ATP-binding Cassette Transporter Family Research Articles

Sitravatinib, a Tyrosine Kinase Inhibitor, Inhibits the Transport Function of ABCG2 and Restores Sensitivity to Chemotherapy-Resistant Cancer Cells in vitro.

Sitravatinib, also called MGCD516 or MG-516, is a broad-spectrum tyrosine kinase inhibitor (TKI) under phase III clinical evaluation. Herein, we explored the activity of sitravatinib toward multidrug resistance (MDR) by emphasizing its inhibitory effect on ATP-binding cassette super-family G member 2 (ABCG2). ABCG2 is a member of ATP-binding cassette (ABC) transporter family and plays a critical role in mediating MDR. Sitravatinb received an outstanding docking score for binding to the human ABCG2 model (PDB code: 6ETI) among thirty screened TKIs. Also, an MTT assay indicated that sitravatinib at 3 μM had the ability to restore the antineoplastic effect of various ABCG2 substrates in both drug-selected and gene-transfected ABCG2-overexpressing cell lines. In further tritium-labeled mitoxantrone transportation study, sitravatinib at 3 μM blocked the efflux function mediated by ABCG2 and as a result, increased the intracellular concentration of anticancer drugs. Interestingly, sitravatinib at 3 μM altered neither protein expression nor subcellular localization of ABCG2. An ATPase assay demonstrated that ATPase activity of ABCG2 was inhibited in a concentration-dependent manner with sitravatinib; thus, the energy source to pump out compounds was interfered. Collectively, the results of this study open new avenues for sitravatinib working as an ABCG2 inhibitor which restores the antineoplastic activity of anticancer drugs known to be ABCG2 substrates.

Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum.

P-glycoprotein (P-gp, ABCB1 member of the ABC (ATP-binding cassette) transporter family) localized in leukemia cell plasma membranes is known to reduce cell sensitivity to a large but well-defined group of chemicals known as P-gp substrates. However, we found previously that P-gp-positive sublines of L1210 murine leukemia cells (R and T) but not parental P-gp-negative parental cells (S) are resistant to the endoplasmic reticulum (ER) stressor tunicamycin (an N-glycosylation inhibitor). Here, we elucidated the mechanism of tunicamycin resistance in P-gp-positive cells. We found that tunicamycin at a sublethal concentration of 0.1 µM induced retention of the cells in the G1 phase of the cell cycle only in the P-gp negative variant of L1210 cells. P-gp-positive L1210 cell variants had higher expression of the ER stress chaperone GRP78/BiP compared to that of P-gp-negative cells, in which tunicamycin induced larger upregulation of CHOP (C/EBP homologous protein). Transfection of the sensitive P-gp-negative cells with plasmids containing GRP78/BiP antagonized tunicamycin-induced CHOP expression and reduced tunicamycin-induced arrest of cells in the G1 phase of the cell cycle. Taken together, these data suggest that the resistance of P-gp-positive cells to tunicamycin is due to increased levels of GRP78/BiP, which is overexpressed in both resistant variants of L1210 cells.

Inhibition of BCRP in Multidrug Resistant Cancer using Novel Inhibitors

Multi‐drug resistance (MDR) occurs when cancer cells become resistant to a diverse array of chemotherapeutics and xenobiotics. Among the many mechanisms of MDR, one of the most prominent is the overexpression of ATP‐binding cassette (ABC) transporters. ABC transporters harness the energy from ATP hydrolysis to transport xenobiotics, drugs, and other toxic compounds out of the cell. When ABC transporters are overexpressed in cancer cells, their efflux activity can lower the intracellular concentration of medicinal compounds to sub‐therapeutic levels. One member of the ABC transporter family, the Breast Cancer Resistance Protein (BCRP, or ABCG2), confers MDR to a variety of cancers. Transient inhibition of BCRP should therefore restore sensitivity of resistant cells to chemotherapeutics. In previous work by our lab, several potential BCRP inhibitors were identified via computational methods. Using cell viability assays, we tested the potential BCRP inhibitors using a BCRP overexpressing cell line. Here we compare the effectiveness of the inhibitors at re‐sensitizing the cells to Mitoxantrone relative to the parental cell line which does not overexpress BCRP. The potential toxicities of the experimental compounds were also assessed using the non‐cancerous HFL1 cell line.

The Genome of Caenorhabditis bovis.

The free-living nematode Caenorhabditis elegans is akey laboratory model for metazoan biology. C.elegans has also become a model for parasitic nematodes despite being only distantly related to most parasitic species. All of the ∼65 Caenorhabditis species currently in culture are free-living, with most having been isolated from decaying plant or fungal matter. Caenorhabditis bovis is a particularly unusual species that has been isolated several times from the inflamed ears of Zebu cattle in Eastern Africa, where it is associated with thedisease bovine parasitic otitis. C.bovis is therefore of particular interest to researchers interested in the evolution of nematode parasitism. However, as C.bovis is not in laboratory culture, it remains little studied. Here, by sampling livestock markets and slaughterhouses in Western Kenya, we successfully reisolated C.bovis from the ear of adult female Zebu. We sequenced the genome of C.bovis using the Oxford Nanopore MinION platform in a nearby field laboratory and used the data to generate a chromosome-scaledraft genome sequence. We exploited this draft genome sequence to reconstruct the phylogenetic relationships of C.bovis to other Caenorhabditis species and reveal the changes in genome size and content that have occurred during its evolution. We also identified expansions in several gene families that have been implicated in parasitism in other nematode species. The high-quality draft genome and our analyses thereof represent a significant advancement in our understanding of this unusual Caenorhabditis species.

The Roles of microRNAs in Multidrug-Resistance Mechanisms in Gastric Cancer.

Multidrug resistance (MDR) is one of the most significant reasons for the chemotherapeutics failure in gastric cancer. Although accumulating investigations and researches have been made to elucidate the mechanisms of multidrug resistance, the detail is far from completely understood. The importance of microRNAs in cancer chemotherapeutic resistance has been demonstrated recently, which provides a new strategy to overcome multidrug resistance. The different mechanisms are related to the phenomena of MDR itself and the roles of miRNAs in these multi-mechanisms by which MDR is acquired. In turn, the aim of this review was to summarize recent publications of microRNAs in regulating MDR in gastric cancer, thereby potentially developing as targeted therapies. Further unraveling the roles of microRNAs in MDR mechanisms including the ATP-binding cassette (ABC) transporter family, autophagy induction, cancer stem cell regulation, hypoxia induction, DNA damage and repair, epigenetic regulation, and exosomes in gastric cancer will be helpful for us to win the battle against it.

Restoration of MARCK enhances chemosensitivity in cancer

PurposeIncreased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1.MethodsCRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU).ResultsWe found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU.ConclusionsCells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.

Tivantinib, A c-Met Inhibitor in Clinical Trials, Is Susceptible to ABCG2-Mediated Drug Resistance.

Tivantinib, also known as ARQ-197, is a potent non-ATP competitive selective c-Met inhibitor currently under phase 3 clinical trial evaluation for liver and lung cancers. In this study, we explored factors that may lead to tivantinib resistance, especially in regards to its interaction with ATP-binding cassette super-family G member 2 (ABCG2). ABCG2 is one of the most important members of the ATP-binding cassette (ABC) transporter family, a group of membrane proteins that play a critical role in mediating multidrug resistance (MDR) in a variety of cancers, including those of the liver and lung. Tivantinib received a high score in docking analysis, indicating a strong interaction between tivantinib and ABCG2, and an ATPase assay indicated that tivantinib stimulated ABCG2 ATPase activity in a concentration-dependent manner. An MTT assay showed that ABCG2 overexpression significantly desensitized both the cancer cells and ABCG2 transfected-HEK293 cells to tivantinib and that this drug resistance can be reversed by ABCG2 inhibitors. Furthermore, tivantinib upregulated the protein expression of ABCG2 without altering the cell surface localization of ABCG2, leading to increased resistance to substrate drugs, such as mitoxantrone. Altogether, these data demonstrate that tivantinib is a substrate of ABCG2, and, therefore, ABCG2 overexpression may decrease its therapeutic effect. Our study provides evidence that the overexpression of ABCG2 should be monitored in clinical settings as an important risk factor for tivantinib drug resistance.

Functional relevance of the multi-drug transporter abcg2 on teriflunomide therapy in an animal model of multiple sclerosis

BackgroundThe multi-drug resistance transporter ABCG2, a member of the ATP-binding cassette (ABC) transporter family, mediates the efflux of different immunotherapeutics used in multiple sclerosis (MS), e.g., teriflunomide (teri), cladribine, and mitoxantrone, across cell membranes and organelles. Hence, the modulation of ABCG2 activity could have potential therapeutic implications in MS. In this study, we aimed at investigating the functional impact of abcg2 modulation on teri-induced effects in vitro and in vivo.MethodsT cells from C57BL/6 J wild-type (wt) and abcg2-knockout (KO) mice were treated with teri at different concentrations with/without specific abcg2-inhibitors (Ko143; Fumitremorgin C) and analyzed for intracellular teri concentration (HPLC; LS-MS/MS), T cell apoptosis (annexin V/PI), and proliferation (CSFE). Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6J by active immunization with MOG35–55/CFA. Teri (10 mg/kg body weight) was given orally once daily after individual disease onset. abcg2-mRNA expression (spinal cord, splenic T cells) was analyzed using qRT-PCR.ResultsIn vitro, intracellular teri concentration in T cells was 2.5-fold higher in abcg2-KO mice than in wt mice. Teri-induced inhibition of T cell proliferation was two fold increased in abcg2-KO cells compared to wt cells. T cell apoptosis demonstrated analogous results with 3.1-fold increased apoptosis after pharmacological abcg2-inhibition in wt cells. abcg2-mRNA was differentially regulated during different phases of EAE within the central nervous system and peripheral organs. In vivo, at a dosage not efficacious in wt animals, teri treatment ameliorated clinical EAE in abcg2-KO mice which was accompanied by higher spinal cord tissue concentrations of teri.ConclusionFunctional relevance of abcg2 modulation on teri effects in vitro and in vivo warrants further investigation as a potential determinant of interindividual treatment response in MS, with potential implications for other immunotherapies.

Possible Intestinal Absorption Enhancers fromCitrus hystrix

Bioavailability of orally administered drugs is regulated by P-gp, a member of the ATP binding cassette transporter families. It expresses at the apical surface of epithelial cells and effluxs out several clinically important drugs resulting in decreased absorption and bioavailability. In recent years, the utilization of bioenhancer to increase the bioavailability of drugs has extensively studied. The objective of this study was to evaluate the potential of the compounds found inCitrus hystrixas a bioenhancer for orally administered drugs by modulation of P-gp function. The modulation effects of fruit extracts and isolated pure compounds on P-gp were investigated by uptake assay of the P-gp substrate calcein-AM in Caco-2, LLC-PK1 and LLC-GA5-COL300 cell lines. The results show that the extract from the flavedo part remarkably increased calcein-AM uptake in Caco-2 and LLC-GA5-COL300 cell lines. Among five furanocoumarins identified, 6’,7’-epoxybergamottin, 6’,7’-dihydroxybergamottin and oxypeucedanin significantly enhanced calcein-AM uptake in LLC-GA5-COL300 in a concentration-dependent manner, indicating strongly inhibition effects on P-gp function. Taken together, 6’,7’-epoxybergamottin, 6’,7’-dihydroxybergamottin and oxypeucedanin could be employed as the potential intestinal bioenhancer to improve the bioavailability of P-gp substrate drugs. However, further studies includingin vivostudies should be performed to confirm these findings.

Cellular cholesterol prediction of mammalian ATP-binding cassette (ABC) proteins based on fuzzy c-means with support vector machine algorithms

Over the years protein interaction and prediction of membrane protein have been a pivotal research area for all researchers. For both prokaryotes and eukaryotes Adenosine Triphosphate-(ATP) binding cassette (ABC) genes plays a significant role. In our analysis, we concentrate on human part of ABC genes. In case of living organisms transport of precise molecules across lipid membranes has been treated as vital part and for that reason a bigger transporter is required to carry out the molecules. Here ABC transporter families are evolved to transport the specific molecules such as sugars, amino acid, peptides, proteins, ions etc. within the plasma membrane. As we know another important component of human being is cholesterol, which is a major component in cell membrane and its main functions are to maintain integrity and mechanical stability. Each and every time, membrane cholesterolsareinteracted with membrane protein in both N-C terminuses and target valid sequence(s) which has relevance in human diseases. In this manuscript we have applied Fuzzy C-Means (FCM) with Support Vector Machine (SVM) algorithm for prediction of cellular cholesterol with ABC genes. Our experiments have been performed well using ABCdata set.

ABC efflux transporters at blood-central nervous system barriers and their implications for treating spinal cord disorders

The barriers present in the interfaces between the blood and the central nervous system form a major hurdle for the pharmacological treatment of central nervous system injuries and diseases. The family of ATP-binding cassette (ABC) transporters has been widely studied regarding efflux of medications at blood-central nervous system barriers. These efflux transporters include P-glycoprotein (abcb1), ‘breast cancer resistance protein’ (abcg2) and the various ‘multidrug resistance-associated proteins’ (abccs). Understanding which efflux transporters are present at the blood-spinal cord, blood-cerebrospinal fluid and cerebrospinal fluid-spinal cord barriers is necessary to determine their involvement in limiting drug transfer from blood to the spinal cord tissue. Recent developments in the blood-brain barrier field have shown that barrier systems are dynamic and the profile of barrier defenses can alter due to conditions such as age, disease and environmental challenge. This means that a true understanding of ABC efflux transporter expression and localization should not be one static value but instead a range that represents the complex patient subpopulations that exist. In the present review, the blood-central nervous system barrier literature is discussed with a focus on the impact of ABC efflux transporters on: (i) protecting the spinal cord from adverse effects of systemically directed drugs, and (ii) limiting centrally directed drugs from accessing their active sites within the spinal cord.

Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family

BackgroundThe western corn rootworm, Diabrotica virgifera virgifera, is a pervasive pest of maize in North America and Europe, which has adapted to current pest management strategies. In advance of an assembled and annotated D. v. virgifera genome, we developed transcriptomic resources to use in identifying candidate genes likely to be involved in the evolution of resistance, starting with members of the ATP-binding cassette (ABC) transporter family.ResultsIn this study, 65 putative D. v. virgifera ABC (DvvABC) transporters were identified within a combined transcriptome assembly generated from embryonic, larval, adult male, and adult female RNA-sequence libraries. Phylogenetic analysis placed the deduced amino-acid sequences of the DvvABC transporters into eight subfamilies (A to H). To supplement our sequence data with functional analysis, we identified orthologs of Tribolium castaneum ABC genes which had previously been shown to exhibit overt RNA interference (RNAi) phenotypes. We identified eight such D. v. virgifera genes, and found that they were functionally similar to their T. castaneum counterparts. Interestingly, depletion of DvvABCB_39715 and DvvABCG_3712 transcripts in adult females produced detrimental reproductive and developmental phenotypes, demonstrating the potential of these genes as targets for RNAi-mediated insect control tactics.ConclusionsBy combining sequence data from four libraries covering three distinct life stages, we have produced a relatively comprehensive de novo transcriptome assembly for D. v. virgifera. Moreover, we have identified 65 members of the ABC transporter family and provided the first insights into the developmental and physiological roles of ABC transporters in this pest species.

Enabling P-glycoprotein inhibition in multidrug resistant cancer through the reverse targeting of a quinidine-PEG conjugate

Previously identified as a key mediator of multidrug resistance, the drug efflux behavior of P-glycoprotein (P-gp) remains a prominent challenge in cancer treatment. P-gp belongs to the ATP-binding cassette transporter family of membrane proteins, and modulates the efflux of many drugs at the cell membrane, resulting in inadequate retention of chemotherapeutic drugs in cancer cells. Here, we explore the FDA-approved drug quinidine as a P-gp inhibitor. Although used clinically for the treatment of malaria, arrhythmia, and pseudobulbar effect, quinidine can induce acquired long QT syndrome and torsade de pointes through its interaction with the Purkinje fibers, which hinders its clinical application as a P-gp inhibitor. We hypothesize that the conjugation of quinidine to a polymer will permit its use as a P-gp inhibitor through mitigation of its distribution into the myocardium. Methoxypolyethylene glycol (mPEG) was conjugated to quinidine through a glycine linker, making a monovalent quinidine-polymer conjugate, which was then evaluated for its interactions with P-gp in vitro. The mPEG-glycine-quinidine conjugate retained its ability to inhibit the function of P-gp (log IC50 of 4.20 nM for quinidine and 4.61 nM for the mPEG-glycine-quinidine conjugate). Additionally, the distribution of quinidine into perfused mouse myocardium was decreased by almost an order of magnitude, strongly supporting our working hypothesis (2.28 × 10−3 μmol/g tissue for quinidine and ~4.10 × 10−4 μmol/g tissue for the conjugate). The results suggest the potential use of such polymer-drug conjugates to reverse multidrug resistance through P-gp inhibition and to mitigate the off-target pharmacologic effects that complicate their clinical use.

A Potent Anti-SpuE Antibody Allosterically Inhibits Type III Secretion System and Attenuates Virulence of Pseudomonas Aeruginosa

Multidrug-resistant gram-negative bacteria infection is particularly severe within the designated ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which underscores the urgent need to explore alternative therapeutic strategies. The type III secretion system (T3SS) is considered to be a key virulence factor in many gram-negative bacteria, and T3SS is in turn regulated by SpuE in P. aeruginosa, which is a spermidine binding protein from an ATP-binding cassette transporter family and highly conserved within ESKAPE pathogens. Here, we identified a potent anti-SpuE antagonistic antibody that allosterically inhibits the expression of T3SS and attenuates virulence of P. aeruginosa. X-ray crystallography and molecular dynamics simulations revealed that binding of antibody to SpuE induces a change in the dynamics of SpuE, which in turn may reduce spermidine uptake by P. aeruginosa. The antibody could serve as a template for developing novel biologics to target a broad spectrum of gram-negative bacteria.

Less and shrunken pollen 1 (LSP1) encodes a member of the ABC transporter family required for pollen wall development in rice (Oryza sativa L.)

Pollen fertility is an agronomic trait that strongly influences rice yield. Recent studies have revealed that the development of the pollen wall is required for pollen fertility and is regulated by several genes. However, the mechanisms underlying pollen and pollen wall development in rice remain largely unknown. In the present study, a point mutation in a gene on chromosome 1 was identified that resulted in the production of less and shrunken pollen (LSP) and led to defects in pollen wall formation. This gene was named LSP1 and was found to encode a member of the adenosine triphosphate-binding cassette (ABC) transporter G subfamily, OsABCG3. Two other loss-of-function mutants of LSP1/OsABCG3, generated using CRISPR/Cas9 technology, showed the same male sterile phenotype. The LSP1/OsABCG3 gene showed a spatio-temporal expression pattern in the developing anthers, and is an ortholog of the Arabidopsis genes AtABCG1 and AtABCG16, which play an important role in pollen wall development. Mutation of LSP1/OsABCG3 affected the expression of several genes involved in pollen and pollen wall formation. These results suggest that LSP1/OsABCG3 is critical for normal pollen fertility and shed light on the molecular mechanisms underlying rice pollen wall development.

Clinical Significance of ABCB1 in Acute Myeloid Leukemia: A Comprehensive Study.

ABCB1 is a member of the ATP binding cassette transporter family and high ABCB1 activity is considered as a poor prognostic factor in acute myeloid leukemia (AML) treated with intensive chemotherapy, its direct relation with drug resistance remains unclear. We evaluated ABCB1 activity in relation with clinical parameters and treatment response to standard chemotherapy in 321 patients with de novo AML. We assessed multiple clinical relationships of ABCB1 activity—ex vivo drug resistance, gene expression, and the ABCB1 inhibitor quinine were evaluated. ABCB1 activity was observed in 58% of AML and was linked to low white blood cell count, high expression of CD34, absence of FLT3-ITD, and absence of mutant NPM1. Moreover, ABCB1 activity was associated with worse overall- and event-free survival. However, ABCB1 activity did not directly lead to ex vivo drug resistance to anthracyclines. We found that ABCB1 was highly correlated with gene expressions of BAALC, CD34, CD200, and CD7, indicating that ABCB1 expression maybe a passenger characteristic of high-risk AML. Furthermore, ABCB1 was inversely correlated to HOX cluster genes and CD33. Thus, low ABCB1 AML patients benefited specifically from anti-CD33 treatment by gemtuzumab ozogamicin in addition to standard chemotherapy. We showed prognostic importance of ABCB1 gene expression, protein expression, and activity. Furthermore, ABCB1 was not directly linked to drug resistance, ABCB1 inhibition did not improve outcome of high ABCB1 AML patients and thus high ABCB1 may represent a passenger characteristic of high-risk AML.

Significance and Mechanisms of P-glycoprotein in Central Nervous System Diseases.

P-glycoprotein (P-gp) is a member of ATP-Binding Cassette (ABC) transporter family. Because of its characteristic luminal surface location, high transport potency and structural specificity, Pgp is regarded as a selective gatekeeper of the Blood Brain Barrier (BBB) to prevent the entry of toxins or unwanted substances into the brain. In recent years, increasing evidence has shown that P-gp is involved in the immune inflammatory response in the Central Nervous System (CNS) disorders by regulating microglia activation, and mediating immune cell migration. Furthermore, Glucocorticoid Receptor (GR) may play a crucial role in P-gp-mediated microglia activation and immune cell migration via GR-mediated mRNA decay. In this article, we will review P-gp structure, distribution, function, regulatory mechanisms, inhibitors and effects of P-gp in the pathogenesis of several CNS diseases and will discuss the role of P-gp in microglia activation, immune cell migration and the relationship with cytokine secretion.

Preliminary Classification of the ABC Transporter Family in Betula halophila and Expression Patterns in Response to Exogenous Phytohormones and Abiotic Stresses

ATP-binding cassette (ABC) transporters comprise a transport system superfamily which is ubiquitous in eukaryotic and prokaryotic cells. In plants, ABC transporters play important roles in hormone transport and stress tolerance. In this study, 15 BhABC transporters encoded by genes identified from the transcriptome of Betula halophila were categorized into four subfamilies (ABCB, ABCF, ABCG, and ABCI) using structural domain and phylogenetic analyses. Upon B. halophila exposure to exogenous phytohormones and abiotic stressors, gene expression patterns and transcriptional responses for each subfamily of genes were obtained using semi-quantitative RT-PCR analysis. The results demonstrated that expression of most genes belonging to ABCB and ABCG subfamilies changed in response to exogenous phytohormone exposures and abiotic stress. These results suggest that BhABC genes may participate in hormone transport and that their expression may be influenced by ABA-dependent signaling pathways involved in abiotic stress responses to various stressors.

Chemo-Mechanical Coupling in the Transport Cycle of a Heme ABC Transporter

The heme importer from pathogenic bacteria is a member of the ATP-binding cassette (ABC) transporter family, which uses the energy of ATP-binding and hydrolysis for extensive conformational changes. Previous studies have indicated that conformational changes after heme translocation are triggered by ATP-binding to nucleotide binding domains (NBDs) and then, in turn, induce conformational transitions of the transmembrane domains (TMDs). In this study, we applied a template-based iterative all-atom molecular dynamics (MD) simulation to predict the ATP-bound outward-facing conformation of the Burkholderia cenocepacia heme importer BhuUV-T. The resulting model showed a stable conformation of the TMD with the cytoplasmic gate in the closed state and the periplasmic gate in the open state. Furthermore, targeted MD simulation predicted the intermediate structure of an occluded form (Occ) with bound ATP, in which both ends of the heme translocation channel are closed. The MD simulation of the predicted Occ revealed that Ser147 on the ABC signature motifs (LSGG[Q/E]) of NBDs occasionally flips and loses the active conformation required for ATP-hydrolysis. The flipping motion was found to be coupled to the inter-NBD distance. Our results highlight the functional significance of the signature motif of ABC transporters in regulation of ATPase and chemo-mechanical coupling mechanism.

Synthesis, in silico and in vitro studies of new 1,4-dihydropiridine derivatives for antitumor and P-glycoprotein inhibitory activity

P-glycoprotein (P-gp) is one of the cell membrane pumps which mediate the efflux of molecules such as anticancer drugs to the extracellular matrix of tumor cells. P_gp is a member of the ATP-binding cassette (ABC) transporter family that is implicated in cancer multidrug resistance (MDR). Since MDR is a contributor to cancer chemotherapy failure, modulation of efflux pumps is a viable therapeutic strategy. In this study, new synthetic 1,4 dihydropiridine (DHP) derivatives containing thiophenyl substitution were tested as inhibitors of P-gp. Efflux assay was conducted to evaluate the intracellular accumulation of Rhodamine123 (Rh123) as a pump substrate. MTT assay, cell cycle analysis and in silico methods were also examined. Flow cytometric analysis revealed that synthetic DHP derivatives (15 µM) increased intracellular concentration of the substrate by 2–3 folds compared with verapamil as a standard P-gp inhibitor. MTT assay on EPG85-257P and its drug-resistant EPG85-257RDB cell line revealed antitumor effects (30–45%) for new DHP derivatives at 15 µM following 72 h incubation. However, MTT test on normal cell line showed negligible toxic effects. Finally combination of synthetic derivatives with doxorubicin showed that these compounds decrease IC50 of doxorubicin in resistant cell lines from 9 to 1.5 µM. Sub-G1 peak-related apoptotic cells showed a stronger effect of synthetic compounds at 5 µM compared with verapamil. Molecular dynamic results showed a high binding affinity between DHP derivative and protein at drug binding site. Findings of these biological tests indicated the antitumor activity and P-gp inhibitory effects of new 1,4-DHP derivatives.