by phenothiazine maleates correlated with their lipophilicity. Within the group of studied derivatives, the compounds substituted with CF(3)- at position 2 of phenothiazine ring were the most active membrane perturbants. No clear relation was found between effects exerted by phenothiazine maleates on model membranes and their ability to modulate P-glycoprotein transport activity.     

Detection of MRP functional activity: calcein AM but not BCECF AM as a Multidrug Resistance-related Protein (MRP1) substrate

Resistance to anticancer drugs has been attributed to an array of cellular changes. The multidrug resistance-related protein (MRP1) is an efflux pump whose overexpression confers resistance to several classes of drugs, such as the anthracyclines, epipodophyllotoxins, and vinca alkaloids. These drugs are mainstays in cancer therapy. MRP1 overexpression is hypothesized to be a causative agent of clinical treatment failure. Consistently accurate methods for detecting this protein are necessary to further understand its biology and delineate its possible clinical relevance. Flow cytometric analysis of multidrug resistance (MDR) is a valuable method to evaluate both antigen expression and function. Using flow cytometry, we assayed MRP1 functional activity in pediatric leukemic blasts and an array of MDR+ and WT cell lines. We conclude that calcein AM, when used in a retention assay with MRP1-specific modulators, is able to reliably detect MRP functional activity. 2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF AM) transport is not indicative of MRP1 overexpression. .     

Sulforaphane and erucin increase MRP1 and MRP2 in human carcinoma cell lines

Multidrug resistance (MDR) transporters have been termed the Phase III detoxification system because they not only export endogenous metabolites but provide protection from xenobiotic insult by actively secreting foreign compounds and their metabolites from tissues. However, MDR overexpression in tumors can lead to drug resistance, a major obstacle in the treatment of many cancers, including lung cancer. Isothiocyanates from cruciferous vegetables, such as sulforaphane (SF) and erucin (ER), are known to enhance the expression of Phase II detoxification enzymes. Here we evaluated the ability of SF and ER to modulate MDR mRNA and protein expressions, as well as transporter activity. The expression of P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and multidrug resistance protein 2 (MRP2) in liver (HepG2), colon (Caco-2) and lung (A549) cancer cells treated with ER or SF was analyzed by Western blotting. Neither SF nor ER affected P-gp expression in any of the cell lines tested. Both SF and ER increased the protein levels of MRP1 and MRP2 in HepG2 cells and of MRP2 in Caco-2 cells in a dose-dependent manner. In A549 lung cancer cells, SF increased MRP1 and MRP2 mRNA and protein levels; ER caused a similar yet smaller increase in MRP1 and MRP2 mRNA. In addition, SF and ER increased MRP1-dependent efflux of 5-carboxyfluorescein diacetate in A549 cells, although again the effect of SF was substantially greater than that of ER. The implication of these findings is that dietary components that modulate detoxification systems should be studied carefully before being recommended for use during chemotherapy, as these compounds may have additional influences on the disposition of chemotherapeutic drugs.     

RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes.

We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al. Biochemistry 39, 9327-9334). Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes. In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes. Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function. Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70%. On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes.     

Gangliosides do not affect ABC transporter function in human neuroblastoma cells

Previous studies have indicated a role for glucosylceramide synthase (GCS) in multidrug resistance (MDR), either related to turnover of ceramide (Cer) or generation of gangliosides, which modulate apoptosis and/or the activity of ABC transporters. This study challenges the hypothesis that gangliosides modulate the activity of ABC transporters and was performed in two human neuroblastoma cell lines, expressing either functional P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1). Two inhibitors of GCS, D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ), very efficiently depleted ganglioside content in two human neuroblastoma cell lines. This was established by three different assays: equilibrium radiolabeling, cholera toxin binding, and mass analysis. Fluorescence-activated cell sorting (FACS) analysis showed that ganglioside depletion only slightly and in the opposite direction affected Pgp- and MRP1-mediated efflux activity. Moreover, both effects were marginal compared with those of well-established inhibitors of either MRP1 (i.e., MK571) or Pgp (i.e., GF120918). t-PPPP slightly enhanced cellular sensitivity to vincristine, as determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide analysis, in both neuroblastoma cell lines, whereas NB-dNJ was without effect. MRP1 expression and its localization in detergent-resistant membranes were not affected by ganglioside depletion. Together, these results show that gangliosides are not relevant to ABC transporter-mediated MDR in neuroblastoma cells.     

ATPase activity of purified and reconstituted multidrug resistance protein MRP1 from drug-selected H69AR cells.

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.     

Purification of the human apical conjugate export pump MRP2 reconstitution and functional characterization as substrate-stimulated ATPase.

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP-binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C-terminal (His)6-tag. Immunoblot and immunofluorescence analyses detected the MRP2-(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2-(His)6-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C4 and were photoaffinity-labelled with 8-azido-[alpha-32P]ATP. MRP2-(His)6 was solubilized from membranes of MRP2-(His)6-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2-(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein-dependent manner with a Km for ATP of 2.1 mM and a Vmax of 25 nmol ADP x mg MRP2-1 x min-1. This ATPase activity was substrate-stimulated by oxidized and reduced glutathione and by S-decyl-glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.     

New phenothiazine-type multidrug resistance modifiers: anti-MDR activity versus membrane perturbing potency

The phenothiazine multidrug resistance (MDR) modulators are chemically diversified but share the common feature to be hydrophobic cationic molecules. Molecular mechanisms of their action may involve interactions with either P-glycoprotein or membrane lipid matrix. In the present work we study the anti-MDR and biophysical membrane effects of new phenothiazine derivatives differing in the type of group substituting phenothiazine ring at position 2 (H-, Cl-, CF(3)-) and in the side chain group (NHCO(2)CH(3) or NHSO(2)CH(3)). Within each phenothiazine subset we found that anti-MDR activity (determined by P-glycoprotein inhibition assessed by flow cytometry) correlates with the theoretically calculated hydrophobicity value (logP) and experimental parameters (determined by calorimetry and fluorescence spectroscopy) of lipid bilayers. It is concluded that the biological and biophysical activity of phenothiazine derivatives depends more on the type of ring substitution than on the nature of the side chain group.     

Differential sensitivity of plant and yeast MRP (ABCC)-mediated organic anion transport processes towards sulfonylureas

The role of ATP-binding cassette (ABC) proteins such as multidrug resistance-associated proteins (MRPs) is critical in drug resistance in cancer cells and in plant detoxification processes. Due to broad substrate spectra, specific modulators of these proteins are still lacking. Sulfonylureas such as glibenclamide are used to treat non-insulin-dependent diabetes since they bind to the sulfonylurea receptor. Glibenclamide also inhibits the cystic fibrosis transmembrane conductance regulator, p-glycoprotein in animals and guard cell ion channels in plants. To investigate whether this compound is a more general blocker of ABC transporters the sensitivity of ABC-type transport processes across the vacuolar membrane of plants and yeast towards glibenclamide was evaluated. Glibenclamide inhibits the ATP-dependent uptake of beta-estradiol 17-(beta-D-glucuronide), lucifer yellow CH, and (2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein. Transport of glutathione conjugates into plant but not into yeast vacuoles was drastically reduced by glibenclamide. Thus, irrespective of the homologies between plant, yeast and animal MRP transporters, specific features of plant vacuolar MRPs with regard to sensitivity towards sulfonylureas exist. Glibenclamide could be a useful tool to trap anionic fluorescent indicator dyes in the cytosol.     

Subcellular localization and activity of multidrug resistance proteins

The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane, where they are thought to reduce the cellular accumulation of toxins over time. Our data demonstrate that members of this family are also expressed in subcellular compartments where they actively sequester drugs away from their cellular targets. The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a perinuclear region positive for lysosomal markers. Fluorescence-activated cell sorting analysis suggests that these three drug transporters do little to reduce the cellular accumulation of the anthracycline doxorubicin. However, whereas doxorubicin enters cells expressing MDR transporters, this drug is sequestered away from the nucleus, its subcellular target, in vesicles expressing each of the three drug resistance proteins. Using a cell-impermeable inhibitor of MRP1 activity, we demonstrate that MRP1 activity on intracellular vesicles is sufficient to confer a drug resistance phenotype, whereas disruption of lysosomal pH is not. Intracellular localization and activity for MRP1 and other members of the MDR transporter family may suggest different strategies for chemotherapeutic regimens in a clinical setting.     

Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells

YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1.     

Involvement of cholesterol in the inhibitory effect of dimethyl-beta-cyclodextrin on P-glycoprotein and MRP2 function in Caco-2 cells

We compared the inhibitory effect of various cyclodextrins (CyDs) on P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) function and examined the contribution of cholesterol to the inhibitory effect of 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD) on the efflux activity of the function in Caco-2 cell monolayers. Of various CyDs, DM-beta-CyD significantly impaired the efflux activity of P-gp and MRP2. DM-beta-CyD released P-gp and MRP2 from the monolayers in the apical side's transport buffer and decreased the extent of cholesterol as well as P-gp and MRP2 in caveolae of Caco-2 cell monolayers, but not caveolin and flotillin-1. On the other hand, DM-beta-CyD did not change MDR1 and MRP2 mRNA levels. Therefore, these results suggest that the inhibitory effect of DM-beta-CyD on P-gp and MRP2 function, at least in part, could be attributed to the release of these transporters from the apical membranes into the medium as secondary effects through cholesterol-depletion in caveolae after treatment of Caco-2 cell monolayers with DM-beta-CyD.     

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein

Dihydro-beta-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-beta-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-beta-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-beta-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-beta-agarofurans.     

Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1

Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha-(32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.     

Modulation of MRP1-like efflux activity in human erythrocytes caused by membrane perturbing agents

The effect of membrane perturbing agents on the efflux (37 degrees C, 60 min) of the fluorescent probe 2', 7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was studied. Several anionic amphiphiles (detergents) markedly inhibited BCPCF efflux (IC50 < or = 40 microM). Most zwitter-ionic amphiphiles were inefficient inhibitors. Non-ionic and cationic amphiphiles had minor effects or increased efflux. Of the aliphatic inhibitors, C12-homologues were the most efficient. Hexanol, ethanol, methyl-beta-cyclodextrin (MbetaCD) and diamide (+ washing) did not influence BCPCF efflux. It is suggested that amphiphiles affect BCPCF efflux by modulating multi-drug resistance protein 1 (MRP1, ABCC1) activity. A negative charge of amphiphiles is essential for the inhibitory effect, while alkyl chain length modulates inhibition. MRP1-mediated BCPCF efflux appears to be relatively insensitive to non-specific plasma membrane modification.