Multidrug Resistance Protein MRP1 Research Articles

Glutathione-dependent interaction of heavy metal compounds with multidrug resistance proteins MRP1 and MRP2

The interactions of three heavy metal-containing compounds, cisplatin (CDDP), arsenic trioxide (As 2O 3), and mercury dichloride (HgCl 2), with the multidrug resistance transporters MRP1 and MRP2 and the involvement of glutathione (GSH)-related processes herein were investigated. In Madin–Darby canine kidney cells stably expressing MRP1 or MRP2, viability, GSH content, calcein efflux and polarized GSH efflux were measured as a function of exposure to CDDP, As 2O 3 and HgCl 2. In isolated Sf9-MRP1 and Sf9-MRP2 membrane vesicles, the interaction with MRP-associated ATPase activity was measured. In the latter model system adduct formation with GSH is not an issue. The data show that (1) CDDP interacts with both MRP1 and MRP2, and GSH appears to play no major role in this process, (2) As 2O 3 interacts with MRP1 and MRP2 in which process GSH seems to be essential, and (3) HgCl 2 interacts with MRP1 and MRP2, either alone and/or as a metal–GSH complex.

Expression of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas

Cholangiocellular carcinomas and gallbladder carcinomas are highly aggressive tumours with a poor prognosis and are generally regarded as chemoresistant tumours. Overexpression of ATP-binding cassette transporters of the multidrug resistance protein (MDR) and multidrug resistance-related protein (MRP) family in cancer cells is a major cause for the multidrug resistance phenotype in vitro and in vivo. To further define the role of MRP family members in biliary tract cancer, we studied the expression and localization of MRP2 and MRP3 in cholangiocellular carcinomas and gallbladder carcinomas. The expression and cellular localization of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas and gallbladder carcinomas were analysed by immunohistochemistry using isoform-specific antibodies. Expression of MRP isoforms was studied in vitro in Mz-ChA-1 cells derived from gallbladder adenocarcinoma by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and immunofluorescence microscopy. Mz-ChA-1 cells constitutively expressed MDR P-glycoproteins, MRP1, MRP2 and MRP3 by RT-PCR, immunoblotting and immunofluorescence microscopy. MRP2 and MRP3 are expressed in the respective apical and basolateral membrane domains. MRP3 was the predominant MRP isoform in gallbladder carcinomas (93%) and cholangiocellular carcinomas (57%), whereas MRP2 expression was detected in only 29% of gallbladder carcinomas and was undetectable in cholangiocellular carcinomas. Our findings suggest that the intrinsic multidrug resistance of cholangiocellular and gallbladder carcinomas seems to be independent of MRP2 expression while the expression of MRP3 may contribute to the MDR phenotype.

Transport of the Dipeptidyl Peptidase-4 Inhibitor Sitagliptin by Human Organic Anion Transporter 3, Organic Anion Transporting Polypeptide 4C1, and Multidrug Resistance P-glycoprotein

Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor recently approved for the treatment of type 2 diabetes, is excreted into the urine via active tubular secretion and glomerular filtration in humans. In this report, we demonstrate that sitagliptin is transported by human organic anion transporter hOAT3 (Km=162 microM), organic anion transporting polypeptide OATP4C1, and multidrug resistance (MDR) P-glycoprotein (Pgp), but not by human organic cation transporter 2 hOCT2, hOAT1, oligopeptide transporter hPEPT1, OATP2B1, and the multidrug resistance proteins MRP2 and MRP4. Our studies suggested that hOAT3, OATP4C1, and MDR1 Pgp might play a role in transporting sitagliptin into and out of renal proximal tubule cells, respectively. Sitagliptin did not inhibit hOAT1-mediated cidofovir uptake, but it showed weak inhibition of hOAT3-mediated cimetidine uptake (IC50=160 microM). hOAT3-mediated sitagliptin uptake was inhibited by probenecid, ibuprofen, furosemide, fenofibric acid, quinapril, indapamide, and cimetidine with IC50 values of 5.6, 3.7, 1.7, 2.2, 6.2, 11, and 79 microM, respectively. Sitagliptin did not inhibit Pgp-mediated transport of digoxin, verapamil, ritonavir, quinidine, and vinblastine. Cyclosporine A significantly inhibited Pgp-mediated transport of sitagliptin (IC50=1 microM). Our data indicate that sitagliptin is unlikely to be a perpetrator of drug-drug interactions with Pgp, hOAT1, or hOAT3 substrates at clinically relevant concentrations. Renal secretion of sitagliptin could be inhibited if coadministered with OAT3 inhibitors such as probenecid. However, the magnitude of interactions should be low, and the effects may not be clinically meaningful, due to the high safety margin of sitagliptin.

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the γ-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.

Characterization of Mice Lacking the Multidrug Resistance Protein Mrp2 (Abcc2)

The multidrug resistance protein Mrp2 is an ATP-binding cassette (ABC) transporter mainly expressed in liver, kidney, and intestine. One of the physiological roles of Mrp2 is to transport bilirubin glucuronides from the liver into the bile. Current in vivo models to study Mrp2 are the transporter-deficient and Eisai hyperbilirubinemic rat strains. Previous reports showed hyperbilirubinemia and induction of Mrp3 in the hepatocyte sinusoidal membrane in the mutant rats. In addition, differences in liver cytochrome P450 and UGT1a levels between wild-type and mutant rats were detected. To study whether these compensatory mechanisms were specific to rats, we characterized Mrp2(-/-) mice. Functional absence of Mrp2 in the knockout mice was demonstrated by showing increased levels of bilirubin and bilirubin glucuronides in serum and urine, a reduction in biliary excretion of bilirubin glucuronides and total glutathione, and a reduction in the biliary excretion of the Mrp2 substrate dibromosulfophthalein. To identify possible compensatory mechanisms in Mrp2(-/-) mice, the expression levels of 98 phase I, phase II, and transporter genes were compared in liver, kidney, and intestine of male and female Mrp2(-/-) and control mice. Unlike in Mrp2 mutant rats, no induction of Mrp3 in Mrp2(-/-) mice was detected. However, Mrp4 mRNA and protein in liver and kidney were increased approximately 6- and 2-fold, respectively. Phenotypic analysis of major cytochrome P450-mediated activities in liver microsomes did not show differences between wild-type and Mrp2(-/-) mice. In conclusion, Mrp2(-/-) mice are a new valuable tool to study the role of Mrp2 in drug disposition.

The insulin-like growth factor-1 receptor inhibitor PPP produces only very limited resistance in tumor cells exposed to long-term selection

The cyclolignan PPP was recently demonstrated to inhibit the activity of insulin-like growth factor-1 receptor (IGF-1R), without affecting the highly homologous insulin receptor. In addition, PPP caused complete regression of xenografts derived from various types of cancer. These data highlight the use of this compound in cancer treatment. However, a general concern with antitumor agents is development of resistance. In light of this problem, we aimed to investigate whether malignant cells may develop serious resistance to PPP. After trying to select 10 malignant cell lines, with documented IGF-1R expression and apoptotic responsiveness to PPP treatment (IC50s less than 0.1 microM), only two survived an 80-week selection but could only tolerate maximal PPP doses of 0.2 and 0.5 microM, respectively. Any further increase in the PPP dose resulted in massive cell death. These two cell lines were demonstrated not to acquire any essential alteration in responsiveness to PPP regarding IGF-1-induced IGF-1R phosphorylation. Neither did they exhibit any increase in expression of the multidrug resistance proteins MDR1 or MRP1. Consistently, they did not exhibit decreased sensitivity to conventional cytostatic drugs. Rather, the sensitivity was increased. During the first half of the selection period, both cell lines responded with a temporary and moderate increase in IGF-1R expression, which appeared to be because of an increased transcription of the IGF-1R gene. This increase in IGF-1R might be necessary to make cells competent for further selection but only up to a PPP concentration of 0.2 and 0.5 microM. In conclusion, malignant cells develop no or remarkably weak resistance to the IGF-1R inhibitor PPP.

St. John's wort and its constituent hyperforin concordantly regulate expression of genes encoding enzymes involved in basic cellular pathways

The effects of St. John's wort and hyperforin on gene expression were analysed in HepG2 cells by Affymetrix microarray hybridization and real time reverse transcription-PCR. Both compounds increased mRNAs of the drug metabolizing enzymes CYP3A4, CYP1A1, CYP1A2 and the flavin containing monooxygenase FMO5, and of the multidrug resistance protein MRP2. CYP4F2 and the reduced nicotinamide adenine dinucleotide dehydrogenase NQO1 were downregulated. Expression of genes mediating cholesterol biosynthesis was decreased, while facilitated glucose transporters and glycolysis genes were induced, indicating increased glucose metabolism. Changes of a considerable number of additional transcripts corresponded to reports on gene regulation by hypoxia. Endoplasmic reticulum stress-regulated genes involved in unfolded protein response and in protection of cells from apoptosis were downregulated. Other calcium binding proteins were affected by both treatments, suggesting an increase in intracellular calcium. St. John's wort and hyperforin concordantly affected expression of genes not only mediating metabolism and transport of exogenous and endogenous compounds, but also involved in energy metabolism, intracellular calcium regulation, cell proliferation and apoptosis.

Inhibition of multidrug resistance proteins MRP1 and MRP2 by a series of α,β-unsaturated carbonyl compounds

To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of α,β-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. α,β-Unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition.

Limited modulation of the transport activity of the human multidrug resistance proteins MRP1, MRP2 and MRP3 by nicotine glucuronide metabolites

The human ATP-binding cassette proteins MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3) are active transporters of antineoplastic drugs as well as conjugated metabolites and other organic anions. In addition to being substrates, many glucuronide, glutathione and sulfate conjugates can also inhibit the transport activities of these MRP-related proteins, sometimes in a glutathione (GSH)-dependent manner. Nicotine is the major addictive component of cigarette smoke. Three glucuronide metabolites of this compound have been identified in vivo: nicotine- N-glucuronide, cotinine- N-glucuronide and trans-hydroxycotinine- O-glucuronide. In this study, we first chemically synthesized trans-hydroxycotinine- O-glucuronide and then tested the ability of this compound, nicotine- N-glucuronide and cotinine- N-glucuronide to modulate the vesicular transport of several organic anions by MRP1, MRP2 and MRP3. We observed that none of the three metabolites at concentrations up to 100 μM significantly affected organic anion transport by MRP1 or MRP2, either in the absence or presence of GSH. MRP3-mediated transport of 17β-estradiol 17-(β- d-glucuronide) and methotrexate were partially inhibited by trans-hydroxycotinine- O-glucuronide (300 μM) (by 70% and 50%, respectively), whereas nicotine- N-glucuronide and cotinine- N-glucuronide had no effect. We conclude that the physiological functions of MRP1, MRP2 and MRP3 are not likely to be substantially affected by nicotine glucuronide metabolites at concentrations achievable in human serum.

Alterations in nuclear hormone receptors (NHR) are associated with changes in the expression of multidrug resistance proteins Mrp2 and Mrp3 in liver and kidney in cholestasis: Lee A Denson, YCHRC, Yale University School of Medicine, New Haven, CT; Alan Bohan, Liver Center, Yale University School of Medicine, New Haven, CT; Himmat J Bajwa, Matthew A Held, YCHRC, Yale University School

Alterations in nuclear hormone receptors (NHR) are associated with changes in the expression of multidrug resistance proteins Mrp2 and Mrp3 in liver and kidney in cholestasis: Lee A Denson, YCHRC, Yale University School of Medicine, New Haven, CT; Alan Bohan, Liver Center, Yale University School of Medicine, New Haven, CT; Himmat J Bajwa, Matthew A Held, YCHRC, Yale University School

Carbamazepine regulates intestinal P-glycoprotein and multidrug resistance protein MRP2 and influences disposition of talinolol in humans.

The antiepileptic drug carbamazepine is known to be an inducer of cytochrome P450 (CYP) 3A4 after binding to the nuclear pregnane X receptor. To evaluate whether it also regulates the multidrug transporter proteins P-glycoprotein (P-gp) and multidrug resistance protein MRP2 in humans, duodenal expression of multidrug resistance gene MDR1 messenger ribonucleic acid (mRNA) and MRP2 mRNA, content of P-gp and MRP2, and disposition of the nonmetabolized P-gp substrate talinolol after intravenous (30 mg) and long-term oral administration (100 mg for 19 days) were assessed in 7 healthy subjects (age, 23-35 years; body weight, 64-93 kg) before and after comedication of carbamazepine (600 mg for 14-18 days). Carbamazepine medication was associated with increased urinary excretion of D-glucaric acid and induction of carbamazepine elimination. Creatinine clearance was not affected. Duodenal expression of both MDR1 mRNA and MRP2 mRNA and the MPR2 protein was significantly induced, whereas the P-gp content was not affected. MDR1 mRNA expression and MPR2 mRNA expression were correlated ( r = 0.873, P <.001). After carbamazepine, metabolic clearance of intravenous talinolol was significantly increased. Residual clearance was significantly decreased in dependence on MDR1 mRNA expression ( r = -0.647, P =.012) and MRP2 mRNA expression ( r = -0.613, P =.020). Oral absorption of talinolol was significantly lower after carbamazepine comedication (53.2% +/- 15.5% versus 62.1% +/- 13.0%, P =.018), and renal clearance and metabolic clearance were significantly increased, correlated in each case with MDR1 mRNA ( r = 0.612, P =.020, and r = 0.554, P =.040, respectively) and MRP2 mRNA ( r = 0.596, P =.025, and r = 0.565, P =.035, respectively). Aside from induction of CYP3A4, carbamazepine acts as an inducer of intestinal MDR1 mRNA, MRP2 mRNA, and MRP2 protein content.

Mutational Analysis of Ionizable Residues Proximal to the Cytoplasmic Interface of Membrane Spanning Domain 3 of the Multidrug Resistance Protein, MRP1 (ABCC1): GLUTAMATE 1204 IS IMPORTANT FOR BOTH THE EXPRESSION AND CATALYTIC ACTIVITY OF THE TRANSPORTER

The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.

Antibody binding capacity for evaluation of MDR-related proteins in acute promyelocytic leukemia: Onset versus relapse expression.

Multidrug resistance (MDR) remains a major obstacle for successful treatment in cancer, in particular in acute leukemia. In acute promyelocytic leukemia (APL), the high sensitivity to anthracyclines appears to be attributable to the low frequency of MDR proteins overexpression at onset even if 30% of patients still relapse and become resistant to therapy. In attempt to explain different blast cell sensitivity, we studied the expression of PGP, MRP1, MRP2, and LRP in 45 cases of APL, comparing onset of disease with relapse. PGP, LRP, and MRP on bone marrow or peripheral blood blast cells were evaluated by flow cytometry using the MRK-16, LRP-56, MRP-m6, and MRP2 antibodies and results expressed by the mean fluorescence index (MFI). The antibody binding capacity (ABC) for each MDR protein was also calculated. At diagnosis, only 2 of 45 patients overexpressed PGP and 1 overexpressed LRP. PGP and LRP overexpressing cases significantly grew up during disease progression and at second relapse mean PGP MFI and mean LRP MFI were significantly higher than at onset (P = 0.001 and P = 0.008, respectively). By analyzing ABC, the same trend was more evident because a significant increment of PGP and LRP was observed at second (P = 0.002 and P = 0.002, respectively), but even at first relapse (P = 0.018 and P = 0.002, respectively). No changes were demonstrated in MRP1 and MRP2 expression in any phase of disease considered. Our data confirm the low expression at diagnosis of proteins related to development of drug resistance in APL. The evidence of a relative easy induction of PGP and LRP, but not of MRP, can be useful in choosing drugs to employ for consolidation or rescue therapy.

Folate concentration dependent transport activity of the Multidrug Resistance Protein 1 (ABCC1)

The Multidrug Resistance Protein MRP1 (ABCC1) can confer resistance to a variety of therapeutic drugs. In addition, MRP1/ABCC1 mediates cellular export of natural folates, such as folic acid and l-leucovorin. In this study we determined whether cellular folate status affected the functional activity of MRP1/ABCC1 mediated efflux of an established substrate, the anthracycline daunorubicin (DNR). As a model system we used the human ovarian carcinoma cell line 2008wt, and its MRP1/ABCC1 transfected subline 2008/MRP1. Both types of these moderate- and high-MRP1/ABCC1 expressing cells displayed efflux of DNR when maintained in standard culture media (2.3 μM folic acid). The initial total cellular DNR efflux rate in 2008/MRP1 cells was ∼2-fold higher compared to 2008wt cells. This efflux consisted of MRP1/ABCC1 mediated transport, possibly non-MRP1 mediated transport, as well as passive diffusion. Benzbromarone, a specific MRP1 inhibitor, decreased the initial efflux rate in 2008/MRP1 cells (4-fold) and in 2008wt cells (2-fold). When 2008/MRP1 cells were challenged for 2 days in folate-free medium, total cellular DNR efflux was decreased to 43% of the initial efflux rate under folate-rich conditions. In 2008wt cells DNR efflux was decreased to 84% of the folate-rich conditions. Benzbromarone did not inhibit DNR efflux after the folate-free period in both cell lines. Repletion of folate by a 2–24 hr exposure to 2.5 μM l-leucovorin or folic acid resulted in a complete restoration of DNR efflux. In contrast, expression of MRP1/ABCC1 protein was not changed significantly during the folate-free period or the repletion-period, nor were cellular ATP or ADP pools. In conclusion, this study demonstrates that the cellular folate status can influence the transport activity of MRP1/ABCC1. These results have potentially important implications in the understanding of the (patho-)physiological roles of MRP1/ABCC1, and possibly other ABC transporter proteins in cellular folate homeostasis and drug resistance.

Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor gamma (PPAR gamma) ligand, 15-deoxy-delta 12,14prostaglandin J2 (15-d-PGJ2).

15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPAR gamma), 15-d-PGJ(2) can transactivate PPAR gamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with formation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPAR gamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ(2) conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little, if any, role in GST inhibition of 15-d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K(i) in the 0.15-2.0 microM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ(2) or 15-d-PGJ(2)-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPAR gamma.

Transport of ethinylestradiol glucuronide and ethinylestradiol sulfate by the multidrug resistance proteins MRP1, MRP2, and MRP3.

Ethinylestradiol (EE) is one of the key constituents of oral contraceptives. Major metabolites of EE in humans are the glucuronide and sulfate conjugates, EE-3-O-glucuronide (EE-G) and EE-3-O-sulfate (EE-S). In the present study, transport of EE-G and EE-S by the human multidrug resistance proteins MRP1, MRP2, and MRP3 was investigated using inside-out membrane vesicles, isolated from Sf9 cells expressing human MRP1, MRP2, or MRP3. Vesicular uptake studies showed that EE-G was not a substrate for MRP1, whereas an ATP-dependent and saturable transport of [(3)H]EE-G was observed in MRP2 (K(m) of 35.1 +/- 3.5 microM) and MRP3 (K(m) of 9.2 +/- 2.3 microM) containing vesicles. EE-S was not transported by either MRP1, MRP2, or MRP3. However, low concentrations of EE-S stimulated MRP2-mediated uptake of ethacrynic acid glutathione. EE-S also stimulated MRP2 and MRP3-mediated uptake of 17beta-estradiol-17beta-D-glucuronide. Interestingly, EE-S stimulated strongly MRP2- and MRP3-mediated uptake of EE-G by increasing its apparent transport affinity, whereas no reciprocal stimulation of EE-S uptake by EE-G was observed. These data indicate that EE-S allosterically stimulates MRP2- and MRP3-mediated transport of EE-G and is not cotransported with EE-G. Our studies demonstrate specific active transport of a pharmacologically relevant drug conjugate by human MRP2 and MRP3, involving complex interactions with other organic anions. We also suggest that caution needs to be taken when using only competition studies as screening tools to identify substrates or inhibitors of MRP-mediated transport.

The ABC Transporters MDR1 and MRP2: Multiple Functions in Disposition of Xenobiotics and Drug Resistance

ATP‐binding cassette (ABC) transporters comprise one of the largest membrane bound protein families. They are involved in transport of numerous compounds. These proteins transport substrates against a concentration gradient with ATP hydrolysis as a driving force across the membrane. Mammalian ABC proteins have important physiological, pharmacological and toxicological functions including the transport of lipids, bile salts, drugs, toxic and environmental agents. The efflux pumps serve both as natural defense mechanisms and influence the bioavailability and disposition of drugs. In general terms, the transporters remove xenobiotics from the cellular environment. For example, in cancer cells, over expression of these molecules may confer to multidrug resistance against cytostatic drugs. In addition, based on diverse structural characteristics and a broad substrate specifity, ABC transport proteins alter the intracellular concentration of a variety of therapeutically used compounds and toxicologically relevant agents. We review the function of the human multidrug resistance protein MDR1, (P‐glycoprotein, ABCB1) and the multidrug resistance protein MRP2 (ABCC2). We focus on four topics namely 1) structure and physiological functions of these transporters, 2) substrates e.g., drugs, xenotoxins, and environmental toxicants including their conjugates, 3) drug–drug interactions, and the role of chemosensitizers which may be able to reverse drug resistance, and 4) pharmacologically and toxicologically relevant genetic polymorphisms in transport proteins and their clinical implications.

Multidrug resistance-associated protein MRP1 expression in human gliomas: chemosensitization to vincristine and etoposide by indomethacin in human glioma cell lines overexpressing MRP1.

The 190 kDa multidrug resistance protein MRP1 is likely to be involved in the multidrug resistance phenotype of human gliomas. MRP1 expression was evaluated in surgical tumor samples from 17 patients with gliomas. In addition, the impact of the MRP's inhibitor, indomethacin, on the chemosensitivity to etoposide (VP16) and vincristine (VCR) of two glioblastoma cell lines expressing MRP1 (GL15 and 8MG) was investigated. When evaluated in tumor samples, MRP1 expression was observed in all of them with more than 90% of stained tumor cells in 14/15 high-grade gliomas. MRP1 was also strongly expressed at the membrane of the vascular endothelial cells in the same 14 tumor samples, suggesting that the permeability to anticancer drugs could be also limited across brain tumor vessels. At concentrations comprised between 5 and 50 microM, indomethacin significantly increased the cytotoxic effect of etoposide in both cell lines but it was more efficient in increasing the cytotoxicity of VCR on GL15 cells, as compared with 8MG cells. These results suggest that the association of indomethacin to VCR or etoposide could be of interest in the clinical management of gliomas.

Interplay between MRP inhibition and metabolism of MRP inhibitors: the case of curcumin.

The multidrug resistance proteins MRP1 and MRP2 are efflux transporters with broad substrate specificity, including glutathione, glucuronide, and sulfate conjugates. In the present study, the interaction of the dietary polyphenol curcumin with MRP1 and MRP2 and the interplay between curcumin-dependent MRP inhibition and its glutathione-dependent metabolism were investigated using two transport model systems. In isolated membrane vesicles of MRP1- and MRP2-expressing Sf9 cells, curcumin clearly inhibited both MRP1- and MRP2-mediated transport with IC(50) values of 15 and 5 microM, respectively. In intact monolayers of MRP1 overexpressing Madin-Darby canine kidney (MDCKII-MRP1) cells, curcumin also inhibited MRP1-mediated activity, although with a 3-fold higher IC(50) value than the one observed in the vesicle model. Interestingly, MRP2-mediated activity was hardly inhibited in intact monolayers of MRP2-overexpressing MDCKII (MDCKII-MRP2) cells upon exposure to curcumin, whereas the IC(50) value in the vesicle incubations was 5 microM. The difference in extent of inhibition of the MRPs by curcumin in isolated vesicles as compared to intact cells, observed especially for MRP2, was shown to be due to a swift metabolism of curcumin to two glutathione conjugates in the MDCKII cells. Formation of both glutathione conjugates was about six times higher in the MDCKII-MRP2 cells as compared with the MDCKII-MRP1 cells, a phenomenon that could be ascribed to the significantly lower glutathione levels in the cell line. The efflux of both conjugates, identified in the present study as monoglutathionyl curcumin conjugates, was demonstrated to be mediated by both MRP1 and MRP2. From dose-response curves with Sf9 membrane vesicles, glutathionylcurcumin conjugates appeared to be less potent inhibitors of MRP1 and MRP2 than their parent compound curcumin. In conclusion, curcumin clearly inhibits both MRP1- and MRP2-mediated transport, but the glutathione-dependent metabolism of curcumin plays a crucial role in the ultimate level of inhibition of MRP-mediated transport that can be achieved in a cellular system. This complex interplay between MRP inhibition and metabolism of MRP inhibitors, the latter affecting the ultimate potential of a compound for cellular MRP inhibition, may exist not only for a compound like curcumin but also for many other MRP inhibitors presently or previously developed on the basis of vesicle studies.

Upregulation in the expression of multidrug resistance protein Mrp1 mRNA and protein by increased bilirubin production in rat

Earlier studies suggest that Mrp1 may mediate ATP-dependent cellular extrusion of unconjugated bilirubin (UCB). We studied the serial responses of expression of Mrp1 mRNA and protein in rats with increased bilirubin production due to hemolysis induced by phenylhydrazine (PHZ) treatment. Mrp1 mRNA was analyzed by quantitative PCR and protein by Western blot. Hepatic expression of Mrp1 mRNA and protein peaked at day 3 of PHZ treatment. Splenic expression of Mrp1 mRNA peaked within 24 h and returned to baseline at day 5 whereas Mrp1 protein expression peaked at day 3. Pretreatment with heme-oxygenase inhibitor, tin mesoporphyrin, blunted the increase in serum UCB and erased the overexpression of Mrp1 both in liver and spleen. Thus, the upregulation of Mrp1 in hemolysis is mediated by UCB and/or other products of heme oxygenase, further supporting a role of Mrp1 in UCB transport and protection from its cellular toxicity.