Inhibition of the P-glycoprotein- and multidrug resistance protein-mediated efflux of anthracyclines and calceinacetoxymethyl ester by PAK-104P

Multidrug resistance phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or Multidrug Resistance-Associated protein (MRP(1)). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. Overexpression of these transporters by tumor cells is thought to be a significant factor in both intrinsic and acquired resistance to anticancer drugs. Consequently a great deal of interest is focused on identifying chemical agents that can either antagonise drug transport by these proteins or that can inhibit the proliferation of tumors cells despite the expression of these transporters. P-glycoprotein-mediated multidrug resistance is reversed by a variety of compounds, but surprisingly, few agents reverse the MRP(1)-mediated multidrug resistance. However, it has recently been shown that 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3, 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P oxide (PAK-104P) was able to inhibit the P-glycoprotein and MRP(1)-mediated efflux of several compounds. Understanding of the interactions between transporters and multidrug resistance reversing agents is important in the design of more effective multidrug resistance modulators. We now examined the effect of PAK-104P on Pgp-and MRP1-mediated efflux of three anthracyclines, daunorubicin, pirarubicin, hydroxydoxorubicin and of calcein acetoxymethyl ester and calcein. Our data show that PAK-104P non-competitively inhibits the P-glycoprotein-mediated efflux of anthracycline derivatives and calcein acetoxymethyl ester with an inhibitory constant K(I)=0. 25+/-0.05 microM. PAK-104P also non-competitively inhibits the MRP(1)-mediated efflux of daunorubicin, pirarubicin, hydroxyrubicin, calcein acetoxymethyl ester and calcein. However, surprisingly, in this case the K(I) values obtained were very different ranging from 0.06 for hydroxyrubicin to 10 microM for calcein. These data strongly suggested the existence of two different mechanisms for the inhibition by PAK-104P of the MRP(1)-mediated efflux of molecules: a first mechanism, involving a low-affinity site for PAK-104P, and which would concern molecules such as calcein, cysteinyl leukotriene LCT(4) etc. whose efflux do not depend on glutathione. A second mechanism involving a high-affinity site for PAK-104P and which would concern molecules such as anthracyclines, calcein acetoxymethyl ester whose efflux depends on the presence of glutathione.     

Interaction of irinotecan (CPT-11) and its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) with human cytochrome P450 enzymes.

The inhibition and mechanism-based inactivation potencies of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; CPT-11) and its active metabolite (7-ethyl-10-hydroxycamptothecin; SN-38) for human cytochrome P450 (P450) enzymes were investigated to evaluate the potential for drug interactions involving CPT-11 using microsomes from insect cells expressing specific human P450 isoforms. The mechanism and potential for interaction were examined by Lineweaver-Burk analysis, and NADPH-, time- and concentration-dependent effects were observed. CPT-11 and SN-38 competitively inhibited CYP3A4 (testosterone 6 beta-hydroxylation) activity with K(i) values of 129 and 121 microM, respectively. CYP2A6 (coumarin 7-hydroxylation) and CYP2C9 (diclofenac 4'-hydroxylation) activities exhibited a mixed type of inhibition comprising competitive and noncompetitive components in response to SN-38, the K(i) values being 181 and 156 microM, respectively. On the other hand, CYP1A2 (phenacetin O-deethylation), CYP2B6 (7-ethoxycoumarin O-deethylation), CYP2C8 (paclitaxel 6 alpha-hydroxylation), CYP2C19 (S-mephenytoin 4'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), and CYP2E1 (chlorzoxazone 6-hydroxylation) were hardly affected by either compound. Furthermore, CPT-11 and SN-38 were suggested to be mechanism-based inactivators of CYP3A4. The k(inact) and K(I) values of CPT-11 and SN-38 were 0.06 min(-1) and 24 microM and 0.10 min(-1) and 26 microM, respectively. However, no inactivation of CYP2A6 and CYP2C9 by SN-38 was observed. These results mean that CPT-11 and SN-38 interact with human P450 isoforms, such as CYP2A6, CYP2C9, and CYP3A4, in vitro and imply that the significant drug interactions involving CPT-11 may be caused by a mechanism-based inactivation of CYP3A4 by SN-38 as an active metabolite of CPT-11 rather than competitive inhibition.     

In Vitro Binding and Partitioning of Irinotecan (CPT-11) and its Metabolite, SN-38, in Human Blood

The binding of CPT-11 and SN-38 to human plasma proteinswas studied by ultrafiltration at 37°C and pH 7.4. In plasma,CPT-11 was 66–60% bound in the range 100–4000ng/ml and SN-38 was 94–96% bound in the range50–200 ng/ml. At these concentrations the plasma bindingof CPT-11 was slightly saturable, but the plasma binding of SN-38was concentration-independent. Albumin was the main carrier ofCPT-11 and SN-38 in plasma. In blood, the binding of CPT-11 wasmoderate (80%), mainly to plasma proteins (47%) anderythrocytes (33%). The binding of SN-38 was high(99%) and most of SN-38 in blood was located in bloodcells (approximately 66%) The simulation of a grade 3hematotoxicity (according to National Cancer Institute's CommonToxicity Criteria grading) on the SN-38 blood distributionyielded an increase in fu (free fraction of drug in plasma) from1.05 to 2.08 and a decrease in C_Bl/C_P from1.66 to 1.14 (both resulting from a decreased cellbinding).     

High-performance liquid chromatographic analysis of the anticancer drug irinotecan (CPT-11) and its active metabolite SN-38 in human plasma

A rapid, sensitive, and specific high-performance liquid chromatography (HPLC) method for the simultaneous determination of irinotecan (CPT-11) and its active metabolite SN-38 in human plasma is described. The analytes are quantified as the totals of their carboxylate and lactone form. The sample pretreatment consisted of a simple protein precipitation with acetonitrile-methanol (1:1, v/v), after which CPT-11 and SN-38 were quantitatively converted to their carboxylate form by adding 0.01 mol/L sodium tetraborate (pH, 9). Chromatography was carried out on a Zorbax SB-C18 column with fluorescence detection. The method has been validated, and stability tests under various clinically relevant conditions have been performed. The lower limit of quantification (LLOQ) was 5.0 ng/mL for CPT-11 and 0.5 ng/mL for SN-38. Standard concentration ranges were linear between 5 and 1,500 ng/mL for CPT-11 and between 0.5 and 100 ng/mL for SN-38. This assay is simple, rapid, and very useful for therapeutic monitoring of CPT-11 and SN-38.     

The transformation of irinotecan (CPT-11) to its active metabolite SN-38 by human liver microsomes Differential hydrolysis for the lactone and carboxylate forms

Irinotecan (CPT-11) is a new camptothecine derivative presently in development for the treatment of several advanced malignancies. It is converted in vivo to a highly potent metabolite, SN-38, by carboxylesterases. All camptothecine derivatives undergo lactonolysis in a pH-dependent reversible manner, generating inactive carboxylate forms. We have investigated in vitro the kinetics of transformation of CPT-11 to SN-38 by human liver microsomes originating from several donors. Microsomes from seven livers were studied individually or as a pooled preparation. CPT-11, either in its lactone or its carboxylate form, was added at a range of concentrations. The SN-38 formed was measured by HPLC with fluorometric detection. In the deacylation-limited carboxylesterase reaction, the linear steady-state kinetics between 10 and 60min were determined. At all concentrations of CPT-11, the steady-state velocity of SN-38 formation as well as the intercept concentrations of SN-38 were about 2-fold higher when the substrate was under the lactone form than under the carboxylate form. We estimated the values (±SD) of K’_m and V _max to be 23.3±5.3μM and 1.43±0.15pmol/min/mg for the lactone and 48.9±5.5μM and 1.09±0.06pmol/min/mg for the carboxylate form of CPT-11, respectively. We conclude that the greater rate of conversion of CPT-11 lactone may contribute to the plasma predominance of SN-38 lactone observed in vivo. The inter-individual variation of SN-38 formation was relatively high (ratio of 4 between extreme values) but no large age- or gender-related differences were seen. The effect of twelve drugs of different therapeutic classes (antibiotics, antiemetics, antineoplastics, antidiarrhoeics, analgesics), which could be administered in association with irinotecan in the clinical setting, was evaluated in this system (drug concentration: 100μM; CPT-11 lactone concentration: 10μM). Loperamide and ciprofloxacine where the only drugs exerting a weak inhibition of CPT-11 conversion to SN-38. Received: 30 December 1996 /Accepted: 30 April 1997     

Inhibition of the multidrug resistance protein 1 (MRP1) by peptidomimetic glutathione-conjugate analogs

Inhibition of multidrug resistance protein 1 (MRP1) mediated cytostatic drug efflux might be useful in the treatment of drug resistant tumors. Because the glutathione (GSH) conjugate of ethacrynic acid (EA), GS-EA, is a good substrate of MRP1, GS-EA derivatives are expected to be good inhibitors of MRP1. To study structure-activity relationships of MRP1 inhibition, a series of novel GS-EA analogs was synthesized in which peptide bonds of the GSH backbone were replaced by isosteric groups [Bioorg Med Chem 10:195-205, 2002]. Several of these compounds were effective inhibitors of MRP1-mediated [(3)H]GS-EA and [(3)H]E(2)17betaG transport, as studied in membrane vesicles prepared from MRP1-overproducing Sf9 cells. The modifications of the peptide backbone have distinct implications for recognition by MRP1: the gamma-glutamyl-cysteine peptide bond is important for binding, whereas the cysteinyl-glycine amide does not seem essential. When the gamma-glutamyl-cysteine peptide bond (C-CO-N) is replaced by a urethane isostere (O-CO-N), an effective competitive MRP1-inhibitor (K(i) = 11 microM) is obtained. After esterification of this compound to improve its cellular uptake, it inhibited MRP1-mediated efflux of calcein from 2008 ovarian carcinoma cells overexpressing MRP1. This compound also partially reversed the resistance of these cells to methotrexate. Because the urethane isostere is stable toward gamma-glutamyl transpeptidase-mediated breakdown, it is an interesting lead-compound for the development of in vivo active MRP1 inhibitors.     

Comparison of furosemide and vinblastine secretion from cell lines overexpressing multidrug resistance protein (P-glycoprotein) and multidrug resistance-associated proteins (MRP1 and MRP2)

Recent studies in our laboratory have shown that the loop diuretic, furosemide, is actively secreted by Caco-2 cells and rat jejunal tissue. This active secretion could be the result of efflux transporters such as P-gp, MRP1 or MRP2 (cMOAT). To determine if any of these transporters is responsible for the secretion of furosemide, we compared directional permeability in the wild-type cell lines, MDCK strains I and II, and LLC-PK1, vs. cell lines that overexpress a single transporter, in both the presence and absence of various inhibitors, for furosemide as compared to vinblastine. Sulfinpyrazone significantly inhibited the transport of vinblastine in MRP2 expressing cells, but not the wild-type controls. Vinblastine could not be confirmed as a substrate of MRP1. We were also unable to demonstrate that any particular transporter affected furosemide in excess of the background effects of endogenous transporters in the parental cell lines. Furosemide secretion from these kidney-derived cell lines is probably not the primary result of any of the well characterized efflux transporters (P-gp, MRP1 or MRP2), although they may still play a role in the observed Caco-2 secretion. This equivocal result acknowledges the difficulty in trying to determine the effect of a single protein in a complicated expression system.     

Monomethylarsenic diglutathione transport by the human multidrug resistance protein 1 (MRP1/ABCC1)

The ATP-binding cassette (ABC) transporter protein multidrug resistance protein 1 (MRP1; ABCC1) plays an important role in the cellular efflux of the high-priority environmental carcinogen arsenic as a triglutathione conjugate [As(GS)(3)]. Most mammalian cells can methylate arsenic to monomethylarsonous acid (MMA(III)), monomethylarsonic acid (MMA(V)), dimethylarsinous acid (DMA(III)), and dimethylarsinic acid (DMA(V)). The trivalent forms MMA(III) and DMA(III) are more reactive and toxic than their inorganic precursors, arsenite (As(III)) and arsenate (As(V)). The ability of MRP1 to transport methylated arsenicals is unknown and was the focus of the current study. HeLa cells expressing MRP1 (HeLa-MRP1) were found to confer a 2.6-fold higher level of resistance to MMA(III) than empty vector control (HeLa-vector) cells, and this resistance was dependent on GSH. In contrast, MRP1 did not confer resistance to DMA(III), MMA(V), or DMA(V). HeLa-MRP1 cells accumulated 4.5-fold less MMA(III) than HeLa-vector cells. Experiments using MRP1-enriched membrane vesicles showed that transport of MMA(III) was GSH-dependent but not supported by the nonreducing GSH analog, ophthalmic acid, suggesting that MMA(III)(GS)(2) was the transported form. MMA(III)(GS)(2) was a high-affinity, high-capacity substrate for MRP1 with apparent K(m) and V(max) values of 11 μM and 11 nmol mg(-1)min(-1), respectively. MMA(III)(GS)(2) transport was osmotically sensitive and inhibited by several MRP1 substrates, including 17β-estradiol 17-(β-D-glucuronide) (E(2)17βG). MMA(III)(GS)(2) competitively inhibited the transport of E(2)17βG with a K(i) value of 16 μM, indicating that these two substrates have overlapping binding sites. These results suggest that MRP1 is an important cellular protective pathway for the highly toxic MMA(III) and have implications for environmental and clinical exposure to arsenic.     

Role of multidrug resistance protein 2 (MRP2) in glutathione-bimane efflux from Caco-2 and rat renal proximal tubule cells.

1. The multidrug resistance protein 2 (MRP2) has been shown to play an important role in the transport of glutathione conjugates in the liver. Its importance in renal excretion, however, is still uncertain and other organic anion transporters may be involved. The objective of the present study was to characterize glutathione conjugate efflux from rat kidney proximal tubule cells (PTC), and to determine the contribution of Mrp2. 2. We used isolated PTC in suspension, as well as grown to monolayer density. For comparison, transport characteristics were also determined in the human intestinal epithelial cell line Caco-2, an established model to study MRP2-mediated transport. The cells were loaded with monochlorobimane (MCB) at 10 degrees C. MCB enters the cells by simple diffusion and is conjugated with glutathione to form the fluorescent glutathione-bimane (GS-B). 3. In primary cultures of rat PTC, no indications for a transporter-mediated mechanism were found. The efflux of GS-B from Caco-2 cells and freshly isolated PTC was time- and temperature-dependent. Furthermore, GS-B transport in both models was inhibited by chlorodinitrobenzene (CDNB), with an inhibitory constant of 46.8+/-0.9 microM in freshly isolated PTC. In Caco-2 cells, the inhibitory potency of CDNB was approximately 20 fold higher. Finally, efflux of GS-B from freshly isolated PTC from Mrp2-deficient (TR(-)) rats was studied. As compared to normal rat PTC, transport characteristics were not different. 4. We conclude that in freshly isolated rat PTC glutathione conjugate excretion is mediated by other organic anion transporters rather than by Mrp2.     

The mechanism for the inhibition of acetylcholinesterases by irinotecan (CPT-11)

Irinotecan (CPT-11) is an anticancer drug that occasionally produces acute cholinergic side effects. Preliminary findings suggest that these are mediated through the inhibition of acetylcholinesterase (AChE). In this study, the inhibition of various AChEs by CPT-11 was studied. The lactone form of CPT-11 resulted in apparent noncompetitive inhibition of electric eel and both human recombinant and erythrocyte AChE with K(i) values of 0.065, 0.19, and 0.29 microM, respectively. The carboxylate form of CPT-11 was approximately 10 times less potent. Apparent noncompetitive inhibition of AChE may arise through several mechanisms, and those relevant to CPT-11 were identified from key experimental findings. First, the inhibition by CPT-11 was found to be instantly reversible in dilution studies. Second, incubation of the enzyme with CPT-11 before the introduction of neostigmine protected the enzyme from inactivation. Third, regeneration of the active enzyme after preincubation with neostigmine was totally suppressed by the addition of 2 microM CPT-11, indicating that CPT-11 is a potent inhibitor of decarbamoylation and, by inference, deacylation. Additional experiments with tacrine revealed functional differences between these two inhibitors. Also, preliminary molecular modeling of the interaction between AChE and CPT-11 indicated that the latter does not bind at the same site as tacrine. Displacement studies with the peripheral site-specific ligand, propidium, confirmed that CPT-11 binds at this site. The rapid reversibility of the inhibition of AChE by CPT-11 and the lower activity of the carboxylate form are likely reasons for the transient nature of the cholinergic toxicity observed clinically.     

ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1

The present study was performed to investigate the ability of the multidrug resistance protein (MRPI) to transport different cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1-transfected cell line (S1(MRP)). As substrates we used 3H-labelled derivatives of the hydrophilic monoquaternary cation N-(4',4'-azo-in-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. ATP- and GSH-dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1-specific monoclonal antibody QCRL-3. MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations > or = 10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 microM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP- and GSH-dependent manner. APDA is not a substrate for MRP1.     

Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters

The 190 kDa multidrug resistance protein 1 (MRP1/ABCC1) is a founding member of a subfamily of the ATP binding cassette (ABC) superfamily of transport proteins and was originally identified on the basis of its elevated expression in multidrug resistant lung cancer cells. In addition to its ability to confer resistance in tumour cells, MRP1 is ubiquitously expressed in normal tissues and is a primary active transporter of GSH, glucuronate and sulfate conjugated and unconjugated organic anions of toxicological relevance. Substrates include lipid peroxidation products, herbicides, tobacco specific nitrosamines, mycotoxins, heavy metals, and natural product and antifolate anti-cancer agents. MRP1 also transports unmodified xenobiotics but often requires GSH to do so. Active efflux is generally an important aspect of cellular detoxification since it prevents the accumulation of conjugated and unconjugated compounds that have the potential to be directly toxic. The related transporters MRP2 and MRP3 have overlapping substrate specificities with MRP1 but different tissue distributions, and evidence that they also have chemoprotective functions are discussed. Finally, MRP homologues have been described in other species including yeast and nematodes. Those isolated from the vascular plant Arabidopsis thaliana (AtMRPs) decrease the cytoplasmic concentration of conjugated toxins through sequestration in vacuoles and are implicated in providing herbicide resistance to plants.     

Bioflavonoid stimulation of glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1).

In tumor cells, the human multidrug resistance protein 1 (MRP1), confers resistance to a broad spectrum of anticancer agents. MRP1 is also expressed in many normal tissues where it acts as an ATP-dependent transporter of organic anions. Reduced glutathione (GSH) is transported by MRP1 with very low affinity, and certain MRP1 substrates are transported in association with this tripeptide. Previous studies have shown that various dietary flavonoids stimulate the ATPase activity of MRP1 and inhibit transport of its conjugated organic anion substrates but are poor reversers of MRP1-mediated drug resistance. In contrast, many of the same flavonoids markedly stimulate GSH transport by MRP1. In the present study, we found that stimulation of GSH transport in inside-out MRP1-enriched membrane vesicles by apigenin, naringenin, genistein, and quercetin was maximum at a concentration of 30 microM. Apigenin was the most efficacious of the four bioflavonoids, showing a maximal 6-fold increase over basal levels of GSH transport. The apparent K(m) and V(max) for GSH uptake in the presence of 30 microM apigenin were 116 microM and 666 pmol mg(-1) min(-1), respectively. Chemosensitivity assays with control-transfected and MRP1-transfected HeLa cell lines showed that the IC(50) values for apigenin, naringenin, genistein, and quercetin were similar, demonstrating that overexpression of MRP1 does not confer resistance to these bioflavonoids. Our results suggest that flavonoids stimulate MRP1-mediated GSH transport by increasing the apparent affinity of the transporter for GSH but provide no evidence that a cotransport mechanism is involved.     

Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.     

Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.     

Characterization of the transport properties of human multidrug resistance protein 7 (MRP7, ABCC10)

Human multidrug resistance protein 7 (MRP7, ABCC10) is a recently described member of the C family of ATP binding cassette proteins (Cancer Lett 162:181-191, 2001). However, neither its biochemical activity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP7 using membrane vesicles prepared from human embryonic kidney 293 cells transfected with MRP7 expression vector. It is shown that expression of MRP7 is specifically associated with the MgATP-dependent transport of 17beta-estradiol-(17-beta-D-glucuronide) (E(2)17betaG). E(2)17betaG transport was saturable, with K(m) and V(max) values of 57.8 +/- 15 microM and 53.1 +/- 20 pmol/mg/min. By contrast, with E(2)17betaG, only modest enhancement of LTC(4) transport was observed and transport of several other established substrates of MRP family transporters was not detectable to any extent. In accord with the notion that MRP7 has a bipartite substrate binding pocket composed of sites for anionic and lipophilic moieties, transport of E(2)17betaG was susceptible to competitive inhibition by both amphiphiles, such as leukotriene C(4) (K(i(app)), 1.5 microM), glycolithocholate 3-sulfate (K(i(app)), 34.2 microM) and MK571 (K(i(app)), 28.5 microM), and lipophilic agents such as cyclosporine A (K(i(app)), 14.4 microM). Of the inhibitors tested, LTC(4) was the most potent, in agreement with the possibility that it is a substrate of the pump. The determination that MRP7 has the facility for mediating the transport of conjugates such as E(2)17betaG indicates that it is a lipophilic anion transporter involved in phase III (cellular extrusion) of detoxification.     

Multidrug resistance protein 1 (MRP1, ABCC1) mediates resistance to mitoxantrone via glutathione-dependent drug efflux

Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50 microM MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]-an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATP-dependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [3H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1-like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)-can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.     

Diverse effects of P-glycoprotein inhibitory agents on human leukemia cells expressing the multidrug resistance protein (MRP)

Multidrug resistance (MDR) to cancer chemotherapy is frequently associated with decreased drug accumulation in cancer cells due to drug expulsion by multidrug transporters such as P-glycoprotein (Pgp) and multidrug resistance protein (MRP). The novel resistance modifying agents PSC 833, 280-446, and LY 335979 are primarily targeted at inhibition of Pgp, and their MRP inhibitory potential is largely unknown. OBJECTIVE: In the present study we addressed the effect of these agents on MRP-derived drug resistance. MATERIALS: Drug-resistant human leukemia cells with Pgp+/MRP- (KG1a/200, K562/150) and Pgp-/MRP+ (HL60/130) phenotypes were maintained in suspension cultures for experimental studies of drug accumulation and drug sensitization by Pgp inhibitors. METHODS: Intracellular accumulation of the fluorescent anthracycline daunorubicin was measured by flow cytometry and fluorescence detection. Daunorubicin dose-response curves were generated by non-linear regression of electronically measured cell counts of 72- - 96-h cultures. The half-maximal growth inhibitory dose (GI50) was used as measure of growth inhibition. RESULTS: All MDR phenotypes studied exercised significant resistance to daunorubicin. PSC 833, 280-446 and LY335979 were equal in sensitizing Pgp+/MRP- cells to daunorubicin-induced growth inhibition (p < 0.0001). The Pgp-/MRP+ cells responded to PSC 833 and 280-446 by increased accumulation of daunorubicin (p = 0.0022 and p = 0.0005, respectively) and sensitization to the drug (p = 0.0009 and p = 0.0007, respectively). Conversely, LY335979 did not affect accumulation of daunorubicin in Pgp-/MRP+ cells nor sensitize these cells to daunorubicin. CONCLUSION: Pgp inhibitory agents have differential effects on MRP-derived drug resistance which could be exploited in treatment of multidrug resistance in cancer patients.     

New insights into the biology and pharmacology of the multidrug resistance protein (MRP) from gene knockout models.

Growing interest in the MRP (multidrug resistance protein) gene stems from its importance in multidrug resistance to chemotherapy, its possible use in gene therapy, and its relationship with the glutathione system. The recent generation of mrp gene knockout models in vitro and in vivo is providing information on the mechanism of action and the physiological function(s) of mrp. The importance of mrp in protection of normal tissues from the toxicity of the anticancer agent etoposide has been established. A total block of mrp has been found to be compatible with life, suggesting that MRP inhibitors can be safely used for treating cancer patients. In some sub-classes of leukocytes, mrp contributes to the transport of leukotriene C4, an endogenous glutathione-S-conjugate. However, the baseline expression of mrp does not appear to contribute to the export of glutathione-S-conjugates of alkylating agents, and thus does not exert a protective role against their toxicity. Besides being capable of exporting certain glutathione-S-conjugates, mrp also catalyzes the co-transport of GSH and drug and, presumably, a presently unknown endogenous metabolite(s).