Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 microM doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin resistance points to the fragility of Pgp1-mediated MXR defence mechanism in fish.     

Human pharmaceuticals modulate P-gp1 (ABCB1) transport activity in the fish cell line PLHC-1

The ubiquitous presence of pharmaceuticals in aquatic systems is a challenging problem as their potential chronic effects on aquatic organisms remain largely unknown. The ATP-binding cassette (ABC) transport proteins contributing to the multidrug/multixenobiotic resistance (MDR/MXR) phenomenon seem to have an important role in the elimination of xenobiotics in aquatic organisms. Modulation of their efflux activities by contaminants may lead to substantial increase in intracellular accumulation and toxic effects of other xenobiotics. The aim of our work was to analyse a series of pharmaceuticals for their potential to modulate the activity of xenobiotic efflux transporters from the ABCB and ABCC sub-family in the Poeciliopsis lucida hepatoma cell (PLHC-1) fish cell line (PLHC-1/wt) and a doxorubicin (DOX) resistant PLHC-1 subclone (PLHC-1/dox) characterized by an elevated expression of the P-glycoprotein (ABCB1). Cellular accumulation of the model fluorescent substrates calcein-AM and rhodamine123 were used to determine an inhibitory effect on P-gp1 and/or MRP-like efflux transporters. 18 out of 33 tested pharmaceuticals showed MXR inhibitory activity with IC50 values occurring in the lower micromolar to millimolar range. Further, cytotoxic effects of pharmaceuticals were evaluated in PLHC-1/dox cells. Co-exposure of resistant cells to model P-gp1 inhibitor cyclosporine A (CyA) resulted in up to five times increased cytotoxicity of pharmaceuticals. In addition, some pharmaceuticals lead to a marked increase in cytotoxicity of doxorubicin, a model P-gp1 substrate. The modulation of toxicity by MDR inhibitors indicates their role in influencing cellular toxicity. In conclusion, the results of our study revealed significant inhibitory effects of environmentally relevant pharmaceuticals on P-gp1 and MRP-like transporters in fish. Our findings correspond well with data from mammalian systems indicating that the specificity and roles of the related efflux transporters may be similar in fish. Furthermore, due to the presence of active and inducible ABC transport proteins, the PLHC-1 cells appear to be a reliable in vitro system for the investigation of MDR/MXR mechanisms in fish.     

Characterization of the multixenobiotic resistance (MXR) mechanism in embryos and larvae of the zebra mussel (Dreissena polymorpha) and studies on its role in tolerance to single and mixture combinations of toxicants

The study of the cellular mechanisms of tolerance of organisms to pollution is a key issue in aquatic environmental risk assessment. Recent evidence indicates that multixenobiotic resistance (MXR) mechanisms represent a general biological defense of many marine and freshwater organisms against environmental toxicants. In this work, toxicologically relevant xenobiotic efflux transporters were studied in early life stages of zebra mussels (Dreissena polymorpha). Expression of a P-gp1 (ABCB1) transporter gene and its associated efflux activities during development were studied, using qRT-PCR and the fluorescent transporter substrates rhodamine B and calcein-AM combined with specific transporter inhibitors (chemosensitizers). Toxicity bioassays with the model P-gp1 chemotherapeutic drug vinblastine applied singly and in combination with different chemosensitizers were performed to elucidate the tolerance role of the P-gp1 efflux transporter. Results evidenced that the gene expression and associated efflux activities of ABC transporters were low or absent in eggs and increased significantly in 1-3 d old trochophora and veliger larvae. Specific inhibitors of Pgp1 and/or MRP transport activities including cyclosporine A, MK571, verapamil and reversin 205 and the musk celestolide resulted in a concentration dependent inhibition of related transport activities in zebra mussel veliger larvae, with IC50 values in the lower micromolar range and similar to those reported for mammals, fish and mussels. Binary mixtures of the tested transporter inhibitors except celestolide with the anticancer drug and P-gp1 substrate vinblastine increased the toxicity of the former compound more than additively. These results indicate that MXR transporter activity is high in early life-stages of the zebra mussel and that may play an important role in the tolerance to environmental contaminants.     

Functional expressions of adenosine triphosphate‐binding cassette transporters during the development of zebrafish embryos and their effects on the detoxification of cadmium chloride and β‐naphthoflavone

Adenosine triphosphate‐binding cassette (ABC) transporters, including ABCB, ABCC and ABCG families represent general biological defenses against environmental toxicants in varieties of marine and freshwater organisms, but their physiological functions at differential developmental stages of zebrafish embryos remain undefined. In this work, functional expressions of typical ABC transporters including P‐glycoprotein (Pgp), multiresistance associated protein 1 (Mrp1) and Mrp2 were studied in zebrafish embryos at 4, 24, 48 and 72 h post‐fertilization (hpf). As a result, both the gene expressions and activities of Pgp and Mrps increased with the development of embryos. Correspondingly, 4–72 hpf embryos exhibited an increased tolerance to the toxicity caused by cadmium chloride (CdCl₂) and β‐naphthoflavone (BNF) with time. Such a correlation was assumed caused by the involvement of ABC transporters in the detoxification of chemicals. In addition, the assumption was supported by the fact that model efflux inhibitors of Pgp and Mrps such as reversine 205 and MK571 significantly inhibited the efflux of toxicants and increased the toxicity of Cd and BNF in zebrafish embryos. Moreover, exposure to CdCl₂ and BNF induced the gene expressions of Pgp and Mrp1 in 72 hpf embryos. Thus, functional expressions of Pgp and Mrps increased with the development of zebrafish embryos, which could cause an increasing tolerance of zebrafish embryos to CdCl₂ and BNF. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.     

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.     

Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human

While P-glycoprotein (PGP, ABCB1) is known to play an important role in drug exclusion at the blood brain barrier (BBB), less is known about the contribution of other members in the ATP-binding cassette (ABC) transporter family to BBB drug efflux, or whether these transporters are expressed differently in humans and in mammalian species of pharmacological interest. We used quantitative real-time PCR to determine mRNA expression levels for the majority of ABC family members in brain and in isolated brain microvessel endothelial capillary cells (BMEC) from human, rat, mouse, pig and cow. We confirmed BBB expression of several well-characterized ABC family members that are implicated in xenobiotic exclusion from the brain, including ABCB1 (PGP), ABCG2 (BCRP), ABCC1 (MRP1), ABCC4 (MRP4), and ABCC5 (MRP5). In addition, we detected high expression and enrichment in BMEC of several less well-characterized ABC transporters in one or more species, including ABCA2-4, ABCB4, ABCB6-8, ABCB10, ABCC3, ABCC6, ABCC10, and ABCE1. We also uncovered species differences in the expression of a number of transporters, including ABCG2 and ABCC4. This study identifies several additional ABC family members that may contribute to xenobiotic efflux at the human BBB, and compares the expression of a broad array of efflux transporters between human and four other species relevant to pharmacological research.     

Gene and protein expression of P-glycoprotein, MRP1, MRP2, and CYP3A4 in the small and large human intestine

The cytochrome P450 3A4 enzyme and the ABC-transporters may affect the first-pass extraction and bioavailability of drugs and metabolites. Conflicting reports can be found in the literature on the expression levels of efflux transporters in human intestine and how they vary along the intestine. The relative levels of mRNA and protein of CYP3A4 and the ABC tranporters Pgp (ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2) were determined using RT-PCR and Western blot for human intestinal tissues (n = 14) from jejunum, ileum and colon. The expression of mRNA for CYP3A4, Pgp, and MRP2 was highest in jejunum and decreased toward more distal regions, whereas MRP1 was equally distributed in all intestinal regions. For CYP3A4, a more significant correlation could be established between mRNA and protein expression than for the ABC transporters. The samples showed considerable interindividual variability, especially at the protein level. The apically located Pgp and MRP2 showed a similar expression pattern along the human intestine as for CYP3A4. The gene expression of MRP1 exhibited a more uniform distribution.     

Effects of MDR1 and MDR3 P-glycoproteins, MRP1, and BCRP/MXR/ABCP on the transport of (99m)Tc-tetrofosmin

Multidrug resistance (MDR1) P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), and breast cancer resistance protein (BCRP/MXR/ABCP) are members of the ATP-binding-cassette (ABC) superfamily of membrane transporters and are thought to function as energy-dependent efflux pumps of a variety of structurally diverse chemotherapeutic agents. We herein report the characterization of (99m)Tc-Tetrofosmin, a candidate radiopharmaceutical substrate of ABC transporters. (99m)Tc-Tetrofosmin showed high membrane potential-dependent accumulation in drug-sensitive KB 3-1 cells and low antagonist-reversible accumulation in MDR KB 8-5 and KB 8-5-11 cells in proportion to levels of MDR1 Pgp expression. In KB 8-5 cells, EC(50) values of the potent MDR antagonists N-(4-[2-(1,2,3, 4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9, 10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), (2R)-anti-5-?3-[4-(10, 11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2 -hydroxypropoxy ?quinoline trihydrochloride (LY335979), and (3'-keto-Bmt')-[Val(2)]-cyclosporin A (PSC 833) were 40, 66, and 986 nM, respectively. Furthermore, only baculoviruses carrying human MDR1, but not MDR3, conferred both a decrease in accumulation of (99m)Tc-Tetrofosmin in host Spodoptera frugiperda (Sf9) cells and a GF120918-induced enhancement. Transport studies with a variety of stably transfected and drug-selected tumor cell lines were performed with (99m)Tc-Tetrofosmin and compared with (99m)Tc-Sestamibi, a previously validated MDR imaging agent. MDR1 Pgp readily transported each agent. To a lesser extent, MRP1 also transported each agent, likely as co-transport substrates with GSH; neither agent was a substrate for the BCRP/MXR/ABCP half-transporter. In mdr1a(-/-) and mdr1a/1b(-/-) mice, (99m)Tc-Tetrofosmin showed approximately 3. 5-fold greater brain uptake and retention compared with wild-type, with no net change in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood-brain barrier. Molecular imaging of the functional transport activity of ABC transporters in vivo with (99m)Tc-Tetrofosmin and related radiopharmaceuticals may enable non-invasive monitoring of chemotherapeutic and MDR gene therapy protocols.     

Role of ABCB1, ABCG2, ABCC2 and ABCC5 transporters in placental passage of zidovudine

Zidovudine (AZT) is one of the most frequently used antiretroviral drugs in prevention of perinatal transmission of HIV. However, safety concerns on AZT use in pregnancy still persist as severe side effects are associated with AZT exposure in children. In our study we aimed to contribute to current knowledge on AZT transplacental transport and to evaluate potential involvement of the main human drug efflux ATP‐binding cassette (ABC) transporters, p‐glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance‐associated proteins 2 and 5 (ABCC2 and ABCC5) in the disposition of AZT between mother and fetus. In order to elucidate this issue we investigated the effect of selected ABC transporters on AZT transepithelial transport across MDCKII cell monolayers. In addition we used the in situ method of dually perfused rat term placenta to further study the role of ABC transporters in AZT transplacental transport. In vitro studies revealed significant effect of ABCB1 and ABCG2 on AZT transport which was subsequently confirmed also on organ level. Lamivudine, an antiretroviral agent commonly co‐administered with AZT, did not affect ABC transporter‐mediated AZT transfer. Copyright © 2016 John Wiley & Sons, Ltd.     

Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCC2 and their impact on drug disposition

The ATP-binding cassette (ABC) transporter superfamily comprises membrane proteins that translocate a variety of substrates across extra- and intra-cellular membranes, and act as efflux proteins. ABC transporters are characterised by the presence of genetic polymorphisms mainly represented by single nucleotide polymorphisms (SNPs), some of which having an impact on their activity. Besides physiological substances, drugs are also substrates of some ABC transporters, mainly ABCB1, ABCC1, ABCC2, ABCC3 and ABCG2. Identifying the impact of these polymorphisms on the pharmacokinetics (PK) of these drugs may have important clinical implications, certainly for those characterised by a narrow therapeutic index and significant inter- and intra-patient PK variability. This review focuses specifically on ABCB1 and ABCC2 and critically analyses important publications dealing with the influence of ABCB1 and/or ABCC2 polymorphisms on drug disposition in humans. For different reasons discussed in this paper, the effect of ABCB1 and/or ABCC2 polymorphisms on drug concentrations in blood is not always easy to interpret and to correlate with pharmacological effects. In contrast, intracellular or target tissue drug concentrations appear more directly influenced by these polymorphisms, as illustrated with intralymphocyte concentrations for immunosupressants and antiretrovirals or with cerebrospinal fluid (CSF) concentrations for antiepileptics and antidepressants. Further research on intracellular and/or target tissue drug concentrations are still needed to better characterise the PK-PG (pharmacogenetics) relationship involving ABC transporters.     

The power of the pump: Mechanisms of action of P-glycoprotein (ABCB1)

Members of the superfamily of ATP-binding cassette (ABC) transporters mediate the movement of a variety of substrates including simple ions, complex lipids and xenobiotics. At least 18 ABC transport proteins are associated with disease conditions. P-glycoprotein (Pgp, ABCB1) is the archetypical mammalian ABC transport protein and its mechanism of action has received considerable attention. There is strong biochemical evidence that Pgp moves molecular cargo against a concentration gradient using the energy of ATP hydrolysis. However, the molecular details of how the energy of ATP hydrolysis is coupled to transport remain in dispute and it has not been possible to reconcile the data from various laboratories into a single model. The functional unit of Pgp consists of two nucleotide binding domains (NBDs) and two trans-membrane domains which are involved in the transport of drug substrates. Considerable progress has been made in recent years in characterizing these functionally and spatially distinct domains of Pgp. In addition, our understanding of the domains has been augmented by the resolution of structures of several non-mammalian ABC proteins. This review considers: (i) the role of specific conserved amino acids in ATP hydrolysis mediated by Pgp; (ii) emerging insights into the dimensions of the drug binding pocket and the interactions between Pgp and the transport substrates and (iii) our current understanding of the mechanisms of coupling between energy derived from ATP binding and/or hydrolysis and efflux of drug substrates.     

Danio rerio ABC transporter genes abcb3 and abcb7 play a protecting role against metal contamination

ATP‐binding cassette (ABC) proteins are efflux transporters and some of them are involved in xenobiotic detoxification. The involvement of four zebrafish ABC transporters in cadmium, zinc and mercury detoxification was characterized in a metal hypersensitive mutant of Escherichia coli. The E. coli tolC mutant expressing ABCB3 or ABCB7 transporters exhibited higher survival ratios and lower metal accumulation under a metal exposure condition than the controls. For instance, in the presence of 8 and 10 μM of HgCl₂, the survival ratios of bacteria expressing ABCB3 were four and six‐times higher than the control whereas the mercury concentrations were 2.5 and 2‐times lower than in the control. This work provides new data on the function of zebrafish ABCB3 and ABCB7 transporters and highlights their significance in metal detoxification. Copyright © 2016 John Wiley & Sons, Ltd.     

Pharmacogenomics of ABC transporters and its role in cancer chemotherapy.

ATP-binding cassette (ABC) genes play a role in the resistance of malignant cells to anticancer agents. The ABC gene products, including ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2, cMOAT), and ABCG2 (BCRP, MXR, ABCP) are also known to influence oral absorption and disposition of a wide variety of drugs. As a result, the expression levels of these proteins in humans have important consequences for an individual's susceptibility to certain drug-induced side effects, interactions, and treatment efficacy. Naturally occurring variants in ABC transporter genes have been identified that might affect the function and expression of the protein. This review focuses on recent advances in the pharmacogenomics of ABC transporters, and discusses potential implications of genetic variants for the chemotherapeutic treatment of cancer.     

Mechanistic study on the intestinal absorption and disposition of baicalein.

The present study aims to investigate the mechanisms of intestinal absorption and disposition of flavonoid baicalein (B) in Caco-2 cell monolayer model, transporter overexpressing membrane, and cellular models. The bidirectional transport studies of B and its metabolite baicalein-7-glucuronide (BG) were conducted at various concentrations and in the absence or presence of the selected transporter inhibitors. To identify specific interactions of BG with ABC transporters, ABC transporter-ATPase assays were carried out on membrane vesicles prepared from Sf9 cells overexpressing human MDR1, MRP1, MRP2, MRP3 and MXR. To further confirm the interactions between BG and specific ABC transporters, inhibition of BG on the transport of substrates of specific transporters were evaluated using membrane vesicles overexpressing MRP1-3 and MXR, or K562MDR cells with overexpressing MDR1. The results showed that B could readily pass through Caco-2 cell monolayer, but with significant glucuronidation and sulfation. The extent of phase II metabolism of B during its transport was in dose-dependent manner. The intracellularly formed glucuronide and sulfate of B were efficiently extruded to both apical and basolateral sides of the Caco-2 monolayer, which were reduced in the presence of MRP inhibitors. Although BG was not permeable from apical to basolateral side, it exhibited significant efflux transport that was inhibited in the presence of MRPs inhibitors. Moreover, BG seemed to activate the ATPase activity of both MRP3 and MXR at a pharmacologically relevant concentration range.