Multidrug Resistance Efflux Transporters Research Articles

Expression of the cobalamin transporters cubam and MRP1 in the canine ileum-Upregulation in chronic inflammatory enteropathy.

Chronic inflammatory enteropathy (CIE) in dogs, a spontaneous model of human inflammatory bowel disease (IBD), is associated with a high rate of cobalamin deficiency. The etiology of hypocobalaminemia in human IBD and canine CIE remains unknown, and compromised intestinal uptake of cobalamin resulting from ileal cobalamin receptor deficiency has been proposed as a possible cause. Here, we evaluated the intestinal expression of the cobalamin receptor subunits, amnionless (AMN) and cubilin (CUBN), and the basolateral efflux transporter multi-drug resistance protein 1 (MRP1) in 22 dogs with CIE in comparison to healthy dogs. Epithelial CUBN and AMN levels were quantified by confocal laser scanning microscopy using immunohistochemistry in endoscopic ileal biopsies from dogs with (i) CIE and normocobalaminemia, (ii) CIE and suboptimal serum cobalamin status, (iii) CIE and severe hypocobalaminemia, and (iv) healthy controls. CUBN and MRP1 expression was quantified by RT-qPCR. Receptor expression was evaluated for correlation with clinical patient data. Ileal mucosal protein levels of AMN and CUBN as well as mRNA levels of CUBN and MRP1 were significantly increased in dogs with CIE compared to healthy controls. Ileal cobalamin receptor expression was positively correlated with age, clinical disease activity index (CCECAI) score, and lacteal dilation in the ileum, inversely correlated with serum folate concentrations, but was not associated with serum cobalamin concentrations. Cobalamin receptor downregulation does not appear to be the primary cause of hypocobalaminemia in canine CIE. In dogs of older age with severe clinical signs and/or microscopic intestinal lesions, intestinal cobalamin receptor upregulation is proposed as a mechanism to compensate for CIE-associated hypocobalaminemia. These results support oral supplementation strategies in hypocobalaminemic CIE patients.

Effective targeting of breast cancer by the inhibition of P-glycoprotein mediated removal of toxic lipid peroxidation byproducts from drug tolerant persister cells

Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of mutations during chemotherapy. Here we show that following in vitro treatment by chemotherapy, epithelial breast cancer cells adopt a transient drug tolerant phenotype characterized by cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and the reversible upregulation of the multidrug resistance (MDR) efflux transporter P-glycoprotein (P-gp). The drug tolerant persister (DTP) state is reversible, as cells eventually resume proliferation, giving rise to a cell population resembling the initial, drug-naïve cell lines. However, recovery after doxorubicin treatment is almost completely eliminated when DTP cells are cultured in the presence of the P-gp inhibitor Tariquidar. Mechanistically, P-gp contributes to the survival of DTP cells by removing reactive oxygen species-induced lipid peroxidation products resulting from doxorubicin exposure. In vivo, prolonged administration of Tariquidar during doxorubicin treatment holidays resulted in a significant increase of the overall survival of Brca1−/−;p53−/− mammary tumor bearing mice. These results indicate that prolonged administration of a P-gp inhibitor during drug holidays would likely benefit patients without the risk of aggravated side effects related to the concomitantly administered toxic chemotherapy. Effective targeting of DTPs through the inhibition of P-glycoprotein may result in a paradigm shift, changing the focus from countering drug resistance mechanisms to preventing or delaying therapy resistance.

Variable p53/Nrf2 crosstalk contributes to triptolide-induced hepatotoxic process

Variable p53/Nrf2 crosstalk contributes to triptolide-induced hepatotoxic process

Metagenomic and Recombination Analyses of Antimicrobial Resistance Genes from Recreational Waters of Black Sea Coastal Areas and Other Marine Environments Unveil Extensive Evidence for Their both Intrageneric and Intergeneric Transmission across Genetically Very Diverse Microbial Communities.

Metagenomic and Recombination Analyses of Antimicrobial Resistance Genes from Recreational Waters of Black Sea Coastal Areas and Other Marine Environments Unveil Extensive Evidence for Their both Intrageneric and Intergeneric Transmission across Genetically Very Diverse Microbial Communities.

Molecular Modeling Strategies of Cancer Multidrug Resistance.

Molecular Modeling Strategies of Cancer Multidrug Resistance.

In-vitro characterization of coding variants with predicted functional implications in the efflux transporter multidrug resistance protein 4 (MRP4, ABCC4).

MRP4 (gene ABCC4) is a polymorphic efflux transporter that has been implicated in drug-induced toxicity. We selected ten commonly observed MRP4 coding variants among Europeans for experimental characterization including nine variants predicted to be deleterious or functional (combined annotation-dependent depletion score >15). We assessed protein localization and activity by quantifying intracellular accumulation of two prototypic substrates, taurocholic acid (TCA) and estradiol 17-β-glucuronide (E217βG), in HEK293T over-expressing MRP4 wildtype or variant where cellular substrate loading was optimized through co-transfection with an uptake transporter. V458M, a novel variant not previously studied, and T1142M, showed reduced activity compared to MRP4 wildtype for E217βG and TCA (P < 0.01), while L18I, G187W, K293E, and R531Q moderately increased activity in a substrate-dependent manner. Protein expression analysis indicated reduced cell surface expression for V458M (P < 0.01) but not T1142M compared to wildtype. Reduced activity may result from altered surface expression (V458M) or intrinsic activity as both variants map within the nucleotide-binding domains of MRP4. G187W showed a trend for reduced surface expression (P = 0.054) despite transport comparable or increased to wildtype suggesting enhanced intrinsic activity. Our findings suggest moderately altered MRP4 activity in six out of nine predicted functional variants with likely different mechanisms and substrate-specific effects. Cell-based studies using multiple known substrates are warranted to more accurately predict functional variants in this clinically important transporter.

Genomic Insight of Alicyclobacillus mali FL18 Isolated From an Arsenic-Rich Hot Spring.

Extreme environments are excellent places to find microorganisms capable of tolerating extreme temperature, pH, salinity pressure, and elevated concentration of heavy metals and other toxic compounds. In the last decades, extremophilic microorganisms have been extensively studied since they can be applied in several fields of biotechnology along with their enzymes. In this context, the characterization of heavy metal resistance determinants in thermophilic microorganisms is the starting point for the development of new biosystems and bioprocesses for environmental monitoring and remediation. This work focuses on the isolation and the genomic exploration of a new arsenic-tolerant microorganism, classified as Alicyclobacillus mali FL18. The bacterium was isolated from a hot mud pool of the solfataric terrains in Pisciarelli, a well-known hydrothermally active zone of the Campi Flegrei volcano near Naples in Italy. A. mali FL18 showed a good tolerance to arsenite (MIC value of 41 mM), as well as to other metals such as nickel (MIC 30 mM), cobalt, and mercury (MIC 3 mM and 17 μM, respectively). Signatures of arsenic resistance genes (one arsenate reductase, one arsenite methyltransferase, and several arsenite exporters) were found interspersed in the genome as well as several multidrug resistance efflux transporters that could be involved in the export of drugs and heavy metal ions. Moreover, the strain showed a high resistance to bacitracin and ciprofloxacin, suggesting that the extreme environment has positively selected multiple resistances to different toxic compounds. This work provides, for the first time, insights into the heavy metal tolerance and antibiotic susceptibility of an Alicyclobacillus strain and highlights its putative molecular determinants.

Nuclear factor-κB signaling inhibitors revert multidrug-resistance in breast cancer cells

Nuclear factor-κB signaling inhibitors revert multidrug-resistance in breast cancer cells

Therapeutic strategies to overcome taxane resistance in cancer.

Therapeutic strategies to overcome taxane resistance in cancer.

S-(+)-Pentedrone and R-(+)-methylone as the most oxidative and cytotoxic enantiomers to dopaminergic SH-SY5Y cells: Role of MRP1 and P-gp in cathinones enantioselectivity

S-(+)-Pentedrone and R-(+)-methylone as the most oxidative and cytotoxic enantiomers to dopaminergic SH-SY5Y cells: Role of MRP1 and P-gp in cathinones enantioselectivity

Quantitative proteomic analysis reveals the mechanisms of polymyxin B toxicity to Escherichia coli

Quantitative proteomic analysis reveals the mechanisms of polymyxin B toxicity to Escherichia coli

Epigenetic Regulation of Multidrug Resistance Protein 1 and Breast Cancer Resistance Protein Transporters by Histone Deacetylase Inhibition

Epigenetic Regulation of Multidrug Resistance Protein 1 and Breast Cancer Resistance Protein Transporters by Histone Deacetylase Inhibition

MarR family proteins are important regulators of clinically relevant antibiotic resistance.

There has been a rapid spread of multidrug-resistant (MDR) bacteria across the world. MDR efflux transporters are an important mechanism of antibiotic resistance in many pathogens among both Gram positive and Gram negative bacteria. These pumps can recognize a variety of chemically and structurally different compounds, including innate and clinically administered antibiotics. Intriguingly, these efflux pumps are often regulated by transcription factors that themselves bind a diverse set of substrates thereby allowing them to regulate the expression of their cognate MDR efflux pumps. One significant family of such transcription factors is the Multiple antibiotic resistance Repressor (MarR) family. Members of this family are well conserved across different bacterial species and in some cases are known to regulate vital bacterial functions. This review focusses on the role of MarR family transcriptional factors in antibiotic resistance within a select group of clinically relevant pathogens.

The multi-factorial nature of clinical multidrug resistance in cancer.

The multi-factorial nature of clinical multidrug resistance in cancer.

Guanidine Riboswitch-Regulated Efflux Transporters Protect Bacteria against Ionic Liquid Toxicity.

Plant cell walls contain a renewable, nearly limitless supply of sugar that could be used to support microbial production of commodity chemicals and biofuels. Imidazolium ionic liquid (IIL) solvents are among the best reagents for gaining access to the sugars in this otherwise recalcitrant biomass. However, the sugars from IIL-treated biomass are inevitably contaminated with residual IILs that inhibit growth in bacteria and yeast, blocking biochemical production by these organisms. IIL toxicity is, therefore, a critical roadblock in many industrial biosynthetic pathways. Although several IIL-tolerant (IILT) bacterial and yeast isolates have been identified in nature, few genetic mechanisms have been identified. In this study, we identified two IILTBacillus isolates as well as a spontaneous IILTEscherichia coli lab strain that are tolerant to high levels of two widely used IILs. We demonstrate that all three IILT strains contain one or more pumps of the small multidrug resistance (SMR) family, and two of these strains contain mutations that affect an adjacent regulatory guanidine riboswitch. Furthermore, we show that the regulation of E. colisugE by the guanidine II riboswitch can be exploited to promote IIL tolerance by the simple addition of guanidine to the medium. Our results demonstrate the critical role that transporter genes play in IIL tolerance in their native bacterial hosts. The study presented here is another step in engineering IIL tolerance into industrial strains toward overcoming this key gap in biofuels and industrial biochemical production processes.IMPORTANCE This study identifies bacteria that are tolerant to ionic liquid solvents used in the production of biofuels and industrial biochemicals. For industrial microbiology, it is essential to find less-harmful reagents and microbes that are resistant to their cytotoxic effects. We identified a family of small multidrug resistance efflux transporters, which are responsible for the tolerance of these strains. We also found that this resistance can be caused by mutations in the sequences of guanidine-specific riboswitches that regulate these efflux pumps. Extending this knowledge, we demonstrated that guanidine itself can promote ionic liquid tolerance. Our findings will inform genetic engineering strategies that improve conversion of cellulosic sugars into biofuels and biochemicals in processes where low concentrations of ionic liquids surpass bacterial tolerance.

Impact of combined MRP1 inhibition and mutant p53-targeting compound APR-246.

e14712 Background: The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. Mutant p53 protein is often expressed at high levels and accompanied with gain-of-function activities that promote tumor development and resistance towards conventional treatment. APR-246 is a mutant p53-reactivating small molecule undergoing a Phase III clinical study in myelodysplastic syndrome (MDS), and several phase II studies. APR-246 is non-enzymatically converted to its active product methylene quinuclidinone (MQ) which binds to cysteine residues in p53. This stimulates proper folding of p53's DNA-binding core domain, leading to cell death. APR-246 also exhibits pro-oxidant activity as the electrophile MQ binds and inactivates important antioxidants such as glutathione and thioredoxin reductase, which both are essential for cellular defense against oxidative and electrophilic stress. Methods: All results are in vitro experiments by LC-MS as well as experiments in cultured cells, including 14C-APR-246/MQ detection, cell viability measurements, LC-MS analysis, enzyme recycling measurements and Western blotting. Results: We have utilized 14C-labelled APR-246 to investigate the effect of APR-246 and its active moiety MQ in tumor cell cultures upon inhibition of efflux transporter multidrug resistance protein 1 (MRP1) or cystine-glutamate transporter (xCT). Transient downregulation or small molecule inhibitors (MK-571, reversan, sulfasalazin) targeting MRP1 or xCT increased drug content and shifted intra- and extracellular thiol status. Missense mutant TP53-carrying cells exhibited higher sensitivity to APR-246 and combination therapies. MRP1 inhibition showed strong synergy with APR-246 and increased intracellular levels of MQ conjugated to glutathione (GS-MQ). We found that GS-MQ conjugate formation is reversible, and suggest that the intrinsic reversibility of MQ adduct formation is an important aspect of the mechanism of action of APR-246. This reversibility may also in part account for the benign safety profile reported from clinical studies with APR-246. Conclusions: Sensitivity to APR-246 is affected by MRP1 efflux activity and the redox status, reflected in ratios of cysteine/cystine and reduced/oxidized glutathione (GSH/GSSG).

Proof of an Outer Membrane Target of the Efflux Inhibitor Phe-Arg-β-Naphthylamide from Random Mutagenesis.

Phe-Arg-β-naphthylamide (PAβN) has been characterized as an efflux pump inhibitor (EPI) acting on the major multidrug resistance efflux transporters of Gram-negative bacteria, such as AcrB in Eschericha coli. In the present study, in vitro random mutagenesis was used to evolve resistance to the sensitizing activity of PAβN with the aim of elucidating its mechanism of action. A strain was obtained that was phenotypically similar to a previously reported mutant from a serial selection approach that had no efflux-associated mutations. We could confirm that acrB mutations in the new mutant were unrelated to PAβN resistance. The next-generation sequencing of the two mutants revealed loss-of-function mutations in lpxM. An engineered lpxM knockout strain showed up to 16-fold decreased PAβN activity with large lipophilic drugs, while its efflux capacity, as well as the efficacy of other EPIs, remained unchanged. LpxM is responsible for the last acylation step in lipopolysaccharide (LPS) synthesis, and lpxM deficiency has been shown to result in penta-acylated instead of hexa-acylated lipid A. Modeling the two lipid A types revealed steric conformational changes due to underacylation. The findings provide evidence of a target site of PAβN in the LPS layer, and prove membrane activity contributing to its drug-sensitizing potency.

Reserpine Is the New Addition into the Repertoire of AcrB Efflux Pump Inhibitors

Acriflavine resistance protein B (AcrB) serves as prototype for multidrug resistance (MDR) efflux transporters of resistance nodulation division (RND) superfamily. AcrB has been proven as potential drug target with many synthetic and natural inhibitors have been identified such as those belonging to pyranopyridine, naphthamide and pimozide classes. The plant derived alkaloid inhibitors represented by reserpine has been found to inhibit both ATP binding cassette and major facilitator efflux transporters. In this study we report the reserpine induced inhibition of RND transporter AcrB. The preliminary docking analysis hints that reserpine shares its binding site with ciprofloxacin, a known substrate of AcrB and could possibly act as competitive inhibitor. For in vitro validation, AcrB from Salmonella typhi was cloned under the control of tac promoter and resulting vector was introduced into E. coli C41(DE3). Under autoinduced conditions, cells overexpressing AcrB transporter were subjected to combined dose of ciprofloxacin and reserpine. The combined exposure resulted in enhanced ciprofloxacin-induced growth inhibition of cells expressing AcrB transporter as compared to control cells transformed with vector of backbone sequence. Time kill analysis further confirmed these findings. To the best of our knowledge, this is first study to show that exposure to reserpine induces inhibition of AcrB. The assay developed in this study allows simple and reproducible detection of substrate/inhibitor effects upon AcrB and related efflux transporters.

Chemosensitivity and survival of non-small cell lung carcinoma patients receiving neoadjuvant therapy depend on the expression of multidrug efflux transporters

The current study aimed to evaluate the impact of neoadjuvant chemotherapy (NACT) on the expression of three major multidrug resistance efflux transporters, P-glycoprotein (P-gp/ABCB1), multidrug resistance associated protein (MRP1/ABCC1) and breast cancer resistance protein (BCRP/ABCG2) in non-small cell lung carcinoma (NSCLC) patients. Particularly, their role as molecular markers of chemosensitivity and prognosis of NSCLC patients receiving NACT was investigated. To that end, we specifically studied mRNA and protein expression of these three efflux transporters in two independent groups, each consisting of 35 NSCLC patients who did or did not receive platinum-based NACT. Alterations in the expression of MDR efflux transporters were statistically analyzed in relation to NACT status, and their associations were evaluated regarding patients’ survival. The frequency of samples with positive MRP1 expression was significantly decreased in the NACT group, regardless of the applied platinum drugs which are known to induce MRP1 expression. On the other hand, the incidence of BCRP expressing tumor specimens, doubly positive BCRP and P-gp as well as triple positive samples increased in the NACT group. Importantly, patients lacking P-gp expression had more favorable prognosis with NACT than without NACT, whereas the status of MRP1 and BCRP did not influence the patients’ survival in both investigated groups. Collectively, we show that decreased MRP1 and increased BCRP expression after NACT could determine the chemosensitivity of NSCLC following adjuvant therapy, whereas P-gp expression status could be considered a prognostic marker for NSCLC patients who can benefit from NACT treatment.

Predicting Inhibitors for Multidrug Resistance Associated Protein-2 Transporter by Machine Learning Approach.

The efflux transporter multidrug resistance associated protein-2 belongs to ATP-binding cassette superfamily which plays an important role in multidrug resistance and drugdrug interactions. Efflux transporters are considered to be important targets for increasing the efficacy of drugs and importance of computational study of efflux transporters for predicting substrates, non-substrates, inhibitors and non-inhibitors is well documented. Previous work on predictive models for inhibitors of multidrug resistance associated Protein-2 efflux transporter showed that machine learning methods produced good results. The aim of the present work was to develop a machine learning predictive model to classify inhibitors and non-inhibitors of multidrug resistance associated protein-2 transporter using a well refined dataset. In this study, the various algorithms of machine learning were used to develop the predictive models i.e. support vector machine, random forest and k-nearest neighbor. The methods like variance threshold, SelectKBest, random forest, and recursive feature elimination were used to select the features generated by PyDPI. A total of 239 molecules consisting of 124 inhibitors and 115 non-inhibitors were used for model development. The best multidrug resistance associated protein-2 inhibitor model showed prediction accuracies of 0.76, 0.72 and 0.79 for training, 5-fold cross-validation and external sets, respectively. It was observed that support vector machine model built on features selected using recursive feature elimination method shows the best performance. The developed model can be used in the early stages of drug discovery for identifying the inhibitors of multidrug resistance associated protein-2 efflux transporter.