Function Of ATP-binding Cassette Transporters Research Articles

Alterations in function and expression of ABC transporters at blood-brain barrier under diabetes and the clinical significances

Diabetes is a systematic metabolic disease, which often develops a number of well-recognized vascular complications including brain complications which may partly result from the dysfunction of blood-brain barrier (BBB). BBB is generally considered as a mechanism for protecting the brain from unwanted actions resulting from substances in the blood and maintaining brain homeostasis via monitoring the entry or efflux of compounds. ATP-binding cassette (ABC) family of transporters including P-glycoprotein (P-GP) and breast cancer-related protein (BCRP), widely expressed in the luminal membrane of the microvessel endothelium and in the apical membrane of the choroids plexus epithelium, play important roles in the function of BBB. However, these transporters are easily altered by some diseases. The present article was focused on the alteration in expression and function of both P-GP and BCRP at BBB by diabetes and the clinical significances.

Alterations in expression and function of ABC transporters and ERM proteins in inflammation

Pathophysiological changes in infection or inflammation are associated with alterations in the production of numerous absorption-, distribution-, metabolism-, and excretion-related proteins. However, little information is available on the effects of inflammation on the expression levels and activities of ATP-binding cassette (ABC) transporters. We examined the effects of acute (on day 7) and chronic (on day 21) inflammation on the expression of ABC transporters in some major tissues in rats. Adjuvant-induced arthritis (AA) in rats was used as an animal model of inflammation. The mRNA levels of mdr1a and mdr1b encoding P-glycoprotein (P-gp) decreased significantly in the liver of AA rats on day 21. Hepatic protein levels of P-gp, Mrp2, and Bcrp decreased significantly in the membrane but not in the homogenate of AA rats after 7 days and after 21 days of treatment with adjuvant. Recently, ezrin/radixin/moesin (ERM) proteins have been found to show linker activity for some transporters and F-actin. We examined the expression levels and functions of ERM proteins in AA because the membrane localization of ABC transporters was decreased without any change in total expression levels. In this review, we summarize our recent work and reports of ERM proteins relevant to ABC transporters.

Efflux pump proteins in antifungal resistance.

It is now well-known that the enhanced expression of ATP binding cassette (ABC) and major facilitator superfamily (MFS) proteins contribute to the development of tolerance to antifungals in yeasts. For example, the azole resistant clinical isolates of the opportunistic human fungal pathogen Candida albicans show an overexpression of Cdr1p and/or CaMdr1p belonging to ABC and MFS superfamilies, respectively. Hence, azole resistant isolates display reduced accumulation of therapeutic drug due to its rapid extrusion and that facilitates its survival. Considering the importance of major antifungal transporters, the focus of recent research has been to understand the structure and function of these proteins to design inhibitors/modulators to block the pump protein activity so that the drug already in use could again sensitize resistant yeast cells. The review focuses on the structure and function of ABC and MFS transporters of Candida to highlight the recent advancement in the field.

Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation.

In oilseed plants, peroxisomal β-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of β-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 μM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid β-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ.

Karanjin interferes with ABCB1, ABCC1, and ABCG2.

The prominent ATP-binding cassette (ABC) transporters ABCB1, ABCC1, and ABCG2 are involved in substance transport across physiological barriers and therefore in drug absorption, distribution, and elimination. They also mediate multi-drug resistance in cancer cells. Different flavonoids are known to interfere with different ABC transporters. Here, the effect of the furanoflavonol karanjin, a potential drug with antiglycaemic, gastroprotective, antifungal, and antibacterial effects, was investigated on ABCB1, ABCC1, and ABCG2-mediated drug transport in comparison to the flavonoids apigenin, genistein, and naringenin. Cells expressing the relevant transporters (ABCB1: UKF-NB-3(ABCB1), UKF-NB-3(r)VCR¹⁰; ABCC1: G62, PC-3(r)VCR²⁰; ABCG2: UKF-NB-3(ABCG2)) were used in combination with specific fluorescent and cytotoxic ABC transporter substrates and ABC transporter inhibitors to study ABC transporter function. Moreover, the effects of the investigated flavonoids were determined on the ABC transporter ATPase activities. Karanjin interfered with drug efflux mediated by ABCB1, ABCC1, and ABCG2 and enhanced the ATPase activity of all three transporters. Moreover, karanjin exerted more pronounced effects than the control flavonoids apigenin, genistein, and naringenin on all three transporters. Most notably, karanjin interfered with ABCB1 at low concentrations being about 1 µM. Taken together, these findings should be taken into account during further consideration of karanjin as a potential drug for different therapeutic indications. The effects on ABCB1, ABCC1, and ABCG2 may affect the pharmacokinetics of co-administered drugs.

The Role of Canalicular ABC Transporters in Cholestasis

Cholestasis, a hallmark feature of hepatobiliary disease, is characterized by the retention of biliary constituents. Some of these constituents, such as bile acids, inflict damage to hepatocytes and bile duct cells. This damage may lead to inflammation, fibrosis, cirrhosis, and eventually carcinogenesis, sequelae that aggravate the underlying disease and deteriorate clinical outcome. Canalicular ATP-binding cassette (ABC) transporters, which mediate the excretion of individual bile constituents, play a key role in bile formation and cholestasis. The study of these transporters and their regulatory nuclear receptors has revolutionized our understanding of cholestatic disease. This knowledge has served as a template to develop novel treatment strategies, some of which are currently already undergoing phase III clinical trials. In this review we aim to provide an overview of the structure, function, and regulation of canalicular ABC transporters. In addition, we will focus on the role of these transporters in the pathogenesis and treatment of cholestatic bile duct and liver diseases.

Marine natural products with P-glycoprotein inhibitor properties.

P-glycoprotein (P-gp) is a protein belonging to the ATP-binding cassette (ABC) transporters superfamily that has clinical relevance due to its role in drug metabolism and multi-drug resistance (MDR) in several human pathogens and diseases. P-gp is a major cause of drug resistance in cancer, parasitic diseases, epilepsy and other disorders. This review article aims to summarize the research findings on the marine natural products with P-glycoprotein inhibitor properties. Natural compounds that modulate P-gp offer great possibilities for semi-synthetic modification to create new drugs and are valuable research tools to understand the function of complex ABC transporters.

The clinical significance of ABCC3 as an imatinib transporter in chronic myeloid leukaemia

The advent of imatinib has revolutionised the treatment of chronic myeloid leukaemia (CML), but in contrast to clinical trial data population studies have demonstrated that ~40% of these patients will develop resistance to imatinib. A proportion of these will progress to accelerated phase and/or blast crisis (BC).1 One cause of therapeutic resistance to imatinib is the emergence of BCR–ABL1 kinase domain mutations and gene amplification, but there are also BCR–ABL1-independent mechanisms of resistance including altered drug uptake and/or efflux.2 The ATP-binding cassette (ABC) transporters actively transport a variety of substances/drugs, and their physiological role as a defence mechanism against xenobiotics has been extensively investigated.2, 3 We and others have previously shown that imatinib is a substrate for the ABC efflux transporters ABCB1 (MDR1, p-glycoprotein) and ABCG2 (BCRP, breast cancer resistance protein), whereas its uptake is dependent on the expression/activity of the influx transporter SLC22A1 (hOCT1, human organic cation transporter-1).3, 4, 5, 6 However, additional transporters can affect treatment outcome in leukaemia, and an interplay between influx and efflux transporters could account for the observed inter-patient variability to drug response. A study by Hu et al.,7 using microarray analysis in acute myeloid leukaemia cell lines showed that hOCT1 mRNA expression was interrelated to the expression of the efflux ABCB1 (MDR1), ABCC3 (MRP3), ABCC4 (MRP4), ABCG2 (BCRP) and the influx OATP1A2 transporters. Radich et al.8 showed that ABCC3 expression was high in CML–BC patients with disease recurrence. None of these studies performed any functional ex vivo or in vitro work. This study was performed to further investigate the expression and function of ABC transporters involved in the cellular efflux of imatinib and their clinical significance in CML.

Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment

The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.

Vemurafenib (PLX4032, Zelboraf®), a BRAF Inhibitor, Modulates ABCB1-, ABCG2-, and ABCC10-Mediated Multidrug Resistance

In this study, we examined the in vitro effects of vemurafenib, a specific inhibitor of V600E mutated BRAFenzyme, on the response of cells overexpressing the ATP binding cassette (ABC) efflux transporters ABCG2, ABCB1, ABCC1 and ABCC10. Vemurafenib, at 5 µM and 20 µM, produced a significant concentration-dependent increase in the cytotoxicity of paclitaxel in cells overexpressing ABCB1 and ABCC10 and mitoxantrone in cells overexpressing ABCG2. Vemurafenib also significantly enhanced the accumulation of paclitaxel in cell lines overexpressing ABCB1 and ABCC10. Vemurafenib significantly increased the intracellular accumulation of mitoxantrone in cells overexpressing ABCG2. In contrast, vemurafenib did not significantly alter the sensitivity of ABCC1 overexpressing HEK/ABCC1 cells to vincristine. Finally, as determined by Western blotting, vemurafenib (20 µM) did not significantly alter the expression of the proteins for ABCG2, ABCC10 or ABCB1. Thus, vemurafenib most likely reverses multidrug resistance by altering the transport function of these aforementioned ABC transporters, as opposed to affecting the expression of ABC proteins. The docking analysis of vemurafenib with the ABCB1 homology model also suggested that vemurafenib binds to the ABCB1 and ABCG2 drug binding site. These findings suggest that combination of specific inhibitors like vemurafenib with chemotherapeutic drugs may be used to overcome multidrug resistance in cells that overexpress ABCB1, ABCC10 and/or ABCG2 transporters.

ATP‐binding cassette transporters in liver

The human ATP-binding cassette (ABC) superfamily consists of 48 members with 14 of them identified in normal human liver at the protein level. Most of the ABC members act as ATP dependent efflux transport systems. In the liver, ABC transporters are involved in diverse physiological processes including export of cholesterol, bile salts, and metabolic endproducts. Consequently, impaired ABC transporter function is involved in inherited diseases like sitosterolemia, hyperbilirubinemia, or cholestasis. Furthermore, altered expression of some of the hepatic ABCs have been associated with primary liver tumors. This review gives a short overview about the function of hepatic ABCs. Special focus is addressed on the localization and ontogenesis of ABC transporters in the human liver. In addition, their expression pattern in primary liver tumors is discussed.

Overlapping Functions of ABC Transporters in Topotecan Disposition as Determined in Gene Knockout Mouse Models

It is established that efflux transporters of the ATP-binding cassette (ABC) superfamily can affect the pharmacokinetics of drugs through mechanisms pertaining to drug absorption, elimination, and distribution. To characterize the role of multiple transporters in topotecan's pharmacokinetics, total (lactone+carboxylate) and lactone forms were measured by liquid chromatography/tandem mass spectrometry (LC/MS-MS) in plasma, bile, urine, and feces following intravenous administration at doses of 1 and 4 mg/kg to eight mouse strains: C57BL/6 [wild-type (WT)], Abcb1(-/-), Abcc2(-/-), Abcc4(-/-), Abcg2(-/-), Abcc2;Abcb1(-/-), Abcc2;Abcg2(-/-), and Abcc4;Abcg2(-/-). Compared with WT mice and at both dose levels, the plasma areas under the curve for topotecan lactone were not significantly different in the Abcc2(-/-), Abcc4(-/-), and Abcb1(-/-) strains, whereas significant differences were found in Abcg2(-/-), Abcc2;Abcb1(-/-) (only at the high dose), Abcc4;Abcg2(-/-), and Abcc2;Abcg2(-/-) mice and ranged from 2.1- to 3.3-fold higher. Consistent with these changes, the fecal and biliary excretion of topotecan was reduced, whereas renal elimination was elevated in Abcg2(-/-)-based strains. Similarly, the Abcc2;Abcb1(-/-) strain also had elevated renal elimination and reduced fecal excretion of topotecan lactone. This was more pronounced at the 4 mg/kg dose level, suggesting possible saturation of Abcg2. The Abcc4 transporter was found not to be a major determinant of topotecan pharmacokinetics. It is concluded that Abcg2 has the most significant effect on topotecan elimination, whereas both Abcb1 and Abcc2 have overlapping functions with Abcg2. As such it is relevant to examine how polymorphisms in these transporters influence topotecan activity in patients and whether coadministration of transport modulators could positively affect efficacy without increasing toxicity.

Impact of ABC transporters, glutathione conjugates in MDR and their modulation by flavonoids: an overview

Overexpression of ATP-binding cassette (ABC) transporter and glutathione conjugates results in efflux of cytotoxic agent from tumor cells leading to multidrug resistance (MDR). Many MDR inhibitors have been identified but none of them have been proven clinically valuable without side effects. Efforts are continue to develop an ideal MDR inhibitor. Recently, herbal modulation of ABC transporter and glutathione conjugates by flavonoids is emerging as popular therapy in MDR. In this review, we have covered structure, function of different ABC transporters and glutathione-mediated MRP overexpression. This review also focuses on the problems with existing MDR inhibitors, modulation of ABC transporter and glutathione-S-transferase by flavonoids.

Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum

BackgroundThe ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance.ResultsWe have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport.ConclusionsThe results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

BIG1, a Brefeldin A–Inhibited Guanine Nucleotide-Exchange Protein Modulates ATP-Binding Cassette Transporter A-1 Trafficking and Function

Cell-surface localization and intracellular trafficking are essential for the function of ATP-binding cassette transporter A-1 (ABCA1). However, regulation of these activities is still largely unknown. Brefeldin A, an uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism. By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells, and inhibited by 60% cholesterol release. In contrast, BIG1 overexpression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through overexpression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 small interfering RNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1. BIG1, through its ability to activate ADP-ribosylation factor 1, regulates cell-surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action.

Reversal of ATP-binding cassette drug transporter activity to modulate chemoresistance: why has it failed to provide clinical benefit?

Enhanced drug extrusion from cells due to the overexpression of the ATP-binding cassette (ABC) drug transporters inhibits the cytotoxic effects of structurally diverse and mechanistically unrelated anticancer agents and is a major cause of multidrug resistance (MDR) of human malignancies. Multiple compounds can suppress the activity of these efflux transporters and sensitize resistant tumor cells, but despite promising preclinical and early clinical data, they have yet to find a role in oncologic practice. Based on the knowledge of the structure, function, and distribution of MDR-related ABC transporters and the results of their preclinical and clinical evaluation, we discuss probable reasons why these inhibitors have not improved the outcome of therapy for cancer patients. We also outline new MDR-reversing strategies that directly target ABC transporters or circumvent relevant signaling pathways.

Role of ABC Transporters in the Pathogenesis of Alzheimer’s Disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of age-related dementia that begins with memory loss and progresses to include severe cognitive impairment. A major pathological hallmark of AD is the accumulation of beta amyloid peptide (Aβ) in senile plaques in the brain of AD patients. The exact mechanism by which AD takes place remains unknown. However, an increasing number of studies suggests that ATP-binding cassette (ABC) transporters, which are localized on the surface of brain endothelial cells of the blood-brain barrier (BBB) and brain parenchyma, may contribute to the pathogenesis of AD. Recent studies have unraveled important roles of ABC transporters including ABCB1 (P-glycoprotein, P-gp), ABCG2 (breast cancer resistant protein, BCRP), ABCC1 (multidrug resistance protein 1, MRP1), and the cholesterol transporter ABCA1 in the pathogenesis of AD and Aβ peptides deposition inside the brain. Therefore, understanding the mechanisms by which these transporters contribute to Aβ deposition in the brain is important for the development of new therapeutic strategies against AD. This review summarizes and highlights the accumulating evidence in the literature which describe the role of altered function of various ABC transporters in the pathogenesis and progression of AD and the implications of modulating their functions for the treatment of AD.

Beyond ABC: Another Mechanism of Drug Resistance in Melanoma Side Population

It has been shown that a side population (SP), which is characterized by high chemical efflux capacity, is present in human melanoma cell lines. However, it was not clear whether patients' samples contain the same subpopulation. In this issue, Luo et al. (2012) report that they have isolated SP cells directly from patients' melanomas. SP cells are resistant to paclitaxel because of the upregulation of ABCB1 and ABCB5. Notably, these cells are also resistant to temozolomide, which is not a substrate for ATP-Binding Cassette (ABC) transporters, in an interleukin (IL)-8-dependent manner. This study provides novel clues for understanding how a small, but critical, subpopulation within melanomas is resistant to therapies.

Zafirlukast antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance

ATP-binding cassette (ABC) transporters are present in the majority of human tumors and are involved in multidrug resistance (MDR). Therefore, compounds that inhibit the function of ABC transporters may improve the efficacy of anticancer agents. Previous research has shown that zafirlukast is a reversal drug for multidrug resistance protein (MRP) 1-mediated MDR. In the present study, we assessed whether zafirlukast could be a reversal agent for other ABC transporter-mediated MDR. Using the MTT assay, we found that zafirlukast enhanced the cytotoxicity of several anticancer drugs that are substrates of breast cancer resistance proteins (BCRP/ABCG2), including mitoxantrone and SN-38. Furthermore, zafirlukast could partially reverse P-glycoprotein-mediated (P-gp/ABCB1) and MRP7 (ABCC10)-mediated MDR at nontoxic doses. Studies on [(3)H]-mitoxantrone accumulation and efflux have shown that zafirlukast increases the intracellular accumulation of [(3)H]-mitoxantrone by directly inhibiting ABCG2-mediated drug efflux. Western blot analysis indicated that zafirlukast did not alter the expression of ABCG2. In addition, a docking model predicted the binding conformation of zafirlukast within the transmembrane region of homology-modeled human ABCG2. Our findings suggest a possible strategy to potentially enhance the activity of anticancer drugs using a clinically approved drug with known side effects and drug-drug interactions.

Flow cytometry‐based assessment of mitoxantrone efflux from leukemic blasts varies with response to induction chemotherapy in acute myeloid leukemia

Accurate prediction of chemotherapy drug resistance would aid treatment decisions in acute myeloid leukemia (AML). The aim of this study was to determine if mitoxantrone efflux from AML blasts would correlate with response to induction chemotherapy. Flow cytometry was used to measure the median fluorescence intensity (MFI) for AML blasts incubated with mitoxantrone [an ATP-binding cassette (ABC) transporter substrate] with or without coincubation with cyclosporine A (a broad-spectrum inhibitor of ABC transporters) and a ratio (MFIR) between the inhibited and uninhibited MFI was calculated. Among 174 AML patient blast samples, the mean MFIR for complete remission (CR) patients was lower than that obtained for induction failure (IF) patients (mean MFIR ± SD 1.62 ± 0.53 for CR after one cycle of chemotherapy vs. 2.22 ± 1.29 for CR after two cycles and 2.59 ± 0.98 for IF, P < 0.001). Logistic regression analysis determined 2.45 as the MFIR threshold above which 29% of patients achieved CR vs. a CR rate of 84% when the MFIR was ≤ 2.45 (P < 0.0001). In AML patients with normal karyotype (n = 80), CR was obtained for 33% of patients with an MFIR > 2.45 vs. 89% of those with MFIR ≤ 2.45 (P < 0.0001). In patients > age 60 (n = 77), 30% vs. 87% of those with MFIR > vs. ≤ 2.45 achieved CR (P < 0.0001). This assay of ABC transporter function can potentially predict response to induction chemotherapy in AML.