Drug transporter expression in human macrophages

Macrophages represent major cellular targets of various drugs, especially antibiotics and anti-viral drugs. Factors that may govern intracellular accumulation of drugs in these cells, especially those related to activity of drug transporters, are consequently likely important to consider. The present study was therefore designed to extensively characterize expression of solute carrier (SLC) and ATP-binding cassette (ABC) transporters in primary human macrophages generated from blood monocytes. Using quantitative polymerase chain reaction assays, these cells were found to exhibit very high or high levels of mRNA expression of concentrative nucleoside transporter (CNT) 3, equilibrative nucleoside transporter 3, monocarboxylate transporter (MCT) 1, MCT4, peptide/histidine transporter (PHT) 1, PHT2, organic anion transporting polypeptide transporter 2B1 and ABC pumps multidrug resistance protein (MRP) 1/ABCC1 and MRP3/ABCC3. By contrast, other transporters, including the efflux pump ABCB1/P-glycoprotein, were found at lower levels or were not expressed. Concomitantly, human macrophages displayed notable uptake of the MCT substrate lactate and of the CNT substrate uridine and also exhibited cellular efflux of the MRP substrate carboxy-2',7'-dichlorofluorescein. Such a functional expression of these transporters has likely to be considered with respect to cellular pharmacokinetics of drugs targeting macrophages.     

Anticancer multidrug resistance mediated by MRP1: recent advances in the discovery of reversal agents

Multidrug resistance protein 1 (MRP1) belongs to the ATP-binding cassette (ABC) transporter family. It is able to transport a broad range of anticancer drugs through cellular membranes, thus limiting their antiproliferative action. Since its discovery in 1992, MRP1 has been the most studied among MRP proteins, which now count nine members. Besides the biological work, which targets structure elucidation, binding sites location, and mode of action, most efforts have been focused on finding molecules which act as MRP1 inhibitors. In this review, we attempt to summarize and highlight studies dealing with modulators of MRP1-mediated multidrug resistance (MDR), which have been accomplished in the last 5 years. The reported MRP1 inhibitors are discussed according to their chemical class. Finally, we try to bring information on structure-activity relationship (SAR) aspects and how modulators might interact with MRP1. This study may facilitate the rational design of future modulators of MDR.     

Expression of ATP-binding cassette membrane transporters in rodent and human sertoli cells: relevance to the permeability of antiretroviral therapy at the blood-testis barrier

The blood-testis barrier (BTB), composed primarily of Sertoli cells, is responsible for protecting developing germ cells from xenobiotic exposure. ATP-binding cassette (ABC) membrane-associated drug efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and the multidrug resistance-associated proteins (Mrps), have been shown to restrict antiretroviral drug permeability at blood-tissue barriers such as the blood-brain barrier. However, it remains unclear whether these transporters are functional at the level of Sertoli cells and can regulate anti-HIV drug permeability at the BTB. This study investigated the functional expression of ABC transporters in a mouse Sertoli cell line system (TM4) and in primary cultures of human Sertoli cells (HSECs). Expression of multidrug resistance Mdr1a/1b/MDR1/P-gp, Mrp1/MRP1, and Mrp4/MRP4 is confirmed by quantitative polymerase chain reaction and immunoblotting analysis in TM4 cells and HSECs. Immunofluorescence studies revealed plasma membrane localization of P-gp, Mrp1/MRP1, and Mrp4/MRP4 in both cell systems. However, Bcrp expression and localization was only detected in rodent cells. Accumulation of 1) rhodamine-6G (R-6G), a fluorescent P-gp substrate, 2) [3H]atazanavir, a HIV protease inhibitor and known P-gp substrate, 3) 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF), a fluorescent Mrp substrate, and 4) [3H]mitoxantrone, a BCRP substrate, by TM4 monolayer cells in the presence of established inhibitors demonstrates that these transporters are functional. In addition, several anti-HIV drugs significantly enhance the accumulation of R-6G, [3H]atazanavir, BCECF, and [3H]mitoxantrone by TM4 cells. This study provides the first evidence of ABC transporter expression and activity in Sertoli cells and suggests that these transporters could play an important role in restricting antiretroviral drug permeability at the BTB.     

急性淋巴细胞白血病患儿甲氨蝶呤转运蛋白及代谢酶基因多态性的研究进展

大剂量甲氨蝶呤治疗儿童急性淋巴细胞白血病的疗效及不良反应具有明显的个体差异。大剂量甲氨蝶呤治疗反应的多样性与甲氨蝶呤相关转运蛋白如溶脂载体转运蛋白、ABC转运蛋白以及甲氨蝶呤代谢相关酶如亚甲基四氢叶酸还原酶、二氢叶酸还原酶、胸腺嘧啶核苷酸合成酶、氨基咪唑胺甲酰转移酶等密切相关。本文就甲氨蝶呤代谢途径,甲氨蝶呤转运蛋白及代谢酶基因多态性对其疗效、不良反应的影响作一综述。     

Species difference in the inhibitory effect of nonsteroidal anti-inflammatory drugs on the uptake of methotrexate by human kidney slices

Simultaneous use of nonsteroidal anti-inflammatory drugs (NSAIDs), probenecid, and other drugs has been reported to delay the plasma elimination of methotrexate in patients. Previously, we have reported that inhibition of the uptake process cannot explain such drug-drug interactions using rats. The present study quantitatively evaluated the possible role of the transporters in such drug-drug interactions using human kidney slices and membrane vesicles expressing human ATP-binding cassette (ABC) transporters. The uptake of methotrexate by human kidney slices was saturable with a K(m) of 45 to 49 microM. Saturable uptake of methotrexate by human kidney slices was markedly inhibited by p-aminohippurate and benzylpenicillin, but only weakly by 5-methyltetrahydrofolate. These transport characteristics are similar to those of a basolateral organic anion transporter (OAT) 3/SLC22A8. NSAIDs and probenecid inhibited the uptake of methotrexate by human kidney slices, and, in particular, salicylate, indomethacin, phenylbutazone, and probenecid were predicted to exhibit significant inhibition at clinically observed plasma concentrations. Among ABC transporters, such as BCRP/ABCG2, multidrug resistance-associated protein (MRP) 2/ABCC2, and MRP4/ABCC4, which are candidates for the luminal efflux of methotrexate, ATP-dependent uptake of methotrexate by MRP4-expressing membrane vesicles was most potently inhibited by NSAIDs. Salicylate and indomethacin were predicted to inhibit MRP4 at clinical plasma concentrations. Diclofenac-glucuronide significantly inhibited MRP2-mediated transport of methotrexate in a concentration-dependent manner, whereas naproxen-glucuronide had no effect. Inhibition of renal uptake (via OAT3) and efflux processes (via MRP2 and MRP4) explains the possible sites of drug-drug interaction for methotrexate with probenecid and some NSAIDs, including their glucuronides.     

Physiological,pharmacological and clinical features of the multidrug resistance protein 2

Multidrug resistance protein 2 (MRP2, ABCC2) is a drug efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily. MRP2 is present predominantly at the biliary pole of hepatocytes and is also expressed in the kidney and intestine. It plays a major role in hepato-biliary elimination of many structurally diverse xenobiotics, including organic anions and drug conjugates, and therefore most likely contributes to pharmacokinetic parameters of these compounds. MRP2 also handles endogenous molecules such as bilirubin, and its overexpression has been shown to confer a multidrug resistance phenotype to tumoral cells. MRP2 expression can be regulated by endogenous substances such as inflammatory cytokines and biliary acids. The MRP2 levels and activity can also be affected by a large panel of xenobiotics, including chemopreventive agents and ligands of the pregnane X receptor, which may be a potential source of drug–drug interactions and drug adverse effects. MRP2 appears therefore as one of the major drug efflux pumps of the organism, whose functional and regulatory features are important to consider, notably for drug disposition.     

The human multidrug resistance-associated protein (MRP) gene family: from biological function to drug molecular design.

The ATP-binding cassette transmembrane proteins play an important role in transport of drugs as well as of biologically active endogenous substances. The human multidrug resistance-associated protein (MRP) subfamily consists of at least six members, exhibiting a wide spectrum of biological functions. MRP1 operates as an ATP-dependent primary active transporter for substrates conjugated with glucuronide, sulfate or glutathione. Leukotriene C4 is an important endogenous substrate for MRP1. Glutathione serves as a cofactor in MRP1-mediated drug transport as well. Genes encoding both MRP1 and the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS) are coordinately regulated in cultured cancer cell lines as well as colorectal cancer tissues from colon cancer patients. The induction of MRP1 and gamma-GCS expression by oxidative stress varies among different cell lines, and p53 mutations are associated with elevated levels of induction. To modulate the transport function of MRP1, we have synthesized novel glutathione derivatives as photoreactive biochemical probes targeting the transporter protein. GIF-0019 restored the cellular sensitivity of MRP1-overexpressing drug-resistant cancer cells to anticancer prostaglandins in vitro, which was characterized by enhanced mRNA levels of the cyclin-dependent kinase inhibitor p21, suppressed c-myc expression and G1 arrest.     

急性髓性白血病膜糖蛋白介导的多药耐药

急性白血病治疗中存在的主要问题是白血病细胞对化疗药物产生耐药性。这种现象由多种耐药机制所致,包括细胞对化疗药物反应所经历的凋亡失败或药物到达及(或)影响细胞内目标的失败。本综述重点讨论后一种机制，特别是细胞内药物转运耐药机制。有报道ATP结合盒(ABC)转运体P-糖蛋白(P—gp)与急性髓性白血病顸后相关。另外更引人瞩目的是ABC转运体多药耐药蛋白(MRP)和穹隆转运体肺耐药蛋白(LRP)的表达在AML也与预后有关。     

The A-B-C of small-molecule ABC transport protein modulators: From inhibition to activation—a case study of multidrug resistance-associated protein 1 (ABCC1)

Several mechanisms of pharmacokinetic, metabolic, and regulatory nature have been elucidated to take part or act in concert in the phenomenon of multidrug resistance (MDR). MDR is characterized by cross-resistance of cells against chemotherapeutic agents, which are used for treatment of e.g., cancer, bacterial infections, or human immunodeficiency virus (HIV) infections. One group of proteins that combines all three stated aspects—the metabolism and distribution of drugs as well as their own regulation—is adenosine triphosphate-binding cassette (ABC) transporters. These efflux pumps use the energy of adenosine triphosphate hydrolysis for drug translocation from the membrane and the cytosol to the extracellular space, often with cotransport of a cosubstrate. Multidrug resistance-associated protein 1 (MRP1, ABCC1) had been discovered as one major key player in cancer-related MDR. The xenobiotic substrates include anthracyclines, vinca alkaloids, podophyllotoxins, as well as glutathione (GSH)-adducts of certain cytostatics. Contrary to other transport proteins involved in cancer-related MDR the activity of MRP1 is related to the GSH content of cells. A modern strategy to overcome MRP1-associated MDR is besides its inhibition the activation of GSH efflux, enforcing cell death due to cellular stress. In addition, it has recently been found that MRP1 contributes to the β-amyloid protein clearance in Alzheimer's disease (AD). Collectively, transport activation of MRP1 is of therapeutic value, and furthermore helps to elucidate the transport protein function and the mechanisms behind it. This review is meant to summarize the known concepts of MRP1 activation, which might contribute to a further understanding of MRP1 in particular and ABC transporters in general.     

The Anopheles gambiae ATP‐binding cassette transporter family: phylogenetic analysis and tissue localization provide clues on function and role in insecticide resistance

The role of ATP‐binding cassette (ABC) transporters in conferring insecticide resistance has received much attention recently. Here we identify ABC transporters differentially expressed in insecticide‐resistant populations of the malaria vector, Anopheles gambiae. Although we found little evidence that the orthologues of the multidrug resistance proteins described in other species are associated with resistance in An. gambiae we did identify a subset of ABC proteins consistently differentially expressed in pyrethroid‐resistant populations from across Africa. We present information on the phylogenetic relationship, primary sites of expression and potential role of ABC transporters in mediating the mosquito's response to insecticides. Furthermore we demonstrate that a paralogous group of eight ABCG transporters, clustered on chromosome 3R, are highly enriched in the legs of An. gambiae mosquitoes, consistent with a proposed role for this ABC subfamily in transport of lipids to the outer surface of the cuticle. Finally, antibodies raised against one of the most highly expressed ABC transporters in adult females, ABCG7 (AGAP009850), localized this transporter to the pericardial cells. These data will help prioritize members of this gene family for further localization and functional validation studies to identify the in vivo function of these transporters in the mosquito and determine whether elevated expression of members of this family contribute to insecticide resistance.     

Variation of MDR proteins expression and activity levels according to clinical status and evolution of CML patients

The involvement of the multidrug resistance (MDR) mediated by ABC transporter proteins P-glycoprotein (Pgp) and multidrug resistance-associated protein-1 (MRP1) overexpressions in patients with chronic myeloid leukemia (CML) are not completely understood. Pgp and MRP1 expressions and activity were analyzed in samples from 158 patients with chronic myeloid leukemia (CML). Using flow cytometry, Pgp expression was more frequently observed in early chronic (P = 0.00) and in advanced (P = 0.02) CML phases when it was compared to MRP1 expression. Variation of MDR expression and activity were observed during the CML evolution in patients previously treated with interferon and imatinib. In the K562-Lucena cell line, Pgp positive, imatinib caused an enhancing in Pgp expression at protein and mRNA levels, whereas in the Pgp negative cell line, this drug was capable of decreasing MDR1/Pgp mRNA levels. Our result emphasizes the importance of understanding the different aspects of MDR status in patients with CML when they are under investigation in determining imatinib resistance.     

A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium''s susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell.     

Inhibition of ABC transporters abolishes antimony resistance in Leishmania Infection

The emergence of antimony (Sb) resistance has jeopardized the treatment of visceral leishmaniasis in various countries. Previous studies have considered the part played by leishmanial parasites in antimony resistance, but the involvement of host factors in the clinical scenario remained to be investigated. Here we show that unlike infection with Sb-sensitive (Sbs) Leishmania donovani, infection with Sb-resistant (Sb r) L. donovani induces the upregulation of multidrug resistance-associated protein 1 (MRP1) and permeability glycoprotein (P-gp) in host cells, resulting in a nonaccumulation of intracellular Sb following treatment with sodium antimony gluconate (SAG) favoring parasite replication. The inhibition of MRP1 and P-gp with resistance-modifying agents such as lovastatin allows Sb accumulation and parasite killing within macrophages and offers protection in an animal model in which infection with Sb r L. donovani is otherwise lethal. The occurrence of a similar scenario in clinical cases is supported by the findings that unlike monocytes from SAG-sensitive kala-azar (KA) patients, monocytes from SAG-unresponsive KA patients overexpress P-gp and MRP1 and fail to accumulate Sb following in vitro SAG treatment unless pretreated with inhibitors of ABC transporters. Thus, the expression status of MRP1 and P-gp in blood monocytes may be used as a diagnostic marker for Sb resistance and the treatment strategy can be designed accordingly. Our results also indicate that lovastatin, which can inhibit both P-gp and MRP1, might be beneficial for reverting Sb resistance in leishmaniasis as well as drug resistance in other clinical situations, including cancer.     

Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro

Members of the multidrug resistance-associated protein (MRP) family of transporters are believed to contribute to cytotoxic drug resistance and chemotherapy failure. We observed frequent MRP4 overexpression in aggressive primary neuroblastoma, a disease for which we have previously shown MRP1 to be a prognostic indicator. High MRP4 expression correlated with MYCN oncogene amplification and was significantly associated with poor clinical outcome. Although MRP4 is known to transport some nucleoside analogues, it has not previously been associated with resistance to drugs used to treat solid tumors. We now show that it mediates substantial resistance in vitro to the topoisomerase I poison irinotecan/CPT-11 and its active metabolite SN-38. These results suggest that MRP4 will be a useful prognostic marker for neuroblastoma and that clinical trials of irinotecan as a neuroblastoma treatment should monitor MRP4 expression. The same may be true for other tumor types expressing high levels of the transporter.