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1.
Food Chem ; 337: 127776, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777574

RESUMO

Staphylococcus aureus is a Gram-positive bacterium responsible for a number of diseases and has demonstrated resistance to conventional antibiotics. This study aimed to evaluate the antibacterial activity of eugenol and its derivatives allylbenzene, 4-allylanisole, isoeugenol and 4-allyl-2,6-dimethoxyphenol against the S. aureus NorA efflux pump (EP) in association with norfloxacin and ethidium bromide. The antibacterial activity of the compounds was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC). A reduction in the MIC of ethidium bromide (a substrate for several efflux pumps) or norfloxacin was used as a parameter of EP inhibition. Molecular modeling studies were used to predict the 3D structure and analyze the interaction of selected compounds with the binding pocket of the NorA efflux pump. Except for 4-allylanisole and allylbenzene, the compounds presented clinically effective antibacterial activity. When associated with norfloxacin against the SA 1199B strain, 4-allyl-2,6-dimethoxyphenol eugenol and isoeugenol caused significant reduction in the MIC of the antibiotic, demonstrating synergistic effects. Similar effects were observed when 4-allyl-2,6-dimethoxyphenol, allylbenzene and isoeugenol were associated with ethidium bromide. Together, these findings indicate a potential inhibition of the NorA pump by eugenol and its derivatives. This in vitro evidence was corroborated by docking results demonstrating favorable interactions between 4-allyl-2,6-dimetoxypheno and the NorA pump mediated by hydrogen bonds and hydrophobic interactions. In conclusion, eugenol derivatives have the potential to be used in antibacterial drug development in strains carrying the NorA efflux pump.


Assuntos
Proteínas de Bactérias/metabolismo , Eugenol/análogos & derivados , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Staphylococcus aureus/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Etídio/farmacologia , Eugenol/metabolismo , Eugenol/farmacologia , Ligação de Hidrogênio , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Norfloxacino/farmacologia , Staphylococcus aureus/efeitos dos fármacos
2.
Xenobiotica ; 51(1): 105-114, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32820679

RESUMO

Mycophenolic acid (MPA) has become a cornerstone of immunosuppressive therapy, in particular for transplant patients. In the gastrointestinal tract, the liver and the kidney, MPA is mainly metabolized into phenyl-ß-d glucuronide (MPAG). Knowledge about the interactions between MPA/MPAG and membrane transporters is still fragmented. The aim of the present study was to explore these interactions with the basolateral hepatic MRP4 transporter. The inhibition of the MRP4-driven transport by various drugs which can be concomitantly prescribed was also evaluated. In vitro experiments using vesicles overexpressing MRP4 showed an ATP-dependent transport of MPAG driven by MRP4 (Michaelis-Menten constant of 233.9 ± 32.8 µM). MPA was not effluxed by MRP4. MRP4-mediated transport of MPAG was inhibited (from -43% to -84%) by ibuprofen, cefazolin, cefotaxime and micafungin. An in silico approach based on molecular docking and molecular dynamics simulations rationalized the mode of binding of MPAG to MRP4. The presence of the glucuronide moiety in MPAG was highlighted as key, being prone to make electrostatic and H-bond interactions with specific residues of the MRP4 protein chamber. This explains why MPAG is a substrate of MRP4 whereas MPA is not.


Assuntos
Glucuronídeos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ácido Micofenólico/análogos & derivados , Transporte Biológico , Hepatócitos/metabolismo , Humanos , Rim/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Acoplamento Molecular , Ácido Micofenólico/metabolismo
3.
J Pharmacol Exp Ther ; 376(1): 29-39, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33127749

RESUMO

Expression and functional changes in the organic anion transporting polypeptide (OATP)-multidrug resistance-associated protein (MRP) axis of transporters are well reported in patients with nonalcoholic steatohepatitis (NASH). These changes can impact plasma and tissue disposition of endo- and exogenous compounds. The transporter alterations are often assessed by administration of a xenobiotic or by transporter proteomic analysis from liver biopsies. Using gene expression, proteomics, and endogenous biomarkers, we show that the gene expression and activity of OATP and MRP transporters are associated with disease progression and recovery in humans and in preclinical animal models of NASH. Decreased OATP and increased MRP3/4 gene expression in two cohorts of patients with steatosis and NASH, as well as gene and protein expression in multiple NASH rodent models, have been established. Coproporphyrin I and III (CP I and III) were established as substrates of MRP4. CP I plasma concentration increased significantly in four animal models of NASH, indicating the transporter changes. Up to a 60-fold increase in CP I plasma concentration was observed in the mouse bile duct-ligated model compared with sham controls. In the choline-deficient amino acid-defined high-fat diet (CDAHFD) model, CP I plasma concentrations increased by >3-fold compared with chow diet-fed mice. In contrast, CP III plasma concentrations remain unaltered in the CDAHFD model, although they increased in the other three NASH models. These results suggest that tracking CP I plasma concentrations can provide transporter modulation information at a functional level in NASH animal models and in patients. SIGNIFICANCE STATEMENT: Our analysis demonstrates that multidrug resistance-associated protein 4 (MRP4) transporter gene expression tracks with nonalcoholic steatohepatitis (NASH) progression and intervention in patients. Additionally, we show that coproporphyrin I and III (CP I and III) are substrates of MRP4. CP I plasma and liver concentrations increase in different diet- and surgery-induced rodent NASH models, likely explained by both gene- and protein-level changes in transporters. CP I and III are therefore potential plasma-based biomarkers that can track NASH progression in preclinical models and in humans.


Assuntos
Coproporfirinas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Coproporfirinas/sangue , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Células Sf9 , Spodoptera
4.
Yakugaku Zasshi ; 140(10): 1235-1242, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999202

RESUMO

The central nervous system (CNS) is segregated from the circulating blood and peripheral tissues by endothelial and epithelial barriers. To overcome refractory CNS diseases, it is important to understand the membrane transport systems of drugs and the endogenous compounds that relate to the pathogenesis of CNS diseases at these barriers. The endothelial barrier in the brain is the blood-brain barrier (BBB). Our studies clarified the efflux transport of prostaglandin E2 (PGE2), a modulator of neural excitation and inflammatory responses, across the BBB via plasma membrane transporters such as organic anion transporter 3 (Oat3) and multidrug resistance-associated protein 4 (Mrp4). This efflux transport was attenuated by peripheral inflammation or cerebral treatment with neuroexcitatory l-glutamate, suggesting that BBB-mediated PGE2 elimination was altered under several pathological conditions. We also examined excitatory amino acid transporter (EAAT) 1 and 3 as l-glutamate efflux transporters of the inner blood-retinal barrier (BRB) and blood-cerebrospinal barrier. It was considered that these efflux membrane transporters participated in the homeostasis of neuroexcitatory and neuroinflammatory responses in the brain and retina. Moreover, we identified connexin 43 (Cx43) hemichannels as a new membrane transport system that is activated under pathological conditions and recognizes several monocarboxylate drugs, such as valproate. As it is expected that the action of these membrane transporters across the CNS barriers is of great importance in understanding the pathology of various neuroexcitatory diseases, our studies should contribute to the establishment of therapeutic strategies for refractory CNS diseases.


Assuntos
Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/metabolismo , Encéfalo/metabolismo , Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/metabolismo , Desenvolvimento de Medicamentos , Proteínas de Membrana Transportadoras/metabolismo , Retina/metabolismo , Animais , Doenças do Sistema Nervoso Central/tratamento farmacológico , Conexina 43/metabolismo , Dinoprostona/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Terapia de Alvo Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo
5.
Ecotoxicol Environ Saf ; 202: 110940, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32800223

RESUMO

Recent evidence indicates that chronic, low-dose exposure to mixtures of pesticides can cause adverse responses in a variety of cells, tissues and organs, although interactions between pesticides circulating in the blood and cancer cells remain largely unexplored. The aim of this study was to investigate the potential of a mixture of four pesticides to induce multidrug resistance against the chemotherapeutic agents cisplatin, 5-fluorouracil and temozolomide in the human U87 glioblastoma cell line, and to explore the molecular mechanisms underlying this resistance. We found that the repeated administration of the pesticide mixture (containing the insecticides chlorpyrifos-ethyl and deltamethrin, the fungicide metiram, and the herbicide glyphosate) induced a strong drug resistance in U87 cells. The resistance was durable and transferred to subsequent cell generations. In addition, we detected a significant over-expression of the ATP-binding cassette (ABC) membrane transporters P-gp/ABCB1 and BRCP/ABCG2 as well as a glutathione-S-transferase (GST)/M1-type cellular detoxification function, known to have important roles in multidrug resistance, thus providing molecular support for the acquired multidrug resistance phenotype and shedding light on the mechanism of resistance. We further determined that there was lower mortality in the resistant brain tumor cells and that the mitochondrial apoptosis pathway was activated at a lower rate after chemotherapy compared to non-resistant control cells. In addition, multidrug-resistant cells were found to have both higher motility and wound-healing properties, suggesting a greater metastatic potential. Our results suggest that the investigation of P-gp, BRCP and GST/M1 multidrug resistance gene expression and/or protein levels in biopsy specimens of brain tumor patients who were at risk of pesticide exposure could be beneficial in determining chemotherapy dose and prolonging patient survival.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Praguicidas/toxicidade , Testes de Toxicidade Crônica , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia
6.
Anticancer Res ; 40(8): 4481-4489, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727778

RESUMO

BACKGROUND/AIM: Cancer stem cells (CSCs) and ABC transporters are associated with treatment resistance and outcomes of cancer patients. We aimed to investigate the prognostic implications of CSC markers and ABC transporters in colorectal cancer (CRC) patients. MATERIALS AND METHODS: We collected 331 CRC samples and evaluated 3 CSC markers (SOX2, LGR5, and ALDH1) and 3 ABC transporters (ABCC2, ABCC3, and ABCG2) by immunohistochemistry. The association between the expression of these protein and patients' prognoses was statistically analyzed. RESULTS: SOX2 was associated with longer overall survival (OS) (p<0.001). ABCG2 was associated with favorable overall survival (OS) p=0.001) and SOX2, and ABCC2 were associated with longer disease-free survival (DFS) (p=0.005 and 0.029, respectively). Multivariate analyses revealed that SOX2 was an independent prognostic factor for DFS [hazard ratio (HR)=2.701, p=0.044]. CONCLUSION: SOX2 and ABCC2 may be promising prognostic markers for CRC patients.


Assuntos
Neoplasias Colorretais/mortalidade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Regulação para Cima , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Análise Serial de Tecidos
7.
Proc Natl Acad Sci U S A ; 117(31): 18497-18503, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32680969

RESUMO

Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Lipossomos/metabolismo , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/ultraestrutura , Conformação Proteica
8.
Nat Commun ; 11(1): 3062, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546788

RESUMO

Anti-tuberculosis (TB) drugs, while being highly potent in vitro, require prolonged treatment to control Mycobacterium tuberculosis (Mtb) infections in vivo. We report here that mesenchymal stem cells (MSCs) shelter Mtb to help tolerate anti-TB drugs. MSCs readily take up Mtb and allow unabated mycobacterial growth despite having a functional innate pathway of phagosome maturation. Unlike macrophage-resident ones, MSC-resident Mtb tolerates anti-TB drugs remarkably well, a phenomenon requiring proteins ABCC1, ABCG2 and vacuolar-type H+ATPases. Additionally, the classic pro-inflammatory cytokines IFNγ and TNFα aid mycobacterial growth within MSCs. Mechanistically, evading drugs and inflammatory cytokines by MSC-resident Mtb is dependent on elevated PGE2 signaling, which we verify in vivo analyzing sorted CD45-Sca1+CD73+-MSCs from lungs of infected mice. Moreover, MSCs are observed in and around human tuberculosis granulomas, harboring Mtb bacilli. We therefore propose, targeting the unique immune-privileged niche, provided by MSCs to Mtb, can have a major impact on tuberculosis prevention and cure.


Assuntos
Antituberculosos/farmacologia , Células-Tronco Mesenquimais/microbiologia , Mycobacterium tuberculosis/patogenicidade , Nicho de Células-Tronco/imunologia , Tuberculose/microbiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células Cultivadas , Dinoprostona/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/farmacologia , Isoniazida/farmacologia , Lisossomos/microbiologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteínas de Neoplasias/metabolismo , Fagossomos/microbiologia , Tuberculose/patologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Fator de Necrose Tumoral alfa/farmacologia
9.
Sci Rep ; 10(1): 7616, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32377003

RESUMO

The transporter Multidrug Resistance Protein 1 (MRP1, ABCC1) is implicated in multidrug resistant (MDR) phenotype of cancer cells. Glutathione (GSH) plays a key role in MRP1 transport activities. In addition, a ligand-stimulated GSH transport which triggers the death of cells overexpressing MRP1, by collateral sensitivity (CS), has been described. This CS could be a way to overcome the poor prognosis for patients suffering from a chemoresistant cancer. The molecular mechanism of such massive GSH transport and its connection to the other transport activities of MRP1 are unknown. In this context, we generated MRP1/MRP2 chimeras covering different regions, MRP2 being a close homolog that does not trigger CS. The one encompassing helices 16 and 17 led to the loss of CS and MDR phenotype without altering basal GSH transport. Within this region, the sole restoration of the original G1228 (D1236 in MRP2) close to the extracellular loop between the two helices fully rescued the CS (massive GSH efflux and cell death) but not the MDR phenotype. The flexibility of that loop and the binding of a CS agent like verapamil could favor a particular conformation for the massive transport of GSH, not related to other transport activities of MRP1.


Assuntos
Glutationa/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Domínios Proteicos
10.
Toxicol Lett ; 327: 9-18, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32201199

RESUMO

Superoxide dismutase 2 (SOD2) is a key enzyme for scavenging reactive oxygen species produced by mitochondria, which plays an important role in maintaining cellular homeostasis. However, its effects on the detoxification capability of liver cells have not been reported. In this study, we found that change in SOD2 expression affects the proliferation of liver cells. Genome-wide microarray analysis showed that SOD2 positively regulates the drug transporter ABCC2, and co-expression analysis suggested that lncRNA CLCA3P participates in the process. Further experiments showed that SOD2 can promote the expression of CLCA3P, which increases the transcription of ABCC2 by interacting with the transcription factor IRF1. By increasing ABCC2 expression SOD2 facilitates drugs efflux of liver cells and thus promotes their survival under a drug-toxic environment. This study elucidates the improvement of the detoxification of liver cells by a regulatory axis, SOD2-CLCA3P-IRF1-ABCC2, and provides novel insight into the modification of human liver cells that can be applied to bioartificial liver system or the study of SOD2 in drug metabolism.


Assuntos
Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Paclitaxel/metabolismo , RNA Longo não Codificante , Superóxido Dismutase/metabolismo , Animais , Antineoplásicos Fitogênicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Superóxido Dismutase/genética
11.
PLoS Pathog ; 16(3): e1008427, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32191775

RESUMO

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Better understanding of the genetic basis of resistance is needed to more effectively monitor, manage, and counter pest resistance to Bt toxins. Here we used CRISPR/Cas9 gene editing to clarify the genetics of Bt resistance and the associated effects on susceptibility to other microbial insecticides in one of the world's most damaging pests, the cotton bollworm (Helicoverpa armigera). We discovered that CRISPR-mediated knockouts of ATP-binding cassette (ABC) transporter genes HaABCC2 and HaABCC3 together caused >15,000-fold resistance to Bt toxin Cry1Ac, whereas knocking out either HaABCC2 or HaABCC3 alone had little or no effect. Inheritance of resistance was autosomal and recessive. Bioassays of progeny from interstrain crosses revealed that one wild type allele of either HaABCC2 or HaABCC3 is sufficient to sustain substantial susceptibility to Cry1Ac. In contrast with previous results, susceptibility to two insecticides derived from bacteria other than Bt (abamectin and spinetoram), was not affected by knocking out HaABCC2, HaABCC3, or both. The results here provide the first evidence that either HaABCC2 or HaABCC3 protein is sufficient to confer substantial susceptibility to Cry1Ac. The functional redundancy of these two proteins in toxicity of Cry1Ac to H. armigera is expected to reduce the likelihood of field-evolved resistance relative to disruption of a toxic process where mutations affecting a single protein can confer resistance.


Assuntos
Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Mariposas , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Controle Biológico de Vetores
12.
Int J Mol Sci ; 21(3)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013182

RESUMO

The drug/proton antiporter AcrB, engine of the major efflux pump AcrAB(Z)-TolC of Escherichia coli and other bacteria, is characterized by its impressive ability to transport chemically diverse compounds, conferring a multi-drug resistance (MDR) phenotype. Although hundreds of small molecules are known to be AcrB substrates, only a few co-crystal structures are available to date. Computational methods have been therefore intensively employed to provide structural and dynamical fingerprints related to transport and inhibition of AcrB. In this work, we performed a systematic computational investigation to study the interaction between representative carbapenem antibiotics and AcrB. We focused on the interaction of carbapenems with the so-called distal pocket, a region known for its importance in binding inhibitors and substrates of AcrB. Our findings reveal how the different physico-chemical nature of these antibiotics is reflected on their binding preference for AcrB. The molecular-level information provided here could help design new antibiotics less susceptible to the efflux mechanism.


Assuntos
Antibacterianos/metabolismo , Carbapenêmicos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antibacterianos/química , Sítios de Ligação , Carbapenêmicos/química , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Ligação Proteica , Termodinâmica
13.
Phytomedicine ; 68: 153148, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32028185

RESUMO

BACKGROUND: Aloe-emodin (AE) is among the primary bioactive anthraquinones present in traditional Chinese medicinal plants such as Rheum palmatum L. Multidrug resistance protein 2 (ABCC2/ MRP2) is an important efflux transporter of substances associated with cellular oxidative stress. However, the effects of traditional Chinese medicine on this protein remain unclear. PURPOSE: The aim of this research is to study the role of ABCC2 in AE-induced hepatotoxicity. METHODS: The expression of ABCC2 protein and mRNA levels were analyzed by Western-Blotting and qRT-PCR, respectively. The intracellular oxidative stress caused by AE was evaluated by quantifying the levels of intracellular reactive oxygen species, malondialdehyde, glutathione reduced and oxidized glutathione. The levels of adenosine triphosphate, mitochondrial membrane potential and mitochondrial DNA were explored to evaluate the effects of AE on mitochondrial function. The effects of AE on cell apoptosis and cell cycle were detected by flow cytometry. To further clarify the key role of ABCC2 in AE induced cytotoxicity, we used pCI-neo-ABCC2 plasmid to over express ABCC2 protein, and small interfering RNA was used to knockdown ABCC2 in HepG2 cells. Additionally, we investigated the impact of AE on ABCC2 degradation pathway and the hepatotoxic effects of AE in mice. RESULTS: AE was found to inhibit ABCC2 transport activity, downregulate ABCC2 expression and altered intracellular redox balance. Induction of oxidative stress resulted in depletion of intracellular glutathione reduced, mitochondria dysfunction and activation of apoptosis. ABCC2 overexpression significantly reduced AE-induced intracellular oxidative stress and cell death, which was enhanced by ABCC2 knockdown. Furthermore, AE was observed to promote ABCC2 degradation through induction of autophagy and hepatotoxicity was induced in mice by promoting ABCC2 degradation. CONCLUSIONS: The inhibition of ABCC2 is a novel effect of AE that triggers oxidative stress and apoptosis. These findings are helpful in understanding the toxicological effects of AE-containing medicinal plants.


Assuntos
Antraquinonas/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Células Hep G2 , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
14.
Int J Med Microbiol ; 310(2): 151400, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32001143

RESUMO

A plethora of toxin-antitoxin systems exist in bacteria and has multilateral roles in bacterial pathogenesis and virulence. Toxin-antitoxin systems have been involved in persister cell formation in Escherichia coli and Mycobacterium but have not been reported to be associated with Staphylococcus aureus persistence. Persistence is the ability of bacterial cells to tolerate unfavorable conditions and multiple stresses. There are less known and more unknown factors that either alleviate or aggravate bacterial persistence phenomenon. For the first time, we reported a new chromosomally encoded tripartite toxin-antitoxin system and its role in S. aureus persister cell formation. The toxin gene is bacteriostatic in action and counterbalanced by antitoxin RNA that could basepair with the toxin mRNA and formed a duplex. The transcriptional regulator positively regulates the toxin expression under certain stress conditions. The toxin ectopic induction increased S. aureus susceptibility to norfloxacin, ciprofloxacin, and ofloxacin. Whole-genome RNA sequencing revealed that MDR efflux pump norA is significantly down-regulated by toxin ectopic induction. The deletion of norA from S. aureus genome reduced resistance toward ciprofloxacin, norfloxacin, and ofloxacin, as well as resulted in a decrease in minimal inhibitory concentration while complementation of norA successfully restored the phenotypes. The persistence assay of the norA mutant revealed that deletion of norA increased persister cell survival in S. aureus. Altogether, we have provided insight into the first tripartite type-I TA system and revealed the role of MDR NorA in the persister cell formation of S. aureus.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Sistemas Toxina-Antitoxina/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Sequenciamento Completo do Genoma
15.
PLoS One ; 15(2): e0228938, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074140

RESUMO

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.


Assuntos
Calcinose/patologia , Microvasos/fisiologia , alfa-2-Glicoproteína-HS/metabolismo , Animais , Calcinose/genética , Difosfatos/metabolismo , Modelos Animais de Doenças , Feminino , Rim/patologia , Fígado/patologia , Magnésio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microvasos/metabolismo , Minerais , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Insuficiência Renal Crônica/complicações , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , alfa-2-Glicoproteína-HS/fisiologia , alfa-Fetoproteínas
16.
Biochem Pharmacol ; 174: 113795, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31926937

RESUMO

Chemoresistance is a major cause of recurrence and poor prognosis in triple-negative breast cancer (TNBC) patients. The essential oil of Rhizoma Curcumae has been recently reported to enhance the chemosensitivity of cancer cells. However, few reports have systematically illuminated the mechanism. Curcumol is the major component of the essential oil of Rhizoma Curcumae. Therefore, we wondered whether curcumol combined with chemotherapy could increase the anticancer effects. In the present study, we evaluated the anticancer effects of doxorubicin and curcumol alone or in combination by a series of growth proliferation and apoptosis assays in TNBC cells. Our results showed that curcumol enhanced the sensitivity of MDA-MB-231 cells to doxorubicin in vitro and in vivo. Through miRNA-seq, we found that miR-181b-2-3p was involved in the curcumol-mediated promotion of doxorubicin-sensitivity in both parental and doxorubicin-resistant MDA-MB-231 (MDA-MB-231/ADR) cells. Further study showed that miR-181b-2-3p suppressed ABCC3 expression by targeting its 3'UTR. More importantly, we identified that overexpression of miR-181b-2-3p sensitized MDA-MB-231/ADR cells to doxorubicin by inhibiting the drug efflux transporter ABCC3. Furthermore, we found that NFAT1 could be activated by curcumol. In addition, ChIP assay results revealed that NFAT1 could directly bind to the promoter region of miR-181b-2-3p. Finally, using PDX models, we identified that curcumol could enhance sensitivity to doxorubicin to suppress tumor growth by the miR-181b-2-3p-ABCC3 axis in vivo. Taken together, our study provides novel mechanistic evidence for curcumol-mediated sensitization to doxorubicin in TNBC, and it highlights the potential therapeutic usefulness of curcumol as an adjunct drug in TNBC patients with doxorubicin-resistance.


Assuntos
Adjuvantes Farmacêuticos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , MicroRNAs/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Sesquiterpenos/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Camundongos Nus , MicroRNAs/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Biochim Biophys Acta Mol Cell Res ; 1867(4): 118647, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31926942

RESUMO

Cisplatin-based chemotherapies have long been considered as a standard chemotherapy in ovarian cancer. However, cisplatin resistance restricts beneficial therapy for patients with ovarian cancer. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) encodes a 15-kDa protein, that is implicated in the post-translational modification of diverse proteins. In this work, we found that ISG15 was downregulated in cisplatin resistant tissues and cell lines of ovarian cancer. Functional studies demonstrated that overexpression of wild type (WT) ISG15, but not nonISGylatable (Mut) ISG15 increased cell responses to cisplatin in resistant ovarian cancer cells. Furthermore, we found that WT ISG15 decreased ABCC2 expression at the protein level. Importantly, overexpression of ABCC2 blocked sensitizing effect of ISG15 on cisplatin. In addition, we identified that hnRNPA2B1 was recruited to 5'UTR of ABCC2 mRNA and promoted its translation, which was blocked by ISG15. We further demonstrated that hnRNPA2B1 could be ISGylated, and ISGylation blocked its recruitment to ABCC2 mRNA, thereby suppressed translation of ABCC2. Altogether, our data support targeting ISG15 might be a potential therapeutic strategy for patients with cisplatin-resistant ovarian cancer.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Citocinas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias Ovarianas/genética , Biossíntese de Proteínas , Ubiquitinas/metabolismo , Regiões 5' não Traduzidas , Adulto , Idoso , Linhagem Celular Tumoral , Citocinas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/metabolismo , Ubiquitinas/genética
18.
Life Sci ; 243: 117291, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31927049

RESUMO

AIMS: Chronic obstructive pulmonary disease (COPD) is a disease with high morbidity and mortality worldwide, which can cause serious social and economic burdens. Allyl isothiocyanate (AITC) is one of the most common natural isothiocyanates and has been shown to have anti-inflammatory and antioxidant biological activities. The purpose of this study was to investigate whether AITC regulated Multidrug resistance-associated protein 1 (MRP1), reactive oxide species (ROS) and reduced glutathione (GSH) levels via Nrf2 and Notch1 signaling pathways to treat COPD and whether there was an interaction between these two pathways. MAIN METHODS: Lung function indexes and histopathological changes in mice were determined by lung function instrument and HE staining, respectively. The protein expression was analyzed using immunohistochemistry and Western blotting. The mRNA expression was measured by RT-PCR in human bronchial epithelial cell line 16HBE. The contents of ROS, GSH and GSSG were detected by kits in 16HBE cells. KEY FINDINGS: The protein expression of Notch1, Hes1, MRP1, Nrf2, and HO-1 in lung tissues of WT mice and untransfected cells were significantly down-regulated in COPD, then significantly ameliorated in treatment groups. The protein expression of MRP1, Notch1 and Hes1 in lung tissues of Nrf2-/- mice were markedly reduced. There was a significant reduction in expression of Nrf2, HO-1 and MRP1 in si-Notch1 transfected cells. Pretreatment with AITC markedly improved oxidative stress and GSH-redox disorder in COPD. SIGNIFICANCE: Our study demonstrates that there is a potential interaction between Nrf2 and Notch1 signaling pathways during treatment of COPD.


Assuntos
Isotiocianatos/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Isotiocianatos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fatores de Transcrição HES-1/metabolismo
19.
Med Sci Monit ; 26: e919566, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31964857

RESUMO

BACKGROUND Chemotherapy is widely used in gastric cancer treatment, but multidrug resistance remains a leading cause of chemotherapy failure. Trop2 is highly expressed in gastric tumor tissues and greatly influences cancer progression. However, little is known about the relationship between Trop2 and drug resistance in gastric cancer. MATERIAL AND METHODS In the present study, Trop2 was knocked down in BGC823 cells and overexpressed in HGC27. CCK-8 assay was performed to explore the relationship of Trop2 expression and cell proliferation treated with anticancer drugs. Flow cytometry was performed to assess the relationship between Trop2 and cell apoptosis after chemotherapy. Subcutaneous xenograft models were generated to explore the curative effect of DDP to GC in vivo. MRP1 and Notch1 expressions were assessed by Western blot. RESULTS Trop2 decreased cell proliferation inhibition and apoptosis after chemotherapeutic treatments. DDP showed stronger therapeutic effects on Trop2-knockdown tumor than control in vivo. MRP1 and Notch1 signaling pathway were confirmed to participate in Trop2-induced drug resistance. CONCLUSIONS Our findings suggest that Trop2 promotes the resistance of gastric cancer to chemotherapy by activating the Notch1 pathway.


Assuntos
Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/farmacologia , Receptor Notch1/deficiência , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/patologia
20.
BMC Pharmacol Toxicol ; 21(1): 7, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969193

RESUMO

BACKGROUND: 2,3,5,4'-tetrahydroxystilbence-2-O-ß-D-glucoside (TSG) is a polyhydroxyphenolic compound, which exhibited a broad spectrum of pharmacological activities, such as anti-inflammatory, anti-depression, anti-oxidation and anti-atherosclerosis. However, the compound had poor bioavailability and the underlying absorption mechanisms had not been studied. Therefore, the purpose of this study was to investigate the intestinal absorption mechanism of TSG. METHODS: This study used Caco-2 cell monolayer model and single-pass intestinal perfusion model to explore the gastrointestinal absorption mechanisms of TSG. The effects of basic parameters such as drug concentration, time and pH on the intestinal absorption of TSG were analyzed by high performance liquid chromatography. The absorption susceptibility of TSG to three inhibitors, P-gp inhibitors verapamil hydrochloride and quinidine, and MRP2 inhibitor probenecid were also assessed. RESULTS: TSG was poorly absorbed in the intestines and the absorption of TSG in stomach is much higher than that in intestine. Both in vitro and in situ experiments showed that the absorption of TSG was saturated with increasing concentration and it was better absorbed in a weakly acidic environment pH 6.4. Moreover, TSG interacts with P-gp and MRP2, and TSG was not only the substrate of the P-gp and MRP2, but also affected the expression of P-gp and MRP2. CONCLUSIONS: It was concluded that the gastrointestinal absorption the most unique active ingredient and considered as the mechanisms of TSG involved processes passive transport and the participation of efflux transporters.


Assuntos
Glucosídeos/farmacologia , Absorção Intestinal/efeitos dos fármacos , Estilbenos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Feminino , Suco Gástrico/metabolismo , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Secreções Intestinais/metabolismo , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ratos Sprague-Dawley
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