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1.
J Immunol Methods ; 483: 112794, 2020 08.
Article in English | MEDLINE | ID: mdl-32428450

ABSTRACT

A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug resistant (MDR) gene deficient mice so that only MDR deficient cells expressing the target molecule would be depleted while target molecule expressing, but MDR sufficient, cells are spared. This allows targeted depletion at a higher degree of specificity than has been previously achieved. We have applied this technique to study trogocytosis, the intercellular transfer of cell surface molecules, but this principle could also be adapted using technology already available for use in other fields of study.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cytotoxicity, Immunologic/drug effects , Genes, MDR/physiology , Immunotoxins/toxicity , Lymphocyte Depletion/methods , ATP Binding Cassette Transporter, Subfamily B/deficiency , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2/deficiency , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , Animals , Female , Graft Survival/drug effects , Heart Transplantation , Histocompatibility Antigens Class II/immunology , Immunoconjugates/toxicity , Immunoglobulin Fab Fragments/toxicity , Kidney Transplantation , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Ribosome Inactivating Proteins, Type 1/toxicity , Spleen/drug effects , Spleen/immunology , Spleen/pathology , Transplantation Tolerance/drug effects
2.
Biomed Pharmacother ; 96: 371-377, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29028589

ABSTRACT

As the first-line drug for breast cancer chemotherapy, doxorubicin (Dox) has strong cardiotoxicity. Meanwhile, prolonged Dox treatment of patients with breast cancer may result in resistance of breast cancer cells to Dox and an increased number of Dox-resistant breast cancer stem cells (BCSCs), thereby easily leading to breast cancer relapse. TanshinoneIIA (Tan IIA) has anti-tumor activity in addition to its cardiovascular protective effect. By preparing Dox resistant human breast cancer MCF-7 cells, here, we wanted to assess a new use of Tan IIA in enhancing the chemosensitivity of breast cancer cells to Dox and investigated its possible mechanisms. The results showed that Tan IIA could enhance the anti-tumor effect of Dox on MCF-7 and MCF-7/dox cells in a dose-dependent manner, especially that of on MCF-7/dox cells. Even nontoxic dose of Tan IIA could also promote intracellular Dox accumulation of MCF-7 and MCF-7/dox cells through down-regulating the expression of efflux ABC transporters including P-gp, BCRP and MRP1, which can effectively eliminated cancerous cells including BCSCs, thereby enhancing the chemosensitivity of breast cancer. Therefore, Tan IIA can be used as a new potential chemotherapeutic sensitizer for the combination treatment of breast cancer.


Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Breast Neoplasms/metabolism , Doxorubicin/pharmacology , Genes, MDR/physiology , Plant Extracts/therapeutic use , Salvia miltiorrhiza , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Down-Regulation/physiology , Doxorubicin/therapeutic use , Female , Gene Expression Regulation, Neoplastic , Genes, MDR/drug effects , Humans , MCF-7 Cells , Plant Extracts/isolation & purification , Plant Extracts/pharmacology
3.
Biomed Pharmacother ; 96: 659-666, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29035832

ABSTRACT

Nowadays, lung cancer, as a health problem in worldwide, has high mortality both in men and women. Despite advances in diagnosis and surgical techniques of lung cancer in recent decades, chemotherapy is still a fundamentally and extensively useful strategy. Vinca alkaloids are a class of important and widely used drugs in the treatment of lung cancer, targeting on the Vinca binding site at the exterior of microtubule plus ends. Either intrinsic or acquired resistance to chemotherapy of Vinca alkaloids has been a major obstacle to the treatment of lung cancer, which arose great interests in studies of understanding and overcoming resistance. In this review, we focused on the application and resistance mechanisms of the Vinca alkaloids such as vinblastine, vincristine, vinorelbine and vinflunine in lung cancer. We reviewed characteristic resistance mechanisms in lung cancer including over-expression of ATP-binding cassette (ABC) transporters P-glycoprotein and structural, functional or expression alterations of ß-tubulin (ßII, ßIII, ßIV) which may devote to the development of acquired resistance to the Vinca alkaloids; multidrug-resistance proteins (MRP1, MRP2, MRP3) and RLIP76 protein have also been identified that probably play a significant role in intrinsic resistance. Lung resistance-related protein (LRP) is contributed to lung cancer therapy resistance, but is not deal with the Vinca alkaloids resistance in lung cancer. Understanding the principle of the Vinca alkaloids in clinical application and mechanisms of drug resistance will support individualized lung cancer therapy and improve future therapies.


Subject(s)
Drug Resistance, Neoplasm/drug effects , Genes, MDR/drug effects , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Vinca Alkaloids/metabolism , Vinca Alkaloids/therapeutic use , Animals , Antineoplastic Agents, Phytogenic/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/therapeutic use , Binding Sites/drug effects , Binding Sites/physiology , Drug Resistance, Neoplasm/physiology , Genes, MDR/physiology , Humans , Microtubules/drug effects , Microtubules/metabolism , Tumor Cells, Cultured , Vinblastine/analogs & derivatives , Vinblastine/metabolism , Vinblastine/pharmacology , Vinca Alkaloids/pharmacology , Vincristine/metabolism , Vincristine/pharmacology , Vincristine/therapeutic use
4.
Microb Pathog ; 108: 122-128, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28457900

ABSTRACT

Acinetobacter baumannii potential to form biofilm and exhibit multiple antibiotic resistances may be responsible in its survival in hospital environment. Accordingly, our study was aimed to determine the correlation between ability of biofilm formation and the frequency of biofilm related genes with antibiotic resistance phenotypes, and also the categorization of their patterns in clinical and environmental isolates. A total of 75 clinical and 32 environmental strains of the A. baumannii were collected and identified via API 20NE. Antibiotic susceptibility was evaluated by disk diffusion and microdilution broth methods. Biofilm formation assay was performed by microtiter plate method. OXA types and biofilm related genes including BlaOXA-51, BlaOXA-23, BlaOXA-24, BlaOXA-58, bap, blaPER-1, and ompA were amplified by PCR. The rate of MDR A. baumannii in clinical isolates (100%) was higher than environmental (81.2%) isolates (p < 0.05). Among 10 antibiotypes, the predominant resistance pattern in clinical and environmental isolates was antibiotypes I (85.3 and 78.1%, respectively). Analysis of the frequency of blaOXA-23 gene revealed a statistically significant difference between clinical (85.3%) and environmental (68.7%) isolates (p < 0.05). The prevalence of strong biofilm producers in clinical and environmental isolates were 31.2%-58.7%, respectively. In the clinical and environmental isolates, the frequencies of ompA, blaRER-1 and bap genes were 100%, 53.3%, 82.7% and 100%, 37.5%, 84.4% respectively. Statistical analysis revealed a significant correlation between the frequency of MDR isolates and biofilm formation ability (p = 0.008). The high frequency of antibiotype I would be indicated that an outbreak has been happened earlier and an endemic strain is currently being settled in the hospital environment. It would be suggested that if there was no difference in the frequency of pattern I and biofilm formation ability between clinical and environmental isolates, it is a critical point representing the higher risk of bacterial transmission from environment to the patients. The resulting data would be assisted in the improvement of disinfection strategies to better control of nosocomial infections. One dominant resistance pattern has shown among clinical and environmental isolates. The frequency of blaOXA-23 had significant difference between clinical and environmental isolates. The presence of bap gene in the A. baumannii isolates was associated with biofilm formation. There was a significant correlation between multiple drug resistance and biofilm formation. The clinical isolates had a higher ability to form strong biofilms compared to the environmental samples.


Subject(s)
Acinetobacter baumannii/genetics , Acinetobacter baumannii/physiology , Biofilms/growth & development , Genes, MDR/genetics , Genes, MDR/physiology , Acinetobacter Infections/microbiology , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/isolation & purification , Adult , Aged , Aged, 80 and over , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacteriological Techniques , Biofilms/drug effects , Cross Infection/microbiology , Cross Infection/prevention & control , DNA, Bacterial/analysis , Drug Resistance, Multiple, Bacterial/genetics , Environmental Microbiology , Female , Humans , Iran , Male , Microbial Sensitivity Tests , Middle Aged
5.
Article in English | MEDLINE | ID: mdl-28373194

ABSTRACT

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Biological Transport/genetics , Biological Transport/physiology , Genes, MDR/genetics , Genes, MDR/physiology , Microbial Sensitivity Tests , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Nucleosides/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicity
6.
Arch Microbiol ; 199(3): 465-474, 2017 Apr.
Article in English | MEDLINE | ID: mdl-27830269

ABSTRACT

Staphylococcus aureus is a serious causative agent of infectious disease. Multidrug-resistant strains like methicillin-resistant S. aureus compromise treatment efficacy, causing significant morbidity and mortality. Active efflux represents a major antimicrobial resistance mechanism. The proton-driven multidrug efflux pump, LmrS, actively exports structurally distinct antimicrobials. To circumvent resistance and restore clinical efficacy of antibiotics, efflux pump inhibitors are necessary, and natural edible spices like cumin are potential candidates. The mode of cumin antibacterial action and underlying mechanisms behind drug resistance inhibition, however, are unclear. We tested the hypothesis that cumin inhibits LmrS drug transport. We found that cumin inhibited bacterial growth and LmrS ethidium transport in a dosage-dependent manner. We demonstrate that cumin is antibacterial toward a multidrug-resistant host and that resistance modulation involves multidrug efflux inhibition.


Subject(s)
Cuminum/chemistry , Drug Resistance, Multiple, Bacterial/drug effects , Genes, MDR/physiology , Methicillin-Resistant Staphylococcus aureus/drug effects , Plant Extracts/pharmacology , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Resistance, Multiple, Bacterial/genetics , Ethidium/metabolism , Genes, MDR/genetics , Microbial Sensitivity Tests , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics
7.
Biochem Pharmacol ; 133: 43-62, 2017 06 01.
Article in English | MEDLINE | ID: mdl-27765485

ABSTRACT

The discovery of penicillin followed by streptomycin, tetracycline, cephalosporins and other natural, semi-synthetic and synthetic antimicrobials completely revolutionized medicine by reducing human morbidity and mortality from most of the common infections. However, shortly after they were introduced to clinical practice, the development of resistance was emerged. The decreasing interest from antibiotic industry in spite of rapid global emergence of antibiotic resistance is a tough dilemma from the pointview of public health. The efficiency of antimicrobial treatment is determined by both pharmacokinetics and pharmacodynamics. In spite of their selective toxicity, antibiotics still cause severe, life-threatening adverse reactions in host body mostly due to defective drug metabolism or excessive dosing regimen. The present article aims at updating current knowledge on pharmacokinetics/pharmacodynamics concepts and models, toxicity of antibiotics as well as antibiotic resistance mechanisms, resistome analyses and search for novel antibiotic resistance determinants with special emphasis given to the-state-of-the-art regarding multidrug efflux pumps and their additional physiological functions in stress adaptation and virulence of bacteria. All these issues are highly linked to each other and not only important for most efficient and prolonged use of current antibiotics, but also for discovery and development of new antibiotics and novel inhibitors of antibiotic resistance determinants of pathogens.


Subject(s)
Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/toxicity , Drug Resistance, Bacterial/drug effects , Genes, MDR/drug effects , Membrane Transport Proteins/metabolism , Animals , Drug Resistance, Bacterial/physiology , Genes, MDR/physiology , Humans
9.
Br J Ophthalmol ; 100(5): 708-12, 2016 May.
Article in English | MEDLINE | ID: mdl-26719489

ABSTRACT

BACKGROUND/AIM: There has been increasing evidence of the emergence of antiseptic resistance mediated by quaternary ammonium compound (QAC) resistance genes, which may reduce the efficacy of disinfection. Although the presence of QAC-positive staphylococci has been shown to be elevated in contact lens wearers, the efficacy of multipurpose solutions (MPS) against such isolates has not been determined. This study investigated the efficacy of four MPS for rigid gas permeable (RGP) lenses against staphylococci-harbouring QAC genes. METHODS: Ability to reduce viability by three or more log reductions of four MPS for RGP lenses was tested against 60 disinfectant-resistant gene-positive staphylococci, comprising 38 coagulase-negative staphylococci (CNS) (17 ITALIC! qacA/B, 7 ITALIC! smr, 5 ITALIC! qacH, 9 habouring two or more genes) and 22 ITALIC! Staphylococcus aureus (16 ITALIC! qacA/B, 4 ITALIC! smr, 2 ITALIC! qacA/B+ ITALIC! smr)). 60 gene-negative isolates of staphylococci (30 CNS and 30 ITALIC! S aureus) were used as controls. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these four MPS were determined. RESULTS: Although there was some variation between solutions, all failed to achieve a 3-log reduction in some ITALIC! S aureus and CNS isolates. Strains harbouring disinfectant-resistant genes were significantly less likely to be reduced by 3 logs by three of the solutions. Overall, the MIC and MBC of the four MPS against gene-positive clinical isolates were significantly higher than those of gene-negative isolates. CONCLUSION: The efficacy of MPS solutions for RGP lenses against staphylococci varied. The presence of disinfectant-resistance genes significantly adversely affected disinfecting capacity of RGP solutions.


Subject(s)
Contact Lens Solutions/pharmacology , Contact Lenses/microbiology , Drug Resistance, Multiple, Bacterial/genetics , Genes, MDR/physiology , Orthokeratologic Procedures , Staphylococcus aureus/drug effects , Antiporters/genetics , Bacterial Proteins/genetics , Colony Count, Microbial , Conjunctiva/microbiology , Humans , Membrane Transport Proteins/genetics , Microbial Sensitivity Tests , Quaternary Ammonium Compounds/pharmacology , Repressor Proteins/genetics , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purification
10.
J Bacteriol ; 198(2): 332-42, 2016 01 15.
Article in English | MEDLINE | ID: mdl-26527645

ABSTRACT

UNLABELLED: The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCE: The structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrAB-TolC-mediated multidrug export.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Ethidium/metabolism , Genes, MDR/physiology , Lipoproteins/metabolism , Membrane Transport Proteins/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Binding Sites , Computational Biology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Deletion , Lipoproteins/genetics , Membrane Transport Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Plasmids , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
11.
J Bacteriol ; 197(15): 2479-88, 2015 Aug 01.
Article in English | MEDLINE | ID: mdl-25962916

ABSTRACT

UNLABELLED: The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine ß-naphthylamide (PAßN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAßN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAßN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAßN. Strong action of PAßN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAßN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAßN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAßN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAßN on membrane integrity are compounded in cells unable to extrude PAßN. IMPORTANCE: The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds.


Subject(s)
Bacterial Outer Membrane Proteins/physiology , Cell Membrane/physiology , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial/physiology , Genes, MDR/physiology , Membrane Transport Proteins/physiology , Animals , Anti-Bacterial Agents/pharmacology , Dipeptides/pharmacology , Drug Resistance, Multiple, Bacterial , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/drug effects , Genes, MDR/genetics , Humans , Microbial Sensitivity Tests , Permeability , Phenotype , Polymyxin B/analogs & derivatives , Polymyxin B/pharmacology , Rabbits
12.
Toxicol Lett ; 235(3): 189-98, 2015 Jun 15.
Article in English | MEDLINE | ID: mdl-25865432

ABSTRACT

ABC transporters play an important role in the disposition of avermectins in several animal species. In this study the interactions of three key avermectins, abamectin, emamectin and ivermectin, with human and mouse homologues of MDR1 (ABCB1/Abcb1a) and MRP (ABCC/Abcc), transporters endogenously expressed by human SH-SY5Y and mouse N2a neuroblastoma cells were investigated. In both cell lines, retention of the fluorescent dye H33342 was found to be significantly increased in the presence of avermectins and cyclosporin A. These effects were shown to be unresponsive to the BCRP inhibitor Ko-143 and therefore MDR1/Mdr1-dependent. Avermectins inhibited MDR1/Mdr1a-mediated H33342 dye efflux, with apparent Ki values of 0.24±0.08 and 0.18±0.02µM (ivermectin); 0.60±0.07 and 0.56±0.02µM (emamectin) and 0.95±0.08 and 0.77±0.25µM (abamectin) in SH-SY5Y and N2a cells, respectively. There were some apparent affinity differences for MDR1 and Mdr1a within each cell line (affinity for ivermectin>emamectin≥abamectin, P<0.05 by One-Way ANOVA), but importantly, the Ki values for individual avermectins for human MDR1 or mouse Mdr1a were not significantly different. MK571-sensitive retention of GSMF confirmed the expression of MRP/Mrp efflux transporters in both cell lines. Avermectins inhibited MRP/Mrp-mediated dye efflux with IC50 values of 1.58±0.51 and 1.94±0.72µM (ivermectin); 1.87±0.57 and 2.74±1.01µM (emamectin) and 2.25±0.01 and 1.68±0.63µM (abamectin) in SH-SY5Y and N2a cells, respectively. There were no significant differences in IC50 values between individual avermectins or between human MRP and mouse Mrp. Kinetic data for endogenous human MDR1/MRP isoforms in SH-SY5Y cells and mouse Mdr1a/b/Mrp isoforms in N2a cells are comparable for the selected avermectins. All are effluxed at concentrations well above 0.05-0.1µM ivermectin detected in plasma (Ottesen and Campbell, 1994; Ottesen and Campbell, 1994) This is an important finding in the light of toxicity seen in the Mdr1-deficient animal models CF-1 mice, Mdr1ab (-/-) double knockout mice and Collie dogs. We also confirm MRP/Mrp-mediated avermectin transport in both N2a and SH-SY5Y cell lines.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Genes, MDR/physiology , Ivermectin/analogs & derivatives , Neuroblastoma/metabolism , ATP-Binding Cassette Transporters/genetics , Animals , Antineoplastic Agents/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm , Gene Expression Regulation , Humans , Ivermectin/metabolism , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism
13.
Article in English | MEDLINE | ID: mdl-25236869

ABSTRACT

The expression levels of key enzymes of the xenobiotic metabolism and excretion pathways concerning biotransformation phases I (cytochrome P4501A), II (glutathione S-transferase) and III (multidrug resistance protein) and of the estrogenic biomarker vitellogenin (vtg) were investigated in primary hepatocytes isolated from male Nile tilapia (Oreochromis niloticus) after exposure to diclofenac and metoprolol, two pharmaceuticals prevalent in the aquatic environment worldwide. The lowest test concentration (4×10(-9) M) was chosen to reflect an environmentally relevant exposure situation. Furthermore concentration dependent effects were investigated. Therefore a series of concentrations higher than the environmentally relevant range were used (10- and 100-fold). Diclofenac significantly induced all chosen biomarkers already at the environmentally relevant concentration indicating that biotransformation and elimination occur via the pathways under investigation. Estrogenic potential of this substance was demonstrated by VTG up-regulation as well. Metoprolol was either less effective than diclofenac or metabolized using different pathways. Key enzymes of the xenobiotic metabolism were less (CYP1A, GST) or not (MDRP) induced and a mild increase in vtg mRNA was detected only for 4×10(-8) M. No concentration-dependency for metoprolol was found.


Subject(s)
Cichlids , Diclofenac/toxicity , Gene Expression Regulation/drug effects , Hepatocytes/drug effects , Metoprolol/toxicity , Water Pollutants, Chemical/toxicity , Adrenergic beta-1 Receptor Antagonists/toxicity , Animals , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Biomarkers , Cells, Cultured , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Estrogens/toxicity , Genes, MDR/genetics , Genes, MDR/physiology , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Vitellogenins/genetics , Vitellogenins/metabolism
14.
Antimicrob Agents Chemother ; 59(1): 724-6, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25367915

ABSTRACT

The global consequence of drug efflux gene overexpression in bacteria has not been specifically analyzed because strains showing high-level expression typically have mutations in genes encoding regulatory proteins that control other genes. Results from a transcriptional profiling study performed with a strain of Neisseria gonorrhoeae that is capable of high-level transcription of the mtrCDE efflux pump operon independently of control by cognate regulatory proteins revealed that its overexpression has ramifications for systems other than drug efflux.


Subject(s)
Genes, MDR/physiology , Neisseria gonorrhoeae/metabolism , Gene Expression Profiling , Gene Expression Regulation, Bacterial/genetics , Gene Expression Regulation, Bacterial/physiology , Genes, MDR/genetics , Neisseria gonorrhoeae/genetics , Real-Time Polymerase Chain Reaction , Transcription, Genetic/genetics , Transcription, Genetic/physiology
15.
Cancer Chemother Pharmacol ; 74(4): 809-17, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25113261

ABSTRACT

PURPOSE: Ceramide is glycosylated to glucosylceramide or lactosylceramide, and this glycosylation is a novel multidrug-resistance (MDR) mechanism. In this work, a short-chain ceramide (C6), lactosylceramide (LacCer), and an inhibitor of ceramide glycosylation (D-threo-1-phenyl-2-decanoylamino-3-1-propanol, PDMP) were evaluated on the proliferation of cervical cancer cells. The participation of glucosylceramide synthase (GCS), P-glycoprotein (P-gp), and multidrug-resistance gene-1 (MDR-1) in the resistance to the antiproliferative effect induced by C6 was also evaluated. METHODS: Cell proliferation was determined by crystal violet staining. GCS and MDR-1 mRNA expression was evaluated by real-time RT-PCR assay. GCS and P-gp protein expressions, as well as Rhodamine 123 uptake, which is a functional test for P-gp efflux activity, were determined by flow cytometry. RESULTS: C6 inhibited proliferation of CaLo and CasKi cells with an IC50 of 2.5 µM; however, 50% proliferation of ViBo cells was inhibited with 10 µM. LacCer increased the proliferation of all cells. When cells were treated with PDMP plus C6, no additional effect on antiproliferation induced by C6 was observed in CaLo and CasKi cells; however, proliferation diminished in comparison with C6 alone in ViBo cells. C6 increased GCS and MDR-1 expression in all cells, as well as P-gp expression in CasKi cells. CONCLUSIONS: Cells that have more capacity to glycosylate ceramide and express a higher level of GCS, MDR-1, and P-gp, are more resistant to the antiproliferative effect induced by C6.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Genes, MDR/physiology , Glucosyltransferases/metabolism , Lactosylceramides/metabolism , Morpholines , Uterine Cervical Neoplasms/metabolism , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Female , Glycosylation/drug effects , Humans , Morpholines/antagonists & inhibitors , Morpholines/metabolism , Multidrug Resistance-Associated Proteins , Tumor Cells, Cultured
16.
PLoS One ; 9(5): e96288, 2014.
Article in English | MEDLINE | ID: mdl-24823362

ABSTRACT

BACKGROUND: Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum ß-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance. PRINCIPAL FINDINGS: Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology. CONCLUSIONS AND SIGNIFICANCE: The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Genes, MDR/physiology , Klebsiella pneumoniae/genetics , Membrane Transport Proteins/genetics , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests
17.
Antimicrob Agents Chemother ; 58(6): 3556-9, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24733458

ABSTRACT

The contribution of drug efflux pumps in clinical isolates of Neisseria gonorrhoeae that express extensively drug-resistant or multidrug-resistant phenotypes has heretofore not been examined. Accordingly, we assessed the effect on antimicrobial resistance of loss of the three gonococcal efflux pumps associated with a known capacity to export antimicrobials (MtrC-MtrD-MtrE, MacA-MacB, and NorM) in such clinical isolates. We report that the MIC of several antimicrobials, including seven previously and currently recommended for treatment was significantly impacted.


Subject(s)
Anti-Bacterial Agents/pharmacology , Genes, MDR/physiology , Gonorrhea/microbiology , Membrane Transport Proteins/genetics , Neisseria gonorrhoeae/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Resistance, Multiple, Bacterial/genetics , Humans , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Mutation , Neisseria gonorrhoeae/drug effects
18.
J Bacteriol ; 195(21): 4975-87, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23995639

ABSTRACT

A hallmark characteristic of biofilms is their extraordinary tolerance to antimicrobial agents. While multiple factors are thought to contribute to the high level of antimicrobial tolerance of biofilms, little is known about the timing of induction of biofilm tolerance. Here, we asked when over the course of their development do biofilms gain their tolerance to antimicrobial agents? We demonstrate that in Pseudomonas aeruginosa, biofilm tolerance is linked to biofilm development, with transition to the irreversible attachment stage regulated by the two-component hybrid SagS, marking the timing when biofilms switch to the high-level tolerance phenotype. Inactivation of sagS rendered biofilms but not planktonic cells more susceptible to tobramycin, norfloxacin, and hydrogen peroxide. Moreover, inactivation of sagS also eliminated the recalcitrance of biofilms to killing by bactericidal antimicrobial agents, a phenotype comparable to that observed upon inactivation of brlR, which encodes a MerR-like transcriptional regulator required for biofilm tolerance. Multicopy expression of brlR in a ΔsagS mutant restored biofilm resistance and recalcitrance to killing by bactericidal antibiotics to wild-type levels. In contrast, expression of sagS did not restore the susceptibility phenotype of ΔbrlR mutant biofilms to wild-type levels, indicating that BrlR functions downstream of SagS. Inactivation of sagS correlated with reduced BrlR levels in biofilms, with the produced BrlR being impaired in binding to the previously described BrlR-activated promoters of the two multidrug efflux pump operons mexAB-oprM and mexEF-oprN. Our findings demonstrate that biofilm tolerance is linked to early biofilm development and SagS, with SagS contributing indirectly to BrlR activation.


Subject(s)
Bacterial Proteins/metabolism , Biofilms/growth & development , Drug Resistance, Bacterial/physiology , Gene Expression Regulation, Bacterial/physiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/physiology , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Biomass , Genes, MDR/genetics , Genes, MDR/physiology , Mutation , Promoter Regions, Genetic , Protein Binding , Pseudomonas aeruginosa/genetics
19.
Curr Cancer Drug Targets ; 13(8): 814-28, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23876050

ABSTRACT

The resistance of tumors to a number of structurally and functionally unrelated chemotherapeutic drugs has been a major obstacle for successful cancer chemotherapy. An important mechanism leading to multidrug resistance (MDR) is the overexpression of the 170 kDa P-glycoprotein (P-gp), which is a member of the ATP-binding cassette (ABC) superfamily of membrane transporters, encoded by the MDR1 gene. Aiming to overcome MDR and due to the clinical failure of P-gp inhibitors, downregulation of MDR1 expression by small molecules has been studied as a possible cancer adjuvant chemotherapy. Here we review the current knowledge on MDR1 gene expression downregulation by small molecules and the mechanisms underlying those effects observed in cancer cell lines, in an attempt to identify targets for future therapeutic interventions.


Subject(s)
Antineoplastic Agents/therapeutic use , Drug Evaluation, Preclinical , Drug Resistance, Multiple , Drug Resistance, Neoplasm/genetics , Genes, MDR/drug effects , Neoplasms/drug therapy , Neoplasms/metabolism , Animals , Genes, MDR/physiology , Humans , Small Molecule Libraries
20.
J Bacteriol ; 195(16): 3651-62, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23749979

ABSTRACT

The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using a lacZ reporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for the mepR and mepA operators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor.


Subject(s)
Amino Acid Substitution , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Genes, MDR/physiology , Staphylococcus aureus/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Binding Sites , Drug Resistance, Multiple, Bacterial/genetics , Genes, MDR/genetics , Models, Molecular , Mutation , Protein Conformation , Reverse Transcriptase Polymerase Chain Reaction , Staphylococcus aureus/genetics
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