Cassette Transporter Proteins Research Articles

Strategies to gain novel Alzheimer's disease diagnostics and therapeutics using modulators of ABCA transporters.

Adenosine-triphosphate-(ATP)-binding cassette (ABC) transport proteins are ubiquitously present membrane-bound efflux pumps that distribute endo- and xenobiotics across intra- and intercellular barriers. Discovered over 40 years ago, ABC transporters have been identified as key players in various human diseases, such as multidrug-resistant cancer and atherosclerosis, but also neurodegenerative diseases, such as Alzheimer’s disease (AD). Most prominent and well-studied are ABCB1, ABCC1, and ABCG2, not only due to their contribution to the multidrug resistance (MDR) phenotype in cancer, but also due to their contribution to AD. However, our understanding of other ABC transporters is limited, and most of the 49 human ABC transporters have been largely neglected as potential targets for novel small-molecule drugs. This is especially true for the ABCA subfamily, which contains several members known to play a role in AD initiation and progression. This review provides up-to-date information on the proposed functional background and pathological role of ABCA transporters in AD. We also provide an overview of small-molecules shown to interact with ABCA transporters as well as potential in silico, in vitro, and in vivo methodologies to gain novel templates for the development of innovative ABC transporter-targeting diagnostics and therapeutics.

Multidrug resistance protein structure of Trypanosoma evansi isolated from buffaloes in Ngawi District, Indonesia: A bioinformatics analysis.

Background and Aim:Trypanosomiasis, also known as surra, is an infectious disease with a wide host spectrum. In Indonesia, this disease is caused by Trypanosoma evansi. Various trypanocidal drugs have been used to treat this pathogen and subsequent disease. Yet, the long-term trypanocidal administration generates drug-resistant T. evansi. Some have identified genetic alterations in T. evansi transporter protein-coding genes that may be responsible for drug resistance. The Multidrug Resistance Protein E (MRPE) gene is a likely candidate gene responsible for the individual resistance. To date, no research has focused on T. evansi MRPE (TevMRPE) in this context. Hence, this research aimed at analyzing and characterizing the TevMRPE gene and protein using a bioinformatics approach.Materials and Methods:T. evansi was isolated from buffalo suffering from surra in Ngawi Regency, Indonesia. Isolated T. evansi was inoculated and cultured in male mice. The T. evansi genome was isolated from mouse blood with a parasitemia degree as high as 105. A polymerase chain reaction procedure was conducted to amplify the putative MRPE coding gene. The amplicon was sequenced and analyzed using MEGA X, BLAST, and I-tasser softwares.Results:The putative TevMRPE coding gene showed sequence similarity as high as 99.79% against the MRPE gene from Trypanosoma brucei gambiense. The protein profile and characteristics depicted that the putative TevMRPE protein was related to a family of Adenosine Triphosphate-Binding Cassette (ABC) transporter proteins. This family of transporter proteins plays a crucial role in the resistance toward several medicines.Conclusion:The obtained gene sequence in this research was identified as the TevMRPE. This gene is homologous to the T. brucei gambiense MRPE gene and possesses ligand active sites for Adenylyl Imidodiphosphate. In addition, MRPE contains enzyme active sites similar to the cystic fibrosis transmembrane conductance regulator. These data suggest that ABC transport proteins, like MRPE, may be necessary to confer trypanocidal drug resistance in T. evansi.

Comparative modeling and structure based drug repurposing of PAX2 transcription factor for targeting acquired chemoresistance in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a pancreatic malignancy suffering from poor prognosis; the worst among all types of cancer. Chemotherapy, which is the standard regime for treatment in most cases, is often rendered useless as drug resistance quickly sets in after prolonged exposure to the drug. The implication of PAX2 transcription factor in regulating several ATP-binding cassette (ABC) transporter proteins that are responsible for the acquisition of drug resistance in PDAC makes it a potential target for treatment purposes. In this study, the 3D structure of PAX2 protein was modeled, and the response of key amino acids to perturbation was identified. Subsequently, kappadione, a vitamin K derivative, was found to bind efficiently to PAX2 with a binding energy of −9.819 kcal/mol. The efficacy of mechanism and mode of binding was studied by docking the protein with DNA in the presence and absence of the drug. The presence of kappadione disrupted DNA binding with key effector resides, preventing the DNA from coming into contact with the binding region essential for protein translation. By occupying the DNA binding region and replacing it with a ligand, the mechanism by which DNA interacts with PAX2 could be manipulated. Inhibition of PAX2-DNA binding using kappadione and other small molecules can prove to be beneficial for combating chemoresistance in PDAC, as proposed through in silico approaches. Communicated by Ramaswamy H. Sarma

Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line

BackgroundOver-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides. Tick cell lines are useful for investigating resistance mechanisms, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Cells were treated with different drug concentrations (25, 50, 100, 150 μM) and incubated for ten days. Cell morphology, viability, metabolic activity and relative expression of ABC (B1, B6, B8 and B10) genes were determined at day 10 post-treatment.ResultsCell morphology determined by light microscopy was altered following treatment with all drugs, but only at high concentrations, while total cell numbers decreased with increasing drug dose. Cell viability determined by trypan blue exclusion was not significantly different from untreated controls (P > 0.1) following treatment with amitraz and permethrin, but high concentrations of fipronil caused decrease (up to 37%, P < 0.01) in viability. At all drug concentrations, fipronil and permethrin induced dose-dependent reduction in cell metabolic activity measured by MTT assay (P < 0.01). Quantitative RT-PCR showed that the drugs significantly affected expression of ABC genes. In particular, fipronil treatment downregulated ABCB1 (P < 0.001) and upregulated ABCB6, ABCB8 and ABCB10 (P < 0.01); amitraz treatment down regulated ABCB1 (significant difference between 25 and 150 μM, P < 0.001) and upregulated ABCB8 and ABCB10 at lower concentrations (25 and 50 μM, P < 0.05); and permethrin upregulated ABCB6, ABCB8 and ABCB10 only at 150 μM (P < 0.01).ConclusionsThe adverse effects on cell viability and metabolic activity, and changes in expression of different ABC transporter genes, detected in IRE/CTVM19 cells following treatment with amitraz, permethrin and fipronil, support the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks.

Meloxicam increases intracellular accumulation of doxorubicin via downregulation of multidrug resistance-associated protein 1 (MRP1) in A549 cells

It has been suggested that selected COX inhibitors can overcome multidrug resistance through the inhibition of ATP‑binding cassette-transporter proteins thereby enhancing the inhibitory effect of doxorubicin on human tumor growth and promoting the actions of cytostatics. However, their effect on lung cancer and the molecular mechanisms involved in the overcoming of multidrug resistance are unclear. In the present study, the ability of meloxicam, a COX-2-specific inhibitor to enhance doxorubicin‑mediated inhibition was investigated in human A549 lung cancer in vivo and in vitro. In order to unravel the molecular mechanisms involved in doxorubicin accumulation, we measured the levels of multidrug resistance-associated protein (MRP)-transporter protein activity and expression by western blotting, since this has been implicated in meloxicam action as well as in chemoresistance. We found that, in A549 cells, meloxicam could increase intracellular accumulation of doxorubicin, a substrate for MRP, through inhibition of cellular export. Western blot analysis indicated that meloxicam reduced the expression of MRP1 and MRP4. The results reported in the present study demonstrate for the first time that the specific COX-2 inhibitor meloxicam can increase the intracellular accumulation of doxorubicin and enhance doxorubicin-induced cytotoxicity in A549 cancer cells by reducing the expression of MRP1 and MRP4.

Abstract C251: Dianotecan: A novel water-soluble CPT prodrug as a potential anticancer agent with less susceptility to multidrug resistance than irinotecan.

Abstract Camptothecin (CPT) and its derivatives are topoisomerase I inhibitors. Among these, irinotecan and topotecan have shown significant clinical activity against a broad range of malignancies. Irinotecan is approved for use in the treatment of advanced colorectal cancer, as first-line therapy in combination with 5-FU or as a salvage agent after failure of 5-fluorouracil-based chemotherapy. However, recent studies have revealed that SN-38 (the active metabolite of irinotecan) is the substrate of BCRP (ABCG2), an ATP-binding cassette (ABC) transport protein which confers multidrug resistance and is also characterized as the marker of stem and progenitor cells. In tumor cell lines over-expressing BCRP, selective BCRP efflux of SN-38 leads to decrease in cellular drug accumulation, resulting in drug resistance phenotype. Here we report discovery of a novel water-soluble CPT prodrug (dianotecan) which can be efficiently metabolized to CPT in vivo after i.v. Injection. In the tumor xenograft studies, dianotecan has been established as a potent inhibitor against a number of human solid tumors including HT-29, S1, NCI-446, MCF-7. Significantly, dianotecan has demonstrated anti-tumor efficacy far superior to irinotecan in the xenograft studies using the BCRP-overexpressing S1-M1-80 cell line. These findings suggest dianotecan, as a potential anticancer agent, is less susceptible to multidrug resistance than irinotecan. The preclinical data of dianotecan will be discussed in the poster. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C251. Citation Format: Wenqiang Zhou, Li-Wu Fu. Dianotecan: A novel water-soluble CPT prodrug as a potential anticancer agent with less susceptility to multidrug resistance than irinotecan. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C251.

Improvement of Daptomycin Yield by Overexpression of ATP-Binding Cassette (ABC) Transport Proteins Genes of Daptomycin Gene Cluster

The effects of the genes of ORF53-55, which code ATP-binding cassette (ABC) transport proteins, on daptomycin production were investigated by overexpression. The yield of daptomycin was promoted significantly when either of the genes of ORF53, ORF54 or ORF55 was overexpressed individually or by a combined manner. The results above suggested that the ORF53, ORF54 and ORF55 genes had positive cooperativity in the biosynthesis of daptomycin. The dry weight and sugar utilization of HP-ORF53-55 is significantly improved as compared with the LC-54-16. Based on these results, it was speculated that the genes of ORF 53, 54 and 55 may play an important positive role in the process of reducing sugar transport, which enhanced the cell growth and daptomycin biosynthesis.

260 Association Between Genetic Polymorphisms of ABCC2 Transporter and the Susceptibility to Maculopapular Eruption Induced by Antituberculosis Drugs

BackgroundATP-binding cassette (ABC) transporter proteins play an important role in drug disposition. Polymorphisms of ABC transporter genes (ABCC2 and ABCB1) may be risk markers for maculopapular eruption (MPE) induced by unusual accumulation of antituberculosis drugs (ATD) itself or metabolites.MethodsWe compared genotype distributions of single nucleotide polymorphisms and haplotypes in ABCC2 and ABCB1 genes between 62 ATD-induced MPE cases and 159 ATD-tolerant controls using multivariate logistic regression analysis.ResultsAmong the 7 selected SNPs of ABCC2, -1549G>A in promoter and IVS3-49C>T in intron were associated with ATD-induced MPE (P = 0.032 and P = 0.029, respectively). ABCC2 haplotype1 [G-C-C-G] was significantly associated with ATD-induced MPE (P = 0.032, OR = 0.35, 95% CI, 0.29-0.95). However, there was no significant association between other genetic polymorphisms in ABCB1 and ATD-induced MPE.ConclusionsThese results suggest that genetic variations of ABCC2 are a potential risk factor for ATD-induced MPE.

Lipopolysaccharide Down-regulates ABCA1 Expression in Foam Cells in a Nucleus Factor-κB Pathway-dependent Manner*

The immnue-inflammatory response mediated by LPS is firmly related to the development of atherosclerosis. ABCA1 has been identified to play a key role in the cellular cholesterol efflux which is regarded to anti-atherosclerosis. To investigate the changes of cholesterol efflux, ATP-binding cassette transporter A1 (ABCA1) mRNA and protein expression in THP-1 macrophage derived foam cells treated with LPS, and to discover the role of TLR4/NF-κB pathway and LXRs in this process. The foam cells were exposed to different concentration of LPS for 24 h or exposed to LPS for different times, and more, the cells were treated with LPS along or together with N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) for 24 h. The mRNA levels of ABCA1, TLR4 and LXRα were measured by reverse transcriptase-polymerase chain reaction,and the protein levels of ABCA1, LXRα and intranuclear NF-κB p65 were measured by Western blotting. Cellular lipid accumulation was determined by high performance liquid chromatography analysis. Cholesterol efflux was determined by FJ-2107P type liquid scintillator. The results show that the expression of ABCA1 were decreased in a dose- and time-dependent manner after treated with LPS, while TLR4 expression and the intranuclear NF-κB p65 protein level were increased by LPS, and these changes can be reversed partly by pretreatment with TPCK. LPS and TPCK can not effect LXRα expression. The results also show that cellular lipid accumulation was increased, while the cellular cholesterol efflux was decreased in THP-1 macrophage derived foam cells after exposured to LPS for 24 h. It was concluded that LPS can down-regulate the expression of ABCA1, promote the accumulation of lipid and decrease cellular cholesterol efflux in THP-1 macrophage derived foam cells, which may be related to the TLR4/NF-κB dependent and LXRα-independent pathway.

Abstract 4244: Dysadherin can enhance tumorigenesis through conferring properties of stem-like cells to hepatocellular carcinoma cells

Abstract Hepatocellular carcinoma (HCC) is associated with a high potential for metastasis and disease recurrence, even after surgical resection. The cancer stem cell (CSC) hypothesis proposes that CSCs are responsible for chemo-resistance, recurrence and metastasis. Dysadherin is a prognostic indicator of metastasis and poor survival in many different cancer types. In this study, we investigated the possible link between dysadherin and CSC in HCC. The transfection of dysadherin cDNA into the liver cancer cell line PLC/PRF/5 enhanced the properties of CSC, including anti-apoptosis, sphere-forming ability, side population (SP) phenotype and tumor initiation ability in vivo. Furthermore, knockdown of dysadherin in the liver cancer cell line SK-Hep1 suppressed its stem cell-like properties. In particular, SP phenotype is mediated by the ATP-binding cassette (ABC) transporter proteins which are responsible for multidrug resistance (MDR) that is a major obstacle in cancer chemotherapy. Dysadherin was also shown to regulate ABCB1 expression. Taken together, these results show that new roles for the dysadherin in cancer progression that was in part dependent on CSC dynamics. Therefore, these findings suggest that dysadherin may be a potential molecular prognostic marker of HCC and may aid in the development of more effective therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4244.

Physiological and Pharmacological Significance of Glutathione-Conjugate Transport

Transport of the glutathione conjugates (GS-E) of electrophilic compounds generated during biotransformation of drugs and environmental pollutants is central to the mechanisms of defense against oxidative/electrophilic stress. In recent years emphasis has been placed on ATP-binding cassette (ABC) transport proteins in the transport of GS-E and their involvement in the detoxification mechanisms, including drug resistance. Recent studies, however, suggested that the majority of GS-E transport in human and rodent cells is mediated by a non-ABC, multifunctional stress-response protein, RLIP76 or RalBP1 (ral-binding GTPase activating protein 1), which also functions as an effector in the Ral-Ras-Rho signaling pathway. In this review, after briefly describing the major discoveries in the field of glutathione (GSH)-conjugate transport, recent findings are presented on the role of RLIP76 in ATP-dependent transport of GS-E, and the relevance of this transport process to the mechanisms of toxicity of xenobiotics, radiation, and endogenous electrophilic toxicants is described. Furthermore, recent studies suggesting a link between RLIP76 mediated GS-E transport and cell cycle signaling are presented.

In vitro evaluation of the effects of gefitinib on the modulation of cytotoxic activity of selected anticancer agents in a panel of human ovarian cancer cell lines

This study was conducted to determine the in vitro optimal combination of selected anticancer agents with gefitinib and evaluate its effect on the expression of correlative biological targets in the cell-signaling pathway. In addition, the effect of gefitinib on the expression of ATP-binding cassette (ABC) transport proteins was evaluated. Growth inhibition assays were conducted in five human ovarian cancer cell lines to evaluate the activity of selected anticancer agents in combination with gefitinib compared to each alone. Enzyme linked immunosorbant assay (ELISA) assessed the presence of pEGFR in treated and untreated cells. Expression of correlative biological targets in the cell-signaling pathway was completed by immunoblotting. RT-PCR was used to characterize the expression ABC transport proteins. This in vitro study confirmed gefitinib did not have significant cytotoxic activity, the combination of gefitinib with other chemotherapy drugs demonstrated improved in vitro cytotoxic activity in platinum sensitive ovarian cancer cell lines. Suppression of pAKT and p-erk activation in cells treated with combination of cisplatin and gefitinib was observed and suggests the role of gefitinib inhibition of proliferative cell signaling pathway. This data suggests that EGFR-inhibitors, such as gefitinib, have the potential to modulate common mechanisms of drug resistance and may have a role in optimizing chemotherapy regimens for the treatment of ovarian cancer.

Pharmacokinetics and bioavailability of a newly synthesized dihydropyridine compound with multidrug resistance reversal activity

(+/-)3-(3-(4,4-diphenylpiperidin-1-yl)propyl) 5-methyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate ((+/-)-DHP-014), is a new 4-aryl-1,4-dihydropyridine that can reverse multidrug resistance mediated by the ATP-binding cassette (ABC) transport proteins, P-glycoprotein, multidrug resistance-associated protein 1 and breast cancer resistance protein; it exhibits negligible calcium channel blocking activity. The objective of this work was to investigate the pharmacokinetics of this new compound in rats. Three intravenous (1, 2 and 5 mg/kg) and two oral (25 and 50 mg/kg) doses were administered to female Sprague-Dawley rats. A two-compartment model with nonlinear elimination best characterized the pharmacokinetic profiles after intravenous and oral administration in rats. The terminal half-life of (+/-)-DHP-014 increased and the systemic clearance significantly decreased at higher doses, indicating nonlinear elimination. The dose-dependent clearance is likely due to saturation of metabolism. The apparent volume of distribution of (+/-)-DHP-014 was 2.0 L/kg in rats and was unchanged with increasing intravenous doses of (+/-)-DHP-014. The estimated oral bioavailability of (+/-)-DHP-014 was 8.2%. The poor bioavailability is likely due to the poor solubility of the compound, as well as to substantial first-pass elimination.

Stress‐ (and Diet‐) Related Regulation of Hepatic Nuclear Receptors and Its Relevance for ABC‐Transporter Functions

Nuclear receptors (NRs) play an important role in maintaining cellular homeostasis. With clearly established roles in fatty acid metabolism and inflammation, peroxisome proliferator activated receptors (PPARs) and other nuclear receptors are essential in liver functioning. However, much less is known about the regulation of NRs themselves during inflammatory processes in the liver. Interestingly PPARs and other NRs are negative acute phase proteins because they become rapidly downregulated during the acute phase response. However, PPARs have important roles in modulating inflammatory responses. One of the mechanisms by which dietary or inflammatory stress is relieved involves the hepatic adenosine triphosphate‐binding cassette (ABC) transporter proteins, which import and export a wide variety of substrates. These ABC transporters are under close control of several NRs. Because NRs play important roles in fatty acid metabolism and inflammation as well as in the regulation of bile production, they are reviewed here with respect to their role in dietary and stress‐related responses of the liver and their impact on the regulation and function of hepatic ABC transporters.

FUNCTIONAL CHARACTERIZATION OF THE ADRENOLEUKODYSTROPHY PROTEIN (ALDP) AND DISEASE PATHOGENESIS

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder characterized by abnormal accumulation of saturated very long chain fatty acids in tissues and body fluids with predominance in brain white matter and adrenal cortex. The clinical phenotype is highly variable ranging from the severe childhood cerebral form to asymptomatic persons. The responsible ALD gene encodes the adrenoleukodystrophy protein (ALDP), a peroxisomal integral membrane protein that is a member of the ATP-binding cassette (ABC) transporter protein family. The patient gene mutations are heterogeneously distributed over the functional domains of ALDP. The extreme variability in clinical phenotype, even within one affected family, indicates that besides the ALD gene mutations other factors strongly influence the clinical phenotype. To understand the cell biology and function of mammalian peroxisomal ABC transporters and to determine their role in the pathogenesis of X-ALD we developed a system for expressing functional ABC protein domains in fusion with the maltose binding protein. Wild type and mutant fusion proteins of the nucleotide-binding fold were overexpressed, purified, and characterized by photoaffinity labeling with 8-azido ATP or 8-azido GTP and a coupled ATP regenerating enzyme assay for ATPase activity. Our studies provide evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter and that ALD gene mutations alter peroxisomal transport function. The established disease model will be used further to study the influence of possible disease modifier proteins on ALDP function.

Preliminary immunocytochemical studies of MDR-1 and MDR-3 Pgp expression in B-cell leukaemias.

P-glycoproteins (Pgps) belong to the family of ATP binding cassette (ABC) transporter proteins. In humans two Pgp genes have been identified; mdr-1 and mdr-3. Classical Multiple Drug Resistance (MDR) is associated with over expression of the mdr-1 gene product, P-170. No role for mdr-3 in MDR has yet been proven. However there is evidence that mdr-3 overexpression may be associated with drug resistance in certain B-cell lymphocytic leukaemias. In an immunocytochemical study we have looked at a selection of B-cell leukaemias for mdr-1 and mdr-3 encoded Pgp expression using monoclonal antibodies specific for the mdr-1 and mdr-3 encoded gene products. In B-CLL patients a differential pattern of MDR-3 positive staining was observed; suggesting that MDR-3 positivity may be associated with a more malignant phenotype in B-CLL. This pattern was not observed with MDR-1 positivity. We also observed MDR-3 positivity in an AML stage M5a patient which is the first report of MDR-3 Pgp expression being detected in AML; suggesting that MDR-3 Pgp expression may be limited to particular subtypes of this disease. Results from B-NHL cases were inconclusive with varying expression of MDR-1 and MDR-3 Pgps observed. Work is currently underway to further explain the significance of these findings.

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium.

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinus dnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them.