Human ATP-binding Cassette Transporter Research Articles

Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.

Differences in Transcript Levels of ABC Transporters Between Pancreatic Adenocarcinoma and Nonneoplastic Tissues

The aim of this study was to evaluate transcript levels of all 49 human ATP-binding cassette transporters (ABCs) in one of the most drug-resistant cancers, namely, the pancreatic ductal adenocarcinoma (PDAC). Association of ABCs levels with clinical-pathologic characteristics and KRAS mutation status was followed as well. Tumors and adjacent nonneoplastic tissues were obtained from 32 histologically verified PDAC patients. The transcript profile of ABCs was assessed using quantitative real-time polymerase chain reaction with a relative standard curve. KRAS mutations in exon 2 were assessed by high-resolution melting analysis and sequencing. Most ABCs were deregulated in PDAC and 10 ABCs were associated with clinical-pathologic characteristics. KRAS mutations did not change the global expression profile of ABCs. The expression of ABC transporters was significantly deregulated in PDAC tumors when compared to nonmalignant tissues. The observed up-regulation of ABCB4, ABCB11, ABCC1, ABCC3, ABCC5, ABCC10, and ABCG2 in tumors may contribute to the generally poor treatment response of PDAC. The up-regulation of ABCA1, ABCA7, and ABCG1 implicates a serious impairment of cellular cholesterol homeostasis in PDAC. On the other hand, the observed down-regulation of ABCA3, ABCC6, ABCC7, and ABCC8 suggests a possible role of stem cells in the development and progression of PDAC.

Abstract 989: Mild ER stress increases human ABC transporter expression and drug transport function in polarized cell monolayers.

Abstract Expression of ATP-binding cassette (ABC) transporter genes such as MDR1 (ABCB1/P-gp) is associated with poor outcome in chemotherapy and a multidrug resistant phenotype in cancer. Endoplasmic reticulum (ER) stress has been implicated in tumor growth but its role in drug resistance remains unclear. Here, we hypothesize that ER stress could increase expression of drug efflux transporters. To test this hypothesis, we developed a transient expression system in which human ABC transporters were ectopically expressed in a cell line monolayer by the BacMam baculovirus. The baculovirus is able to infect several mammalian cell lines including LLC-PK1, T-84, and MA104 without affecting the tightness of the host cell monolayers. In LLC-PK1 cells infected by BacMam vectors, induction of mild ER stress by pre-incubation (pre-conditioning) with thapsigargin (0.5 μg/ml) or tunicamycin (1 μg/ml), which are well-characterized ER-stress inducers, increased ABCB1 (P-gp) and ABCG2 (BCRP) levels. Drug transport assays showed that P-gp expressing cells pre-conditioned with thapsigargin showed increased directional transport of [3H] paclitaxel across the cell monolayer. The effect of thapsigargin was inhibited by the presence of the Erk inhibitor PD098059 but not the P38(MAPK) inhibitor SB203580, or the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002, suggesting that expression of ABC transporter is influenced by mild ER stress response and, at least in part, by the ERK/MAPK signaling pathway. Although pre-conditioning with tunicamycin can increase P-gp expression, it also inhibits N-glycosylation of P-gp. Collectively, our findings indicate that mild ER stress responses up-regulate drug transporters introduced into cells by BacMam vectors. Citation Format: King Leung Fung, Jessica Pixley, Darrell J. Talbert, Khyati Kapoor, Suresh Ambudkar, Michael M. Gottesman. Mild ER stress increases human ABC transporter expression and drug transport function in polarized cell monolayers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 989. doi:10.1158/1538-7445.AM2013-989

Abstract LB-222: Chemical and structural flexibility contribute to the broad substrate and modulator specificity of multidrug resistance-linked P-glycoprotein (ABCB1).

Abstract The human ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein, P-gp) mediates efflux of cytotoxic anticancer agents from cells, thereby conferring resistance to chemotherapeutic agents in most cancers. One of the most important features of ABC drug transporters, including P-gp, is their ability to recognize a variety of chemically dissimilar hydrophobic or amphipathic compounds. In this report, we elucidate the molecular basis of the promiscuous nature of this transporter. We modeled the structure of human P-gp using the X-ray structure of mouse P-gp as a template, with docked substrates (cyclosporine A, valinomycin and nilotinib) and modulators (tariquidar and 5′-Fluorosulfonylbenzonyl 5′-adenosine) in the pocket where the QZ59-peptide inhibitors bind mouse P-gp. Single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, M949, F978, V982 and A985 residues at the drug binding site with cys in a cys-less P-gp were expressed in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cys-less wild-type P-gp. Several drugs including cyclosporine A, valinomycin, nilotinib and tariquidar partially or completely lose the ability to inhibit the photolabeling of mutant P-gps with the transport substrate [125I]-Iodoarylazidoprazosin, indicating that these drugs cannot bind at their primary binding site. However, the drugs can modulate the ATP hydrolysis of the mutant P-gps, demonstrating that they bind at alternate sites. The transport of six fluorescent substrates in HeLa cells overexpressing triple mutants (Y307C/Q725C/V982 and Y307A/Q725A/Y953A) is also not significantly altered, showing that substrates bound at alternate sites are still transported. In aggregate, our results suggest that a large flexible pocket in the transmembrane domains in P-gp is able to bind chemically diverse compounds through a versatile allocation of hydrogen-bond donors and acceptors and hydrophobic residues. When residues of the primary drug-binding site are mutated, substrates and modulators bind to alternate sites on the transporter, revealing an exceptional chemical flexibility. We propose that both the chemical and structural flexibility provide the molecular basis for interaction of P-gp and possibly other ABC drug transporters with a wide range of substrates and modulators. Citation Format: Suresh V. Ambudkar, Eduardo E. Chufan, Khyati Kapoor, Stewart R. Durell, Hong-May Sim. Chemical and structural flexibility contribute to the broad substrate and modulator specificity of multidrug resistance-linked P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-222. doi:10.1158/1538-7445.AM2013-LB-222

Abstract 2193: Characterization of the aggresome targeted Q141K ABCG2/BCRP variant.

Abstract ABCG2 is a member of the G subfamily of human ABC transporters. It is an ATP-dependent protein and is a half-transporter that must dimerize for function. In various tumor cell lines, ABCG2 was involved in resistance to chemotherapy, by transporting numerous anticancer agents including tyrosine kinase inhibitors. Studies show the transporter has an important role in normal tissue protection, including the blood-brain and maternal-fetal barriers. It has also been observed that ABCG2 may play a protective role with respect to exposure to smoke or dietary carcinogens. A common variant in human ABCG2, the polymorphism Q141K, impairs expression and function, thereby reducing drug clearance and increasing the toxicity of chemotherapy and other substrate drugs. Although the activity of the Q141K variant is regularly described, biochemical data remain rare. Q141K has an impaired trafficking to cell surface compared to the WT protein and is sequestered in the aggresome. Here we characterized the Q141K and WT protein in human cells, studying post-translational modification, biochemistry, dimerization status, half-life, and endocytosis. Glycosylation and phosphorylation are equivalent, as is ATP binding and hydrolysis, and substrate binding using IAAP competition assays. Cell treatment with romidepsin, an HDAC inhibitor approved by FDA as an anticancer agent, restored the impaired variant trafficking, leading to an increase in drug efflux. The restoration of ABCG2 Q141K function has potential applications in oncology and in medicine, in improving normal tissue protection and drug elimination, as well as in cancer prevention since several carcinogens are substrates for ABCG2. Citation Format: Agnes Basseville, Suneet Shukla, Akina Tamaki, Robert Robey, Suresh V. Ambudkar, Susan E. Bates. Characterization of the aggresome targeted Q141K ABCG2/BCRP variant. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2193. doi:10.1158/1538-7445.AM2013-2193

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.

The Expression Profile of Atp-Binding Cassette Transporter Genes in Breast Carcinoma

ATP-binding cassette (ABC) transporters contribute to development of resistance to anticancer drugs via ATP-dependent drug efflux. A major goal of our study was to investigate associations between the expression of ABC transporters and outcome of breast carcinoma patients. Transcript levels of all 49 human ABC transporters were determined in post-treatment tumor and non-neoplastic tissue samples from 68 breast carcinoma patients treated by neoadjuvant chemotherapy. Six ABC transporters were then evaluated in independent series of 100 pretreatment patients. ABCA5/6/8/9/10, ABCB1/5/11, ABCC6/9, ABCD2/4, ABCG5 and ABCG8 were significantly downregulated and ABCA2/3/7/12, ABCB2/3/8/9/10, ABCC1/4/5/10/11/12, ABCD1/3, ABCE1, ABCF1/2/3 and ABCG1 were upregulated in post-treatment tumors compared with non-neoplastic tissues. Significant associations of intratumoral levels of ABCC1 and ABCC8 with grade and expression of hormonal receptors were found in both sets of patients. ABCA12, ABCA13 and ABCD2 levels were significantly associated with the response to neoadjuvant chemotherapy in post-treatment patients. Protein expression of ABCA12, ABCC8 and ABCD2 in tumor tissues of patients with breast carcinoma was observed by immunoblotting for the first time. ABCA12, ABCA13, ABCC1, ABCC8 and ABCD2 present potential modifiers of progression and response to the chemotherapy of breast carcinoma.

Expression levels of PEPT1 and ABCG2 play key roles in 5-aminolevulinic acid (ALA)-induced tumor-specific protoporphyrin IX (PpIX) accumulation in bladder cancer

A detection method widely used of late in cancer surgery is 5-aminolevulinic acid-based photodynamic diagnosis (ALA-PDD), which relies on the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX) after the administration of ALA. In this regard, we recently reported that peptide transporter PEPT1 and human ATP-binding cassette transporter ABCG2 are key players in regulating intracellular PpIX levels. In the present study, we re-evaluated in vivo the expression of genes involved in the porphyrin biosynthesis pathway. Using quantitative real-time (qRT)-PCR, we measured the mRNA levels in a clinical specimen of bladder cancer from a patient who had been subjected to ALA-PDD. We confirmed that PEPT1 and ABCG2 are major contributors to the regulation of tumor-specific PpIX accumulation. qRT-PCR analysis revealed a predominantly high level of PEPT1 mRNA and a very low level of ABCG2 mRNA in the bladder cancer, corresponding to the roles of these genes in vitro. These findings were further confirmed by immunohistochemical studies with PEPT1- and ABCG2-specific antibodies. The induction of PEPT1 gene and the suppression of ABCG2 gene expression are among the key molecular mechanisms underlying tumor-specific PpIX accumulation after the administration of ALA in bladder cancer.

Tethered proteoliposomes containing human ABC transporter MRP3: New perspectives for biosensor application based on transmembrane proteins

While transmembrane proteins and transporters comprise one of the largest protein families, their use in biosensors like biochips or lab-on-a-chip devices has so far been limited by their demanding requirements of a stable and compartmentalized lipid environment. A possible remedy lies in the tethering of proteoliposomes containing the reconstituted transmembrane protein to the biosensoric surface. As a proof of concept, we reconstituted the human ABC transporter MRP3 into biotinylated proteoliposomes and tethered those to a gold surface coated with streptavidin on a biotinylated self-assembled thiol monolayer. The tethering process was investigated by quartz crystal microbalance with dissipation monitoring. The final assembly of tethered proteoliposomes exhibited biological activity in terms of drug-stimulated ATP hydrolysis and substrate translocation. The presented facile immobilization approach can be easily extended to other transmembrane proteins as it does not require any modification of the protein and will open up transmembrane proteins for future application in biosensors.

ABCG2 is not able to catalyze glutathione efflux and does not contribute to GSH-dependent collateral sensitivity

ABCG2 is a key human ATP-binding cassette (ABC) transporter mediating cancer cell chemoresistance. In the case of ABCC1, another multidrug transporter, earlier findings documented that certain modulators greatly increase ABCC1-mediated glutathione (GSH) efflux and, upon depletion of intracellular GSH, induce “collateral sensitivity” leading to the apoptosis of multidrug resistant cells. Recently, it has been suggested that ABCG2 may mediate an active GSH transport. In order to explore if ABCG2-overexpressing cells may be similarly targeted, we first looked for the effects of ABCG2 expression on cellular GSH levels, and for an ABCG2-dependent GSH transport in HEK293 and MCF7 cells. We found that, while ABCG2 overexpression altered intracellular GSH levels in these transfected or drug-selected cells, ABCG2 inhibitors or transport modulators did not influence GSH efflux. We then performed direct measurements of drug-stimulated ATPase activity and 3H-GSH transport in inside-out membrane vesicles of human ABC transporter-overexpressing Sf9 insect cells. Our results indicate that ABCG2-ATPase is not modulated by GSH and, in contrast to ABCC1, ABCG2 does not catalyze any significant GSH transport. Our data suggest no direct interaction between the ABCG2 transporter and GSH, although a long-term modulation of cellular GSH by ABCG2 cannot be excluded.

Molecular Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Cystic fibrosis is a monogenic genetic disease due to mutations in the gene coding for a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR controls the flow of chloride ions through the apical membrane of epithelia, thus regulating the transepithelial movement of both water and ions, needed for the production of healthy secretions. Mutations in the CFTR gene affect the proper folding, trafficking and function of CFTR, resulting in the formation of thick and viscous mucus that accumulates in different organs, notably in the lungs where it predisposes to persistent bacterial infections.CFTR belongs to the ATP binding cassette (ABC) transporter superfamily. Human ABC transporters share a common structural architecture, which minimally consists of two transmembrane domains (TMDs), forming in CFTR the pore for chloride flow, and two nucleotide binding domains (NBDs), for ATP binding and hydrolysis.The lack of crystal structures hampers a global understanding of the structure and function of CFTR, and thus the development of approaches directly targeting defective CFTR. Here, we present molecular models of CFTR in different conformational states, built on available structural data. We focus in particular on closed state conformations, and on the interactions at the NBD interface. The models are used, together with available experimental data, to infer the roles played by specific residues in the gating transitions, allowing hypothesis testing through mutagenesis and functional studies.

Pharmacogenetics of human ABC transporter ABCC11: new insights into apocrine gland growth and metabolite secretion.

Cell secretion is an important physiological process that ensures smooth metabolic activities and tissue repair as well as growth and immunological functions in the body. Apocrine secretion occurs when the secretory process is accomplished with a partial loss of cell cytoplasm. The secretory materials are contained within secretory vesicles and are released during secretion as cytoplasmic fragments into the glandular lumen or interstitial space. The recent finding that the non-synonymous single nucleotide polymorphisms (SNP) 538G > A (rs17822931; Gly180Arg) in the ABCC11 gene determines the type of earwax in humans has shed light on the novel function of this ABC (ATP-binding cassette) transporter in apocrine glands. The wild-type (Gly180) of ABCC11 is associated with wet-type earwax, axillary osmidrosis, and colostrum secretion from the mammary gland as well as the potential risk of mastopathy. Furthermore, the SNP (538G > A) in the ABCC11 gene is suggested to be a clinical biomarker for the prediction of chemotherapeutic efficacy. The aim of this review article is to provide an overview on the discovery and characterization of genetic polymorphisms in the human ABCC11 gene and to explain the impact of ABCC11 538G > A on the apocrine phenotype as well as the anthropological aspect of this SNP in the ABCC11 gene and patients’ response to nucleoside-based chemotherapy.

Functional polymorphisms of xenobiotics metabolizing enzymes—a research topic

The human genome harbors an impressive number of genes encoding enzymes that primarily metabolize or transport drugs or other xenobiotics (XMEs). Genetic and functional variation in these genes is tremendous and has complex consequences, depending, for example, on whether enzyme structure or expression is affected, or whether the produced metabolite is pharmacologically or toxicologically active or not. Despite numerous impressive examples of the impact of genetic variation on pharmacokinetics and drug response, today's knowledge is incomplete regarding most XME genes and fragmentary even for many well-investigated XMEs. This is one of the reasons why clinical pharmacogenetic studies are often controversial and clinical application in personalized medicine is presently limited. Advanced technology and ongoing large-scale projects are rapidly uncovering the existing genetic variation in all populations on earth, ultimately enabling the personal genome in the very near future. A wealth of mostly rare novel variants is awaiting functional characterization either by high-throughput expression/phenotyping techniques or by prediction using improved algorithms to estimate functional relevance. With this Research Topic we would like to give an up-to-date overview about the current knowledge in this field by covering both, known hard facts as well as cutting-edge advancement in novel genetic and genomic variation of XMEs and their functional consequences. Five major subtopics which include 20 research or review papers are included in this E-book. These are the following: History and current knowledge of XMEs Clinical application of CYP2C19 pharmacogenetics toward more personalized medicine (Lee, 2013, review). Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance (Zanger and Klein, 2013, review). Pharmacogenetics of human ABC transporter ABCC11: new insights into apocrine gland growth and metabolite secretion (Ishikawa et al., 2013, review). Pharmacogenomics of cytochrome P450 3A4: recent progress toward the “missing heritability” problem (Klein and Zanger, 2013, review). Clinical implications of XME gene variants ABCB1 4036A>G and 1236C>T polymorphisms affect plasma efavirenz levels in South African HIV/AIDS patients (Swart et al., 2012, research article). Genetic variations in drug-induced liver injury (DILI): resolving the puzzle (Stephens et al., 2012, opinion). MDMA, methamphetamine, and CYP2D6 pharmacogenetics: what is clinically relevant? (de la Torre et al., 2012, review). Molecular interactions between NAFLD and xenobiotic metabolism (Naik et al., 2013, review). Toward a clinical practice guide in pharmacogenomics testing for functional polymorphisms of drug-metabolizing enzymes. Gene/drug pairs and barriers perceived in Spain (Agundez et al., 2012, perspective). Inter/intraethnic variability of XME gene variants Characterization of the genetic variation present in CYP3A4 in three South African populations (Drogemoller et al., 2013, research article). Frequencies of 23 functionally significant variant alleles related with metabolism of antineoplastic drugs in the Chilean population: comparison with Caucasian and Asian populations (Roco et al., 2012, research article). Pharmacogenomic diversity among Brazilians: influence of ancestry, self-reported color, and geographical origin (Suarez-Kurtz et al., 2012, review). Regulation of XME gene expression Impact of the interaction between 3′-UTR SNPs and microRNA on the expression of human xenobiotic metabolism enzyme and transporter genes (Wei et al., 2012, research article). Molecular mechanisms of genetic variation and transcriptional regulation of CYP2C19 (Helsby and Burns, 2012, review). Pharmacogenetics in cancer therapy Impact of genetic polymorphisms on chemotherapy toxicity in childhood acute lymphoblastic leukemia (Gervasini and Vagace, 2012, review). Multilocus genotypes of relevance for drug metabolizing enzymes and therapy with thiopurines in patients with acute lymphoblastic leukemia (Stocco et al., 2013, review). Functional polymorphisms in xenobiotic metabolizing enzymes and their impact on the therapy of breast cancer (Vianna-Jorge et al., 2013, review). High-resolution melting analysis of the common c.1905+1G>A mutation causing dihydropyrimidine dehydrogenase deficiency and lethal 5-fluorouracil toxicity (Borras et al., 2013, research article). Polymorphisms of phase I and phase II enzymes and breast cancer risk (Justenhoven, 2012, review). Analysis of the functional polymorphism in the cytochrome P450 CYP2C8 gene rs11572080 with regard to colorectal cancer risk (Ladero et al., 2012, research article).

Michaelis–Menten kinetic analysis of drugs of abuse to estimate their affinity to human P-glycoprotein

The pharmacokinetics of various important drugs are known to be significantly influenced by the human ABC transporter P-glycoprotein (P-gp), which may lead to clinically relevant drug–drug interactions. In contrast to therapeutic drugs, emerging drugs of abuse (DOA) are sold and consumed without any safety pharmacology testing. Only some studies on their metabolism were published, but none about their affinity to the transporter systems. Therefore, 47 DOAs from various classes were tested for their P-gp affinity using human P-gp (hP-gp) to predict possible drug–drug interactions. DOAs were initially screened for general hP-gp affinity and further characterized by modeling classic Michaelis–Menten kinetics and assessing their Km and Vmax values. Among the tested drugs, 12 showed a stimulation of ATPase activity. The most intensive stimulating DOAs were further investigated and compared with the known P-gp model substrates sertraline and verapamil. ATPase stimulation kinetics could be modeled for the entactogen 3,4-methylenedioxy-α-ethylphenethylamine (3,4-BDB), the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), the abused alkaloid glaucine, the opioid-like drugs N-iso-propyl-1,2-diphenylethylamine (NPDPA), and N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA), with Km and Vmax values within the same range as for verapamil or sertraline. As a consequence interactions with other drugs being P-gp substrates might be considered to be very likely and further studies should be encouraged.

The Potential ofSutherlandia frutescensfor Herb-Drug Interaction

In Africa, Sutherlandia frutescens is a popular medicinal herb widely consumed by people living with human immunodeficiency virus/AIDS. Concomitant use with antiretroviral drugs has generated concerns of herb-drug interaction (HDI). This study investigated the inhibitory effects of the crude extracts of S. frutescens on the major cytochrome P450 isozymes with the use of pooled human liver microsomes. Its effect on the metabolic clearance of midazolam using cryopreserved hepatocytes was also monitored. The potential of S. frutescens to inhibit human ATP-binding cassette transporters (P-gp and BCRP) and the human organic anion transporting polypeptide (OATP1B1 and OATP1B3) activity was assessed using cell lines overexpressing the transporter proteins. S. frutescens showed inhibitory potency for CYP1A2 (IC(50) = 41.0 µg/ml), CYP2A6 (IC(50) = 160 µg/ml), CYP2B6 (IC(50) = 20.0 µg/ml), CYP2C8 (IC(50) = 22.4 µg/ml), CYP2C9 (IC(50) = 23.0 µg/ml), CYP2C19 (IC(50) = 35.9 µg/ml), and CYP3A4/5 (IC(50) = 17.5 µg/ml [with midazolam1'-hydroxylation]; IC(50) = 28.3 µg/ml [with testosterone 6β-hydroxylation]). Time-dependent (irreversible) inhibition by S. frutescens was observed for CYP3A4/5 (K(I) = 296 µg/ml, k(inact) = 0.063 min(-1)) under the conditions of this study. S. frutescens also delays the production of midazolam metabolites in the hepatocytes, decreasing its clearance by 40%. Furthermore, S. frutescens inhibited P-gp (IC(50) = 324.8 µg/ml), OATP1B1 (IC(50) = 10.4 µg/ml), and OATP1B3 (IC(50) = 6.6 µg/ml). The result indicates the potential for HDI between S. frutescens and the substrates of the affected enzymes, if sufficient in vivo concentration of the extract is attained.

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.

Substrate translocation and stimulated ATP hydrolysis of human ABC transporter MRP3 show positive cooperativity and are half-coupled

ABC transporters are involved in countless processes from lipid excretion over cellular detoxification to multidrug resistance of cancer cells. The latter is especially conferred by the ABCC subfamily also called multidrug resistance-associated proteins (MRPs) that excrete a variety of amphipathics including anticancer drugs by ATP-dependent transport. As the mechanisms of substrate translocation and ATP hydrolysis are still unclear for MRPs, we investigated the kinetics of both processes with focus on cooperativity and coupling between ATPase activity and substrate transport using purified MRP3 in proteoliposomes. Although the ATP-dependent uptake of amphipathics and the hydrophilic 5(6)-carboxy-2′-7′-dichlorofluorescein (CDCF) into the lumen of proteoliposomes showed affinity constants similar to those reported for cell-based assays, the maximal uptake rates were up to 250 times higher. Moreover, all substrates showed cooperative interactions of two subunits. Upon stimulation with amphipathics, ATPase activity of MRP3 increased from 80nmol/(mgmin) to 180nmol/(mgmin) showing positive cooperativity with a Hill coefficient of 2. While Hill coefficient and maximal ATPase activity were found to be substrate independent, the affinity constants are characteristic for a given substrate and correspond to the value for transport. Therefore, cooperative interactions of the two nucleotide binding domains (NBDs) in MRP3 are mediated by substrate binding to the transmembrane domains (TMDs). In contrast to amphipathic substrates, CDCF did not stimulate ATPase activity despite being transported in an ATP-dependent manner. This indicates that ATP hydrolysis and substrate translocation are half-coupled in MRP3 as CDCF shuttles on a basal TMD activity resulting from the basal ATPase activity.

ABCB5 expression and cancer stem cell hypothesis in oral squamous cell carcinoma

IntroductionThe vast majority of oral cancers are squamous cell carcinomas (OSCC). The effectiveness of adjuvant cytostatic chemotherapy for OSCC is frequently restricted due to an inducible cellular mechanism called multidrug resistance (MDR) and a putative cancer stem cell (CSC) compartment in human carcinogenesis expressing multidrug efflux pumps. The novel human ATP-binding cassette (ABC) transporter ABCB5 [subfamily B (MDR/TAP) member 5] acts as an energy-dependent drug efflux transporter and marks tumour cells of a putative CSC compartment. However, to date, there is no link between ABCB5 expression and OSCC. Materials and methodsExpression of ABCB5 was analysed in OSCC specimen (n=191) and cancer cell lines (BICR3, BICR56) by immunohistochemistry, real-time polymerase chain reaction (RT-PCR) analysis and western blotting. Scanned images were digitally analysed using ImageJ and the immunomembrane plug-in. ABCB5 expression on protein level was correlated with clinical characteristics and impact on survival. ABCB5 was co-labelled with CD44 in immunohistochemical and immunofluorescence double labelling experiments. Expression subgroups were identified by receiver operating characteristics (ROC) analysis. ResultsHigh ABCB5 expression was significantly associated with tumour progression and recurrence of the tumour. Multivariate analysis demonstrated high ABCB5 expression as an independent prognostic factor (p=0.0004). Immunohistochemical and immunofluorescence double labelling experiments revealed ABCB5 expression by CD44+ cancer cells. ABCB5 specificity was confirmed by western blot and RT-PCR analysis. ConclusionsFor the first time, this study provides evidence that ABCB5 expression in OSCC might be associated with tumour formation, metastasis and a putative CSC compartment. One of the principal mechanisms for protecting putative cancer stem cells is through the expression of multifunctional efflux transporters from the ABC gene family, like ABCB5. This provides one mechanism in which putative cancer stem cells could survive and may lead to tumour relapse. Knowledge of expression profiles of ABC transporters and other genes involved in MDR will likely help therapeutic optimisation for cancer patients in clinic. However, this hypothesis requires further in vitro and in vivo studies.

The histidin-loop is essential for transport activity of human MDR3. A novel mutation of MDR3 in a patient with progressive familial intrahepatic cholestasis type 3

Experimental evidence has been provided that a histidine-loop within the nucleotide binding domain of ABC transporter is essential for efficient function of this class of transporter proteins. Here we report the first patient with a mutation of the putative histidine‐loop of a human ABC transporter, the multi drug resistance protein 3 (MDR3). The patient presented at the age of 4years with a history of severe pruritus, elevated serum gamma-glutamyltransferase and bile acid levels since several years suggesting the diagnosis of progressive familial intrahepatic cholestasis type 3 (PFIC-3) due to defects in MDR3. Liver biopsy demonstrated an apparently normal MDR3 expression, however, genetic analysis revealed a novel homozygous mutation in the ABCB4 gene (c.3691C>T) in the patient. This mutation was associated with a change of histidine to tyrosine at amino acid position 1231 of MDR3 (p.H1231Y). As shown by sequence alignment, this amino acid corresponds to the highly conserved histidine of the “H-loop”, which is critical for ATP-hydrolysis, suggesting an essential role of histidine 1231 of human MDR3.

Expression levels of multidrug resistance-associated protein 4 (MRP4) in human leukemia and lymphoma cell lines, and the inhibitory effects of the MRP-specific inhibitor MK-571 on methotrexate distribution in rats

In the development of anti-blood cancer drugs, the chronic myelocytic leukemia (KU812), acute myelocytic leukemia (KG-1) and lymphoma (U937) cell lines are commonly used in preclinical pharmacology studies as human cancer xenograft models in mice. In the present study, mRNA expression levels of typical human ATP-binding cassette (ABC) transporters in these human blood cancer cell lines were analyzed by real-time polymerase chain reaction (RT-PCR). Based on the results, the expression level of multidrug resistance-associated protein 4 (MRP4) was found to be extremely high in KU812 cells compared with those of other transporters. Additionally, MRP4 expression levels were found to be relatively high in U937, KG-1 and a blood cell line derived from a healthy subject (RPMI 1788). In addition, to elucidate the contribution of MRP4 to the methotrexate (MTX) distribution in normal blood cells and tissues, [3H]MTX was intravenously (i.v.) administered to two groups of rats. Animals in one group received [3H]MTX only; the other group was concomitantly administered i.v. MK-571, a typical inhibitor of MRP transporters. No marked difference was observed between the two groups; the Kp values (tissue concentration/plasma concentration) of the concomitant group showed slightly higher values compared with those of the MTX alone group in erythrocytes (1.4 times, P<0.001), spleen (1.3 times, P<0.05) and thymus (1.2 times, P<0.05), respectively. Although in the present study we could not evaluate the direct involvement of MRP4 in blood cancer cells in which MRP4 expression was excessively high, these results suggest a possible functional role of MRP4 in blood cancer cells and albeit only slightly in normal blood cells/tissues.