ATP-binding Cassette Transporter Family Research Articles

Downregulation of ABCG2 protein inhibits migration and invasion in U251 glioma stem cells.

ABCG2 is a member of the ATP-binding cassette transporter family, which has been detected in a wide variety of human aggressive tumors, including glioma stem cells (GSCs), glioma tissues of higher grades, and implanted glioma xenografts. Previous research has implied that ABCG2 might be associated closely with invasion and spread in tumors. However, the specific roles and mechanisms of ABCG2 in regulating the migration and invasion of GSCs remain unclear. In this study, we aimed to identify the effects and mechanisms of ABCG2 on invasion by GSCs. Our results showed that downregulation of ABCG2 protein significantly inhibited the migration and invasion potentials of U251 GSCs. Further research on the underlying mechanism showed that the effects of ABCG2 downregulation on inhibiting the migration and invasion of U251 GSCs were through significantly decreasing the activity of matrix metalloproteinase-9, but not the expression of matrix metalloproteinase-9 protein. These findings show that ABCG2 plays an important role in regulating the migration and invasion of GSC, and represents a potential novel therapeutic agent to target the progression of GSCs.

Regulations of P-Glycoprotein/ABCB1/MDR1in Human Cancer Cells

Multidrug resistance (MDR) in cancer cells is a phenotype whereby cells display reduced sensitivity to anticancer drugs, based on a variety of mechanisms, including an increase in drug efflux, the reduction of drug uptake, the activation of cell growth and survival signaling, the promotion of DNA repair, and the inhibition of apoptosis signaling. Increased expression of the plasma membrane drug efflux pumps, the ATP-binding cassette (ABC) transporters, is involved in MDR. P-Glycoprotein/ABCB1 is a member of the ABC transporter family, and facilitates the efflux of various anticancer drugs, including anthracyclines,vincaalkaloids, epipodophyllotoxins, taxanes, and kinase inhibitors, from cells. P-Glycoprotein is also expressed in normal tissues and cells, including the kidney, liver, colon, and adrenal gland, to transport and/or secrete substrates and at the blood-brain, blood-placenta, and blood-testis barriers to protect these tissues from toxic substances. To understand the mechanistic functions of P-glycoprotein and to overcome MDR, investigators have identified the substrates and competitive inhibitors of P-glycoprotein. Recently, we and other groups reported associations between cellular signaling pathways and the expression, stability, degradation, localization, and activity of P-glycoprotein. The present review summarizes the currently available information about the transcriptional and posttranslational regulation of P-glycoprotein expression and function.

PD173074, a selective FGFR inhibitor, reverses MRP7 (ABCC10)-mediated MDR

Multidrug resistance protein 7 (MRP7, ABCC10) is a recently identified member of the ATP-binding cassette (ABC) transporter family, which adequately confers resistance to a diverse group of antineoplastic agents, including taxanes, vinca alkaloids and nucleoside analogs among others. Clinical studies indicate an increased MRP7 expression in non-small cell lung carcinomas (NSCLC) compared to a normal healthy lung tissue. Recent studies revealed increased paclitaxel sensitivity in the Mrp7−/− mouse model compared to their wild-type counterparts. This demonstrates that MRP7 is a key contributor in developing drug resistance. Recently our group reported that PD173074, a specific fibroblast growth factor receptor (FGFR) inhibitor, could significantly reverse P-glycoprotein-mediated MDR. However, whether PD173074 can interact with and inhibit other MRP members is unknown. In the present study, we investigated the ability of PD173074 to reverse MRP7-mediated MDR. We found that PD173074, at non-toxic concentration, could significantly increase the cellular sensitivity to MRP7 substrates. Mechanistic studies indicated that PD173074 (1μmol/L) significantly increased the intracellular accumulation and in-turn decreased the efflux of paclitaxel by inhibiting the transport activity without altering expression levels of the MRP7 protein, thereby representing a promising therapeutic agent in the clinical treatment of chemoresistant cancer patients.

MicroRNAs and their relevance to ABC transporters

MicroRNAs (miRNAs) are small noncoding RNAs, which regulate the expression of their target genes post-transcriptionally by RNA interference. They are involved in almost all cellular processes, including proliferation, differentiation, apoptosis, cell survival and the maintenance of tissue specificity. Recent findings also suggest that efflux pumps of the ABC (ATP-binding cassette) transporter family are subject to miRNA-mediated gene regulation. Moreover, it seems that ABC transporters are embedded in a concerted and miRNA-guided network of concurrently regulated proteins that mediate altered drug transport and cell survival in changing environmental conditions. In this review, we summarize recent findings of miRNAs interacting with ABC transporters, which have been connected with drug distribution as well as with drug resistance. Additionally, we specify findings of complex miRNA-protein pathways conferring increased drug export and cell survival.

Intestinal drug transport: ex vivo evaluation of the interactions between ABC transporters and anthelmintic molecules

The family of ATP-binding cassette (ABC) transporters is composed of several transmembrane proteins that are involved in the efflux of a large number of drugs including ivermectin, a macrocyclic lactone (ML) endectocide, widely used in human and livestock antiparasitic therapy. The aim of the work reported here was to assess the interaction between three different anthelmintic drugs with substrates of the P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP). The ability of ivermectin (IVM), moxidectin (MOX) and closantel (CST) to modulate the intestinal transport of both rhodamine 123 (Rho 123), a P-gp substrate, and danofloxacin (DFX), a BCRP substrate, across rat ileum was studied by performing the Ussing chamber technique. Compared to the controls, Rho 123 efflux was significantly reduced by IVM (69%), CST (51%) and the positive control PSC833 (65%), whereas no significant differences were observed in the presence of MOX (30%). In addition, DFX efflux was reduced between 59% and 72% by all the assayed drug molecules, showing a higher potency than that observed in the presence of the specific BCRP inhibitor pantoprazole (PTZ) (52%). An ex vivo intestinal transport approach based on the diffusion chambers technique may offer a complementary tool to study potential drug interactions with efflux transporters such as P-gp and BCRP.

7-Ketocholesteryl-9-carboxynonanoate enhances ATP binding cassette transporter A1 expression mediated by PPARγ in THP-1 macrophages

BackgroundATP binding cassette transporter A1 (ABCA1) is a member of the ATP-binding cassette transporter family. It plays an essential role in mediating the efflux of excess cholesterol. It is known that peroxisome proliferator-activated receptor gamma (PPARγ) promoted ABCA1 expression. We previously found 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) upregulated ABCA1 partially through CD36 mediated signals. In the present study, we intended to test if PPARγ signally is involved in the upregulation mediated by oxLig-1. Methods and resultsFirst, we docked oxLig-1 and the ligand-binding domain (LBD) of PPARγ by using AutoDock 3.05 and subsequently confirmed the binding by ELISA assay. Western blotting analyses showed that oxLig-1 induces liver X receptor alpha (LXRα), PPARγ and consequently ABCA1 expression. Furthermore, oxLig-1 significantly enhanced ApoA-I-mediated cholesterol efflux. Pretreatment with an inhibitor for PPARγ (GW9662) or/and LXRα (GGPP) attenuated oxLig-1-induced ABCA1 expression. Under PPARγ knockdown by using PPARγ–shRNA, oxLig-1-induced ABCA1 expression and cholesterol efflux in THP-1 macrophages was blocked by 62% and 25% respectively. ConclusionsThese observations suggest that oxLig-1 is a novel PPARγ agonist, promoting ApoA-I-mediated cholesterol efflux from THP-1 macrophages by increasing ABCA1 expression via induction of PPARγ.

An Experimental Approach To Evaluate the Impact of Impaired Transport Function on Hepatobiliary Drug Disposition Using Mrp2-Deficient TR– Rat Sandwich-Cultured Hepatocytes in Combination with Bcrp Knockdown

Breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) are members of the ATP binding cassette (ABC) transporter family located in the canalicular membrane of hepatocytes that mediate biliary excretion of many drugs and endogenous compounds. BCRP and MRP2 have overlapping substrate profiles. Predicting drug disposition in the setting of altered transport function has important clinical significance. This investigation was designed to establish an in vitro model system to evaluate the impact of impaired Mrp2 and Bcrp function on hepatobiliary drug disposition. To achieve Bcrp knockdown by RNA interference (RNAi), sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR–) and wild-type (WT) rats were infected with adenoviral vectors to express shRNA targeting Bcrp (Ad-siBcrp) at multiplicity of infection (MOI) of 1–10. MOI of 5 was identified as optimal. At MOI of 5, viral infection as well as WT or TR– status was statistically significant predictors of the rosuvastatin (RSV) biliary excretion index (BEI), consistent with the known role of Bcrp and Mrp2 in the biliary excretion of RSV in vivo in rats. Relative to WT rat SCH, marginal mean BEI (%) of RSV in TR– rat SCH decreased by 28.6 (95% CI: 5.8–51.3). Ad-siBcrp decreased marginal mean BEI (%) of RSV by 13.3 (7.5–9.1) relative to SCH infected with adenoviral vectors expressing a nontargeting shRNA (Ad-siNT). The BEI of RSV was almost ablated in TR– rat SCH with Bcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT rat SCH (45.4 ± 6.6%). These results demonstrated the feasibility of Bcrp knockdown in TR– rat SCH as an in vitro system to assess the impact of impaired Bcrp and Mrp2 function. At MOI of 5, viral infection had minimal effects on RSV total accumulation, but significantly decreased marginal mean taurocholate total accumulation (pmol/mg of protein) and BEI (%) by 9.9 (7.0–12.8) and 7.5 (3.7–11.3), respectively, relative to noninfected SCH. These findings may be due to off-target effects on hepatic bile acid transporters, even though no changes in protein expression levels of the hepatic bile acid transporters were observed. This study established a strategy for optimization of the knockdown system, and demonstrated the potential use of RNAi in SCH as an in vitro tool to predict altered hepatobiliary drug disposition when canalicular transporters are impaired.

Identification of a Novel Haemophilus parasuis-Specific B Cell Epitope Using Monoclonal Antibody against the OppA Protein

Monoclonal antibody (MAb) 1B3 against Haemophilus parasuis (H. parasuis) was generated by fusing SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with the whole-bacterial-cell suspension of H. parasuis HS80 (serotype 5). The MAb 1B3 showed strong reactivity with 15 serotype reference strains of H. parasuis using Dot blot and Western blot analysis. Immunoprecipitation and protein spectral analysis indicated that MAb 1B3 recognized by Oligopeptide permease A (OppA) belongs to the ATP binding cassette transporter family. In addition, a linear B-cell epitope recognized by MAb 1B3 was identified by the screening of a phage-displayed 12-mer random peptide library. Sequence analysis showed that MAb 1B3 was recognized by phages-displaying peptides with the consensus motif KTPSEXR (X means variable amino acids). Its amino acid sequence matched 469KTPAEAR475 of H. parasuis OppA protein. A series of progressively truncated peptides were synthesized to define the minimal region that was required for MAb 1B3 binding. The epitope was highly conserved in OppA protein sequences from the isolated H. parasuis strains, which was confirmed by alignment analysis. Furthermore, the minimal linear epitope was highly specific among 75 different bacterial strains as shown in sequence alignments. These results indicated MAb 1B3 might be potentially used to develop serological diagnostic tools for H. parasuis.

Enhancing the ATPase Activity of the Cystic Fibrosis Transmembrane Conductance Regulator CFTR in Live Cells Reduces Chloride Flux

Maintaining the fluid balance in tissues such as the lung airways, the intestine, and many exocrine glands is of vital importance. This task is fulfilled essentially by the cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), a channel for passive diffusion of chloride and bicarbonate. Deregulation of CFTR causes multiple disorders, the most prominent of which is cystic fibrosis (CF). CFTR is also implicated in autosomal dominant polycystic kidney disease and secretory diarrheas. Owing to the increasing number of diseases recognized to be associated with CFTR dysfunction, the interest in CFTR potentiators and inhibitors has grown. However, their function at the molecular level is not yet understood. On the one hand, this is due to the complex function of CFTR which, as a member of the ATP binding cassette (ABC) transporter family, is the only known ion channel that links ATP binding and hydrolysis to channel gating; on the other hand, it is due to fact that chloride efflux is generally measured in cells, whereas ATP hydrolysis was so far measured in reconstituted proteoliposomes exhibiting significant functional attrition. Here we take a new approach to answer the intriguing question as to whether chloride channel inhibitors and potentiators enhance or reduce ATP hydrolysis by CFTR. For this purpose we measured ATPase activity and chloride efflux, both, in live cells, under similar experimental conditions. ATPase activity was measured by microphsiometry using silicon chip technology and anion flux was measured using an iodide electrode. Most importantly we demonstrate that chloride flux inhibitors enhance ATPase activity of CFTR, whereas chloride channel potentiators do not stimulate ATPase activity, but tend to reduce cell metabolism. These findings have implications for the synthesis of new potentiators and inhibitors.

Small and Innovative Molecules as New Strategy to Revert MDR

Multidrug resistance (MDR) is a complex phenomenon principally due to the overexpression of some transmembrane proteins belonging to the ATP binding cassette (ABC) transporter family. Among these transporters, P-glycoprotein (P-gp) is mostly involved in MDR and its overexpression is the major cause of cancer therapy failure. The classical approach used to overcome MDR is the co-administration of a P-gp inhibitor and the classic antineoplastic drugs, although the results were often unsatisfactory. Different classes of P-gp ligands have been developed and, among them, Tariquidar has been extensively studied both in vitro and in vivo. Although Tariquidar has been considered for several years as the lead compound for the development of P-gp inhibitors, recent studies demonstrated it to be a substrate and inhibitor, in a dose-dependent manner. Moreover, Tariquidar structure–activity relationship studies were difficult to carry out because of the complexity of the structure that does not allow establishing the role of each moiety for P-gp activity. For this purpose, SMALL molecules bearing different scaffolds such as tetralin, biphenyl, arylthiazole, furoxane, furazan have been developed. Many of these ligands have been tested both in in vitro assays and in in vivo PET studies. These preliminary evaluations lead to obtain a library of P-gp interacting agents useful to conjugate chemotherapeutic agents displaying reduced pharmacological activity and appropriate small molecules. These molecules could get over the limits due to the antineoplastic-P-gp inhibitor co-administration since pharmacokinetic and pharmacodynamic profiles are related to a dual innovative drug.

Sphingosine-1-phosphate transport and its role in immunology

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite with many important functions in cellular and systemic physiology, including the immune system. As it cannot traverse the membrane, it is exported from cells by transporters. Several members of the ATP-binding cassette (ABC) transporter family, ABCA1, ABCC1, ABCG2 and potentially ABCA7 have been identified as S1P transporters. In addition spinster 2 (SPNS2), a protein from the major facilitator superfamily (MFS), was identified as a S1P transporter. Here we review the current knowledge on S1P transport and discuss how this process creates S1P gradients in the body that are important in various functions of the immune system.

Functional Assay for Characterizing Human P-Glycoprotein Transport using the Pore Forming Peptide Gramicidin A

P-glycoprotein (P-gp), a member of the ATP-Binding Cassette (ABC) transporter family, actively transports chemotherapeutics out of cancer cells, leading to reduced efficacy of these drugs. Thus the development of functional assays for better characterization of P-gp is important in finding ways to combat multidrug resistance (MDR) in cancer treatment. We establish a novel approach that takes advantage of the single molecule sensitivity of the patch clamp technique and provides access to the intracellular environment as well as direct control of the cellular transmembrane potential. Briefly, the assay measures whole cell current in the presence of ion channel forming peptide gramicidin A (gA). Since gA is a substrate of P-gp, changes in gA current report the efflux of gA by P-gp. For instance, gA current in chemosensitive parental cells that did not express P-gp, increased with time in a dose-dependent manner. In contrast, P-gp expressing resistant cells exhibited significantly reduced gA current due to its efflux by P-gp. Consistently, P-gp inhibitors restored gA current in P-gp expressing cells to a level comparable to that in parental cells. HeLa cells transduced with non-functional mutant (E556Q/E1201Q) P-gp displayed large gA currents comparable to control HeLa cells. This can be attributed to the loss of transport activity due to defective ATP hydrolysis by this mutant P-gp. Additionally fitting the gA currents data to a model that quantifies the kinetic constants showed that P-gp efflux activity was correlated with the intracellular ATP concentration. Moreover, to our surprise, membrane depolarization enhanced P-gp activity whereas hyperpolarization reduced its activity. Collectively our findings demonstrate that whole cell gA current can be used to monitor P-gp function.

Probing Drug-Binding Pathways in P-Glycoprotein with Ensemble Docking

P-glycoprotein (P-gp) is one of the key members of the ATP-binding cassette (ABC) transporters family and the major contributor of multidrug resistance against a broad spectrum of drugs in cancer cells. Powered by ATP, P-gp acts as an efflux pump to export a diverse set of hydrophobic molecules, including drugs, out of the cell. These hydrophobic substrates are assumed to enter the translocation pathway of P-gp either via the intracellular side of the cell or directly from the cytoplasmic leaflet of the membrane. In order to characterize the mechanism, residues facilitating drug entry, and translocation, we have taken an approach combining the strengths of molecular dynamics (MD) and molecular docking. A 100 ns MD simulation of membrane-bound inward facing P-gp with Mg2+ and ATP was performed and subsequently used in ensemble docking. Doxorubicin, one of the drug substrates of P-gp, was docked into the defined active sites of 10,000 different snapshots representing the conformational flexibility of the protein in the membrane. Clustered docked poses identified the binding sites and yielded a picture of the sequence of binding events of the substrate within the translocation pathway. Interestingly, a few of the highly populated clusters were located within the region that interacts with the cytoplasmic leaflet of the membrane supporting the recruitment of the substrate laterally and directly from the membrane. Further MD simulations and energetic calculations of representative substrate poses are then used to characterize their real-time binding behavior and dynamics within the intracellular lumen and at the cytoplasmic leaflet of the membrane. This study, for the first time, explored and utilized the dynamic ensemble of conformations of P-gp in molecular docking and dissected important and complex features of its translocation pathway.

Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.

ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus

The Madagascar periwinkle (Catharanthus roseus) is highly specialized for the biosynthesis of many different monoterpenoid indole alkaloids (MIAs), many of which have powerful biological activities. Such MIAs include the commercially important chemotherapy drugs vinblastine, vincristine, and other synthetic derivatives that are derived from the coupling of catharanthine and vindoline. However, previous studies have shown that biosynthesis of these MIAs involves extensive movement of metabolites between specialized internal leaf cells and the leaf epidermis that require the involvement of unknown secretory processes for mobilizing catharanthine to the leaf surface and vindoline to internal leaf cells. Spatial separation of vindoline and catharanthine provides a clear explanation for the low levels of dimers that accumulate in intact plants. The present work describes the molecular cloning and functional identification of a unique catharanthine transporter (CrTPT2) that is expressed predominantly in the epidermis of young leaves. CrTPT2 gene expression is activated by treatment with catharanthine, and its in planta silencing redistributes catharanthine to increase the levels of catharanthine-vindoline drug dimers in the leaves. Phylogenetic analysis shows that CrTPT2 is closely related to a key transporter involved in cuticle assembly in plants and that may be unique to MIA-producing plant species, where it mediates secretion of alkaloids to the plant surface.

Inhibition of ABCB1 Expression Overcomes Acquired Docetaxel Resistance in Prostate Cancer

Docetaxel is the first-line standard treatment for castration-resistant prostate cancer. However, relapse eventually occurs due to the development of resistance to docetaxel. To unravel the mechanism of acquired docetaxel resistance, we established docetaxel-resistant prostate cancer cells, TaxR, from castration-resistant C4-2B prostate cancer cells. The IC50 for docetaxel in TaxR cells was about 70-fold higher than parental C4-2B cells. Global gene expression analysis revealed alteration of expression of a total of 1,604 genes, with 52% being upregulated and 48% downregulated. ABCB1, which belongs to the ATP-binding cassette (ABC) transporter family, was identified among the top upregulated genes in TaxR cells. The role of ABCB1 in the development of docetaxel resistance was examined. Knockdown of ABCB1 expression by its specific shRNA or inhibitor resensitized docetaxel-resistant TaxR cells to docetaxel treatment by enhancing apoptotic cell death. Furthermore, we identified that apigenin, a natural product of the flavone family, inhibits ABCB1 expression and resensitizes docetaxel-resistant prostate cancer cells to docetaxel treatment. Collectively, these results suggest that overexpression of ABCB1 mediates acquired docetaxel resistance and targeting ABCB1 expression could be a potential approach to resensitize docetaxel-resistant prostate cancer cells to docetaxel treatment.

Identification of serum biomarkers for occupational medicamentosa-like dermatitis induced by trichloroethylene using mass spectrometry

Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is an autoimmune disease and it has become a serious occupational health hazard. In the present study, we collected fasting blood samples from patients with OMLDT (n=18) and healthy volunteers (n=33) to explore serum peptidome patterns. Peptides in sera were purified using weak cation exchange magnetic beads (MB-WCX), and analyzed by matrix-assisted laser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) and ClinProTools bioinformatics software. The intensities of thirty protein/peptide peaks were significantly different between the healthy control and OMLDT patients. A pattern of three peaks (m/z 2106.3, 2134.5, and 3263.67) was selected for supervised neural network (SNN) model building to separate the OMLDT patients from the healthy controls with a sensitivity of 95.5% and a specificity of 73.8%. Furthermore, two peptide peaks of m/z 4091.61 and 4281.69 were identified as fragments of ATP-binding cassette transporter family A member 12 (ABCA12), and cationic trypsinogen (PRRS1), respectively. Our findings not only show that specific proteomic fingerprints in the sera of OMLDT patients can be served as a differentiated tool of OMLDT patients with high sensitivity and high specificity, but also reveal the novel correlation between OMLDT with ABC transports and PRRS1, which will be of potential value for clinical and mechanistic studies of OMLDT.

Effects of Selected OATP and/or ABC Transporter Inhibitors on the Brain and Whole-Body Distribution of Glyburide

Glyburide (glibenclamide, GLB) is a widely prescribed antidiabetic with potential beneficial effects in central nervous system injury and diseases. In vitro studies show that GLB is a substrate of organic anion transporting polypeptide (OATP) and ATP-binding cassette (ABC) transporter families, which may influence GLB distribution and pharmacokinetics in vivo. In the present study, we used [(11)C]GLB positron emission tomography (PET) imaging to non-invasively observe the distribution of GLB at a non-saturating tracer dose in baboons. The role of OATP and P-glycoprotein (P-gp) in [(11)C]GLB whole-body distribution, plasma kinetics, and metabolism was assessed using the OATP inhibitor rifampicin and the dual OATP/P-gp inhibitor cyclosporine. Finally, we used in situ brain perfusion in mice to pinpoint the effect of ABC transporters on GLB transport at the blood-brain barrier (BBB). PET revealed the critical role of OATP on liver [(11)C]GLB uptake and its subsequent impact on [(11)C]GLB metabolism and plasma clearance. OATP-mediated uptake also occurred in the myocardium and kidney parenchyma but not the brain. The inhibition of P-gp in addition to OATP did not further influence [(11)C]GLB tissue and plasma kinetics. At the BBB, the inhibition of both P-gp and breast cancer resistance protein (BCRP) was necessary to demonstrate the role of ABC transporters in limiting GLB brain uptake. This study demonstrates that GLB distribution, metabolism, and elimination are greatly dependent on OATP activity, the first step in GLB hepatic clearance. Conversely, P-gp, BCRP, and probably multidrug resistance protein 4 work in synergy to limit GLB brain uptake.

Molecular cloning and expression analysis of PDR1-like gene in ginseng subjected to salt and cold stresses or hormonal treatment

The plant pleiotropic drug resistance (PDR) family of ATP-binding cassette (ABC) transporters is potentially involved in diverse biological processes. Currently, little is known about their actual physiological functions. A Panax ginseng PDR transporter gene (PgPDR1) was cloned and the cDNA has an open reading frame of 4344 bp. The deduced amino acid sequence contained the characteristic domains of PDR transporters: Walker A, Walker B, and ABC signature. Genomic DNA hybridization analysis indicated that one copy of PgPDR1 gene was present in P. ginseng. Subcellular localization showed that PgPDR1-GFP fusion protein was specifically localized in the cell membrane. Promoter region analysis revealed the presence of cis-acting elements, some of which are putatively involved in response to hormone, light and stress. To understand the functional roles of PgPDR1, we investigated the expression patterns of PgPDR1 in different tissues and under various conditions. Quantitative real-time PCR (qRT-PCR) and Western blotting analysis showed that PgPDR1 was expressed at a high level in the roots and leaves compared to seeds and stems. The expression of PgPDR1 was up-regulated by salicylic acid (SA) or chilling, down-regulated by ABA, and regulated differently at transcript and protein levels by MeJA. These results suggest that PgPDR1 might be involved in responding to environmental stresses and hormones.

English

The plant pleiotropic drug resistance (PDR) family of ATP-binding cassette (ABC) transporters has comprehensively been researched in relation to transport of antifungal agents and resistant pathogens. In our study, analyses of the whole family of PDR genes present in the potato genome were provided. This analysis resolves discrepancies of potato PDR proteins and provides an expression analysis of all annotated potato PDR genes based on RNA-seq data. The results indicate that the potato genome contains 76 encoding PDR proteins and that these genes show a specific expression patterns, both at the organ level and in response to various hormonal treatment. These data provide some clues for future molecular genetic analysis of this important subfamily of ABC transporters. In addition, potato PDR genes may also play some important roles in the transportation of antifungal agents and resistant pathogens. Key words: ABC transporter, potato, pleiotropic drug resistance (PDR), RNA-seq.