Multidrug Resistance Protein Transporter Research Articles

ODP560 Thyroid Hormones membrane receptor, Integrin αvβ3, as a potential prognostic marker of chemotherapy response in breast cancer

Abstract Despite the development of novel therapies, breast cancer (BC) tumors are treated with conventional therapy eventually. Due to tumor heterogeneity, it is necessary to define new biomarkers to identify groups with different molecular characteristic and on this basis improve the treatment decision. The impact of thyroid status in BC growth has previously been studied. In particular, alterations of thyroid function during chemotherapy for BC treatment have been found and it has been demonstrated that T3 induces chemosensitization of BC cells. However, the relationship between thyroid hormones (THs) membrane receptor, integrin αvβ3, and BC response to chemotherapy remains unclear. The aim of this study is to evaluate the relationship between THs and integrin αvβ3 expression as prognostic value in BC patients. The mRNA expression of ITGB3 in the TCGA-PanCancer Atlas BC dataset (1084 patients) was associated with worse overall survival. Moreover, ITGB3 mRNA expression was significantly correlated with genes encoding multidrug resistance transporter proteins MDR1 and BCRP, (Pearson, R score >0.3 and p<0,05). The diagnostic role of ITGB3 in BC was assessed by receptor operating characteristic (ROC) curve analysis. ITGB3 mRNA levels were higher in non-responder patients treated with anthracycline (AUC=0,673, p=1.8e-10) and taxanes (AUC=0.675, p=1.9e-07). To confirm the association between the integrinαvβ3-mediated effects and multidrug resistance protein transporters, we conducted in vitro assays ofMDA-MB-231 cells treated with physiological concentrations of THs and evaluated their action on MDR1 protein levels and Rhodamine efflux as an indicator of the transporter activity. We found that THs induce MDR1 protein expression and transporter activation and this effect is blocked by the presence of the integrin selective inhibitor cilengitide. Furthermore, in MDA-MB-231 doxorubicin-resistant cells THs induce MDR1 and BCRP greater protein expression than in MDA-MB-231 parental cells and this effect is abrogated by cilengitide. Therefore, we found that integrin β3 is correlated with worse overall survival and response to anthracyclines and taxanes pointing out its role as acancer biomarker with potential clinical utility. In patients with high integrin β3 mRNA expression, we found a significant correlation with proteins involved in drug transport such as MDR1 and BCRP. We also demonstrated that THs through integrin αvβ3 modulate the expression and activity of drug transporters. These results pointout the role of THs, and their membrane receptor, in BC response to treatments and introduce a new biomarker with potential clinical significance. Presentation: No date and time listed

Mebendazole as a Candidate for Drug Repurposing in Oncology: An Extensive Review of Current Literature.

Anticancer treatment efficacy is limited by the development of refractory tumor cells characterized by increased expression and activity of mechanisms promoting survival, proliferation, and metastatic spread. The present review summarizes the current literature regarding the use of the anthelmintic mebendazole (MBZ) as a repurposed drug in oncology with a focus on cells resistant to approved therapies, including so called “cancer stem cells”. Mebendazole meets many of the characteristics desirable for a repurposed drug: good and proven toxicity profile, pharmacokinetics allowing to reach therapeutic concentrations at disease site, ease of administration and low price. Several in vitro studies suggest that MBZ inhibits a wide range of factors involved in tumor progression such as tubulin polymerization, angiogenesis, pro-survival pathways, matrix metalloproteinases, and multi-drug resistance protein transporters. Mebendazole not only exhibits direct cytotoxic activity, but also synergizes with ionizing radiations and different chemotherapeutic agents and stimulates antitumoral immune response. In vivo, MBZ treatment as a single agent or in combination with chemotherapy led to the reduction or complete arrest of tumor growth, marked decrease of metastatic spread, and improvement of survival. Further investigations are warranted to confirm the clinical anti-neoplastic activity of MBZ and its safety in combination with other drugs in a clinical setting.

Effect of different concentrations of Gegen Qinlian decoction on expression of P-glycoprotein and multi-drug resistance protein transporter in Caco-2 cells

To investigate the effects of Gegen Qinlian decoction on the expression of P-glycoprotein (P-gp) and multi-drug resistance protein (MRP) in epithelial cells of human colon adenocarcinoma Caco-2 cells．The effects of different concentrations of Gegen Qinlian decoction on the expression levels of p-gp and MRP1-6 mRNA in Caco-2 cells were detected by real time quantitative reverse transcriptase PCR (qRT-PCR).12 h after drug treatment (5.00 g·L⁻¹), the expression levels of MDR1 and MRP1-6 were significantly down-regulated at concentration of 5.00 g·L⁻¹; the mRNA expression levels of MDR1，MRP1，MRP2，MRP4，MRP5 and MRP6 were significantly down-regulated at concentration of 2.50 g·L⁻¹; only the expression levels of MRP2 and MRP5 were significantly affected at concentration of 1.00 g·L⁻¹. The results showed that the expression levels of MDR1 and MRP1-6 mRNA in Caco-2 cells could be down-regulated in a dose-dependent manner. Gegen Qinlian decoction may reduce drug efflux by down-regulating the mRNA expression of cell transporters in Caco-2 cell, and increase the time of drug action, thereby enhancing the bioavailability of chemotherapeutic drugs.

Functional diversification of sea urchin ABCC1 (MRP1) by alternative splicing.

The multidrug resistance protein (MRP) family encodes a diverse repertoire of ATP-binding cassette (ABC) transporters with multiple roles in development, disease, and homeostasis. Understanding MRP evolution is central to unraveling their roles in these diverse processes. Sea urchins occupy an important phylogenetic position for understanding the evolution of vertebrate proteins and have been an important invertebrate model system for study of ABC transporters. We used phylogenetic analyses to examine the evolution of MRP transporters and functional approaches to identify functional forms of sea urchin MRP1 (also known as SpABCC1). SpABCC1, the only MRP homolog in sea urchins, is co-orthologous to human MRP1, MRP3, and MRP6 (ABCC1, ABCC3, and ABCC6) transporters. However, efflux assays revealed that alternative splicing of exon 22, a region critical for substrate interactions, could diversify functions of sea urchin MRP1. Phylogenetic comparisons also indicate that while MRP1, MRP3, and MRP6 transporters potentially arose from a single transporter in basal deuterostomes, alternative splicing appears to have been the major mode of functional diversification in invertebrates, while duplication may have served a more important role in vertebrates. These results provide a deeper understanding of the evolutionary origins of MRP transporters and the potential mechanisms used to diversify their functions in different groups of animals.

Inhibition of ABC transport proteins by oil sands process affected water

The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI+ and ESI− mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O+, SO+, and NO+ chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2−), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates this effect and inhibition of the activity of MRPs by OSPW from Base Mine Lake does not occur at environmentally relevantconcentrations.

Glutathione and multidrug resistance protein transporter mediate a self-propelled disposal of bismuth in human cells

Glutathione and multidrug resistance protein (MRP) play an important role on the metabolism of a variety of drugs. Bismuth drugs have been used to treat gastrointestinal disorder and Helicobacter pylori infection for decades without exerting acute toxicity. They were found to interact with a wide variety of biomolecules, but the major metabolic pathway remains unknown. For the first time (to our knowledge), we systematically and quantitatively studied the metabolism of bismuth in human cells. Our data demonstrated that over 90% of bismuth was passively absorbed, conjugated to glutathione, and transported into vesicles by MRP transporter. Mathematical modeling of the system reveals an interesting phenomenon. Passively absorbed bismuth consumes intracellular glutathione, which therefore activates de novo biosynthesis of glutathione. Reciprocally, sequestration by glutathione facilitates the passive uptake of bismuth and thus completes a self-sustaining positive feedback circle. This mechanism robustly removes bismuth from both intra- and extracellular space, protecting critical systems of human body from acute toxicity. It elucidates the selectivity of bismuth drugs between human and pathogens that lack of glutathione, such as Helicobacter pylori, opening new horizons for further drug development.

Mechanisms of Action of Herbal Cholagogues

Plant secondary metabolic compounds with the cholagogue mode of action are important therapeutic agents for the treatment of cholestasis and hepatobiliary disorders. Herbal cholagogues target different components of the complex bile production and secretion system, and exert their action via diverse routes, such as cholecystokinin-dependent and independent gallbladder contraction, up-regulation of the bile acid synthesis, stimulation of the bile salt export pump, multidrug resistance protein transporter system, and osmotic bile flow.

MRP transporters as membrane machinery in the bradykinin-inducible export of ATP

Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

Multidrug Resistance Protein Transporter and Ins(1,4,5)P3-Sensitive Ca2+-Signaling Involved in Adenosine Triphosphate Export via Gq Protein–Coupled NK2-Receptor Stimulation With Neurokinin A

The purpose of this study is to identify the membrane transport machinery and cell signaling involved in the neurokinin A–inducible release of adenosine triphosphate (ATP) as an autocrine/paracrine signal from cultured guinea-pig taenia coli (T. coli) smooth muscle cells (SMCs). ATP release evoked by neurokinin A was inhibited by L-659877, a NK2-receptor antagonist; by modulators for Ins(1,4,5)P3-sensitive Ca2+-signaling, U-73122, thapsigargin, and 2-APB; and by W-7, a calmodulin inhibitor, and staurosporine, a protein kinase C (PKC) inhibitor, but not by wortmannin, a phosphoinositide 3-kinase inhibitor. The evoked release was suppressed by a multidrug resistance protein (MRP)-transporter inhibitors, MK-571, indomethacin, and benzbromarone, but not by CFTR-inh 172, a CFTR-Cl− channel blocker, and α-glycyrrhetinic acid, a gap junction hemichannel blocker. Neurokinin A caused a marked accumulation of Ins(1,4,5)P3 and an increase in [Ca2+]i in the cultured cells. These findings suggest that stimulation of Gq/11 protein– coupled NK2 receptor with neurokinin A caused a substantial release of ATP from cultured T. coli SMCs and that the evoked release may be mediated by Ins(1,4,5)P3-sensitive Ca2+-signaling, further by PKC and Ca2+/calmodulin signals, and finally by an activation of MRP transporters as the membrane device.

Current target ranges of mycophenolic acid exposure and drug-related adverse events: A 5-year, open-label, prospective, clinical follow-up study in renal allograft recipients

Background: Two recent randomized clinical trials—Fixed Dose Versus Concentration Controlled and the Apomygre—evaluating the benefit of therapeutic drug monitoring of mycophenolate mofetil (MMF) in renal allograft recipients reported conflicting results. In both studies, target mycophenolic acid (MPA) AUC 0-14;12h ranges (ie, values used to guide MMF dosing) were derived from a previous study establishing target MPA AUC 0-12h ranges in cyclosporine-treated patients between 30 and 60 mg/L . h -1. Both studies found an association between MPA exposure and acute rejection. However, only one of the studies found concentrationcontrolled MMF dosing to be significantly associated with less biopsy-proven acute-rejection episodes compared with fixed dosing. No reduced incidence of MMF-related adverse events (AEs) was observed in either of the 2 trials when MMF concentration-controlled and fixed dosing were compared. Objective: The aim of this study was to assess the clinical utility of target MPA AUC 0-12h ranges between 30 and 60 mg/L . h -1 in associating drug exposure with AEs within different time windows after transplantation, thereby identifying patients at increased risk for MMF-related AEs. The effects of single nucleotide polymorphisms (SNPs) of the uridine glucuronosyltransferase 1A9 (UGT1A9) and MRP2 genes (ie, coding for the UGT1A9 and the multidrug resistance protein transporter MRP2)—both involved in MPA metabolism—on stratified MPA exposure were assessed by applying the current advised target MPA AUC 0-12h ranges. Methods: We conducted a 5-year clinical follow-up study in renal allograft recipients in whom MPA exposure was measured at 7 days, 6 weeks, 3 months, 1, 3, and 5 years post transplantation using abbreviated AUC measurements. MMF dose adjustments were based on clinical indications (eg, persistent leukopenia, chronic afebrile diarrhea, BK-polyomavirus infection of the renal allograft). Clinicians were blinded to the results of the AUC measurements. Results: One hundred white de novo renal allograft recipients (59 men, 41 women; mean [SD] age 51.4 [13.8] years) were included in this study. Ninety-eight patients received a renal allograft from a deceased donor. Significantly more episodes of leukopenia were associated with MPA AUC 0-12h ranges >60 mg/L . h -1 (P = 0.03). Anemia was also significantly associated with higher MPA exposure ranges (incidence of anemia was 40.8%, 52.2%, and 64.3% for MPA AUC 0-12h ranges <30, 30-60, and >60 mg/L . h -1, respectively; P = 0.004). Mean MPA AUC 0-12h was significantly higher in the time window immediately preceding or following leukopenia (mean [SD] 59.7 [31.0] vs 46.5 [26.0] mg/L . h -1; P = 0.004) and anemia (mean [SD] hemoglobin <12 g/L . d -1: 52.5 [30.0] vs 42.2 [21.2] mg/L . h -1, P = 0.002; hemoglobin <10 g/L . d -1: 56.2 [32.5] vs 45.6 [24.7] mg/L . h -1, P = 0.005). No association was found between incident episodes

The Effects of Antidepressants on the Hypothalamic-Pituitary-Adrenal Axis

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis has been found in some psychiatric disorders, especially in older patients with severe depression. Altered feedback inhibition, as demonstrated by increased circulating cortisol and nonsuppresssion of cortisol following administration of dexamethasone, may be to blame. Two glucocorticoid receptors control the HPA axis, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). MR regulates normal HPA fluctuations and the GR regulates in times of stress. Long-term antidepressant treatment in humans has been shown to upregulate both GR and MR in the brain, whereas short-term treatment has been shown to downregulate GR and MR. After 6-9 weeks of treatment GR function returns to normal, and the MR stays upregulated. Chronic antidepressant treatment in rodents has been shown to reduce HPA activity, even in the absence of GR or MR upregulation. These effects of antidepressants on HPA regulation may be attributed in part to regulation of the multidrug resistance protein transporter, P-glycoprotein. Finding relationships between antidepressant action and HPA regulation leads to the conclusion that the disruption of the HPA may be more a contributing factor to depression than other biological abnormalities.

Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2)

BackgroundThe purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition.MethodsThe intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined.ResultsThe absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent Km values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 μM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family.ConclusionsThe current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro. However, the involvement of other transporters cannot be ruled out based on these studies. Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects.

Pharmacokinetics and interactions of a novel antagonist of chemokine receptor 5 (CCR5) with ritonavir in rats and monkeys: role of CYP3A and P-glycoprotein.

The mechanisms of pharmacokinetic interactions of a novel anti-human immunodeficiency virus (anti-HIV-1) antagonist of chemokine receptor 5 (CCR5) [2-(R)-[N-methyl-N-(1-(R)-3-(S)-((4-(3-benzyl-1-ethyl-(1H)-pyrazol-5-yl)piperidin-1-yl)methyl)-4-(S)-(3-fluorophenyl)cyclopent-1-yl)amino]-3-methylbutanoic acid (MRK-1)] with ritonavir were evaluated in rats and monkeys. MRK-1 was a good substrate for the human (MDR1) and mouse (Mdr1a) multidrug resistance protein transporters and was metabolized by CYP3A isozymes in rat, monkey, and human liver microsomes. Both the in vitro MDR1-mediated transport and oxidative metabolism of MRK-1 were inhibited by ritonavir. Although the systemic pharmacokinetics of MRK-1 in rats and monkeys were linear, the oral bioavailability increased with an increase in dose from 2 to 10 mg/kg. The area under the plasma concentration-time curve (AUC) of MRK-1 was increased 4- to 6-fold when a 2 or 10 mg/kg dose was orally coadministered with 10 mg/kg ritonavir. Further pharmacokinetic studies in rats indicated that P-glycoprotein (P-gp) inhibition by ritonavir increased the intestinal absorption of 2 mg/kg MRK-1 maximally by approximately 30 to 40%, and a major component of the interaction likely resulted from its reduced systemic clearance via the inhibition of CYP3A isozymes. Oral coadministration of quinidine (10 and 30 mg/kg) increased both the extent and the first-order rate of absorption of MRK-1 (2 mg/kg) by approximately 40 to 50% and approximately 100 to 300%, respectively, in rats, thus further substantiating the role of P-gp in modulating the intestinal absorption of MRK-1 in this species. At the 10 mg/kg MRK-1 dose, however, the entire increase in its AUC upon coadministration with ritonavir or quinidine could be attributed to a reduced systemic clearance, and no effects on intestinal absorption were apparent. In contrast to rats, the effects of P-gp in determining the intestinal absorption of MRK-1 appeared less significant in rhesus monkeys at either dose.

High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival.

Multidrug resistance protein (MRP) activity was investigated in 44 newly diagnosed acute myeloid leukaemia (AML) patients using a functional assay based on efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by both MRP1 and MRP2. Elevated MRP transport was detected in 29% of cases, but was not significantly correlated with sex, age, white blood cell count at diagnosis or karyotype. In contrast, it was associated with secondary AML (P = 0.002), CD34 positivity (P = 0.041) and P-glycoprotein activity (P = 0.01). There was a lower rate of complete remission in MRP-positive patients versus MRP-negative patients (23% versus 81%; P = 0.001); overall survival was also better for MRP-negative patients (P = 0.004). These data indicate a probable role for MRP activity in the clinical outcome of AML.

The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites.

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.

Direct binding of chloroquine to the multidrug resistance protein (MRP): Possible role for MRP in chloroquine drug transport and resistance in tumor cells

Multidrug resistance protein (MRP) transports a range of compounds that include glutathione S-conjugates, amphiphilic anionic drugs, and natural-product toxins. However, the mechanism of MRP drug binding and transport is presently unclear. We recently demonstrated the direct binding of a quinoline-based photoactive drug, N-{4-[1-hydroxy-2-(dibutylamino)ethyl] quinolin-8-yl}-4-azidosalicylamide (IAAQ), to MRP at a biologically relevant site [Vezmar et al., Biochem Biophys Res Commun 241: 104–111, 1997]. In the present report, we demonstrated that the lysosomotropic or antimalarial drug chloroquine is a substrate for MRP. Specifically, our results showed that chloroquine, similar to leukotriene C 4 (LTC 4) and 3-(3-(2-(7-chloro-2-quinolinyl)ethenyl-phenyl)((3-(dimethyl amino-3-oxo propyl)thio)methyl)thio) propanoic acid (MK 571), inhibits the photoaffinity labeling of MRP by IAAQ. Furthermore, cell growth assays showed MRP-expressing multidrug-resistant cells (H69/AR and HL60/AR) to be more resistant to chloroquine than their parental cells (i.e., ic 50 of 121 μM versus 28 μM chloroquine for H69/AR and H69, respectively). Moreover, MK 571, an LTD 4 receptor antagonist, reversed the resistance of H69/AR cells to chloroquine. Drug transport studies using [ 14C]chloroquine demonstrated that MRP-expressing cells accumulate less drug than the parental drug-sensitive cells. The reduced accumulation of [ 14C]chloroquine in resistant cells was ATP dependent and was due to enhanced drug efflux. Taken together, the results of this study show that MRP modulates the transport of chloroquine by direct binding.