RESUMEN
AIMS: Estradiol 17ß-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. MAIN METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. KEY FINDINGS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SIGNIFICANCE: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.
Asunto(s)
Colestasis , Endocitosis , Estradiol , Hepatocitos , Fosfatidilinositol 3-Quinasas , Transducción de Señal , Ácido Tauroquenodesoxicólico , Animales , Colestasis/metabolismo , Colestasis/inducido químicamente , Colestasis/prevención & control , Ratas , Transducción de Señal/efectos de los fármacos , Estradiol/metabolismo , Estradiol/farmacología , Estradiol/análogos & derivados , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Endocitosis/efectos de los fármacos , Ácido Tauroquenodesoxicólico/farmacología , Ácido Tauroquenodesoxicólico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , Femenino , Masculino , Proteína Quinasa C/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismoRESUMEN
Chemotherapy is a widely used strategy to treat cancer, a disease that causes millions of deaths each year. However, its efficacy is reduced by the overexpression of ABC transporters, which are proteins that expel the drugs used in chemotherapy and involved in the multidrug resistance (MDR). Glycolipids have been identified as potential inhibitors of ABC transporters. Algae of the genus Sargassum contain high levels of glycolipids, making them a promising therapeutic alternative against the MDR phenotype. Sargassum filipendula glycolipids were obtained by exhaustive maceration with chloroform/methanol, purified by column and thin layer chromatography, and then characterized by FTIR, NMR, and LC-MS. Cell viability by PI labeling and inhibition of ABC transporters were analyzed by flow cytometry. Assessment of resistance reversal was determined by MTT assay. Ten sulfoquinovosylglycerol-type compounds were found, and six of them are reported for the first time. In particular, moiety 4 (GL-4) showed strong and moderate inhibitory activity against ABCC1 and ABCB1 transporters respectively. Treatment of GL-4 in combination with the antineoplastic drug vincristine sensitized Lucena-1â cell model to drug and reversed the MDR phenotype. This is the first report of glycolipids isolated from S. filipendula capable of inhibiting ABC transporters and thus overcoming acquired drug resistance.
Asunto(s)
Antineoplásicos , Filipendula , Neoplasias , Sargassum , Humanos , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/farmacología , Sargassum/metabolismo , Resistencia a Antineoplásicos , Resistencia a Múltiples Medicamentos , Antineoplásicos/farmacología , Antineoplásicos/química , Neoplasias/metabolismo , Línea Celular TumoralRESUMEN
Evidence from preclinical and clinical studies demonstrate that pregnancy is a physiological state capable of modifying drug disposition. Factors including increased hepatic metabolism and renal excretion are responsible for impacting disposition, and the role of membrane transporters expressed in biological barriers, including the placental- and blood-brain barriers, has received considerable attention. In this regard, the brain disposition of drugs in the mother and fetus has been the subject of studies attempting to characterize the mechanisms by which pregnancy could alter the expression of ATP-binding cassette (ABC) and solute carrier (SLC) transporters. This chapter will summarize findings of the influence of pregnancy on the maternal and fetal expression of ABC and SLC transporters in the brain and the consequences of such changes on the disposition of therapeutic drugs.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Placenta , Femenino , Embarazo , Humanos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Placenta/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Feto , Barrera Hematoencefálica/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
Multidrug resistance (MDR) and induction of metastasis are some of the puzzles encountered during cancer chemotherapy. The MDR phenotype is associated with overexpression of ABC transporters, involved in drug efflux. Metastasis originates from the epithelial-mesenchymal transition (EMT), in which cells acquire a migratory phenotype, invading new tissues. ABC transporters' role during EMT is still elusive, though cells undergoing EMT exhibit enhanced ABCB1 expression. We demonstrated increased ABCB1 expression but no change in activity after TGF-ß-induced EMT in A549 cells. Moreover, ABCB1 inhibition by verapamil increased snail and fibronectin expression, an event associated with upregulation of ABCB1, evidencing coincident cell signaling pathways leading to ABCB1 and EMT-related markers transcription, rather than a direct effect of transport. Additionally, for the first time, increased ABCC1 expression and activity was observed after EMT, and use of ABCC1 inhibitors partially inhibited EMT-marker snail, although increased ABCC1 function translated into collateral sensibility to daunorubicin. More investigations must be done to evaluate the real benefits that the gain of ABC transporters might have on the process of metastasis. Considering ABCC1 is involved in the stress response, affecting intracellular GSH content and drug detoxification, this transporter could be used as a therapeutic target in cancer cells undergoing EMT.
Asunto(s)
Transición Epitelial-Mesenquimal , Neoplasias , Humanos , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Factor de Crecimiento Transformador betaRESUMEN
Limited information is available about the effect of mid-pregnancy viral infections on the placental expression of efflux transporters and offspring behavior. We hypothesized that maternal exposure to polyinosinic-polycytidylic acid [poly(I:C)], a synthetic double-stranded RNA viral mimic, would impair placental cell turnover, the expression of selected ABC transporters and adult offspring behavior. C57BL/6 mice were administered poly(I:C) (10 mg/Kg;ip) or vehicle at gestational day (GD) 13.5 (mid-pregnancy). Dams were euthanized for blood collection 4 h after injection, fetal and placental collection at GD18.5 or allowed to deliver spontaneously at term. At GD 13.5, poly(I:C) induced an acute pro-inflammatory response characterized by an increase in maternal plasma levels of IL-6, CXCL-1 and CCL-2/MCP-1. At GD 18.5, poly(I:C) decreased cell proliferation/death in the labyrinthine and increased cell death in the junctional zones, characterizing a disruption of placental cell turnover. Abca1 and Abcg1 immunolabelling was decreased in the labyrinthine zone, whereas Abca1, Abcg1 and breast cancer resistance transporter (Bcrp) expression increased in the junctional zone. Moreover, adult offspring showed motor and cognitive impairments in the Rotarod and T-water maze tests. These results indicate that viral infection during mid-pregnancy may disrupt relevant placental efflux transporters, as well as placental cell turnover and offspring behavior in adult life.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Disfunción Cognitiva , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Disfunción Cognitiva/metabolismo , Femenino , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/metabolismo , Placenta/metabolismo , Poli I-C/farmacología , EmbarazoRESUMEN
This study investigated whether norepinephrine (NE) and epinephrine (E) interfere in the response of head and neck squamous cell carcinoma (SCC) cell lines to cisplatin and explored the mechanisms of chemoresistance. Head and neck SCC-derived cell lines SCC-9, Cal27, SCC-25, and FaDu were stimulated with NE or E and treated with the inhibitory concentration of cisplatin for 24 h. As for adrenergic receptors (ADRB) inhibition, cells were treated with propranolol. The results showed that, when combined with NE, cisplatin effectiveness against SCC-9 and Cal27 but not SCC-25 and FaDu cells were notably reduced. E did not affect the response of the cells to cisplatin. Further experiments were performed with the responsive SCC-9 and SCC-25 cell lines and the hormone NE. The time course assay showed that stimulation of oral SCC cells with NE decreased the cleavage of caspase-3 and expression of multidrug resistance protein 1 (MDR-1) but only transiently affected ATP-binding cassette (ABC) subfamily G, isoform 2 protein (ABCG2) expression. The expression of cleaved caspase-3 and Bcl-2 were, respectively, decreased and increased by the combination of NE and cisplatin in SCC-9 and Cal27 cells. NE-induced resistance was reverted by previous treatment with propranolol. Expressions of ABCG2, and p-Akt but not of MDR-1, were enhanced by NE plus cisplatin when compared to cisplatin only in both cell lines. Migratory activity of oral SCC cells challenged with cisplatin was not affected by NE. These findings reveal for the first time that stress hormone NE induces resistance of oral cancer cells to cisplatin in vitro through the ADRB/Akt/ABCG2 pathway, pumping the drug out of the cell and inhibiting apoptosis.
Asunto(s)
Antineoplásicos , Neoplasias de la Boca , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Hormonas/farmacología , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Norepinefrina/farmacologíaRESUMEN
Human papillomavirus (HPV) is the major pathogen for cervical lesions. The evasion mechanism of the immune response and persistence of HPV infection can be influenced by polymorphisms (SNPs) in genes associated with transporter associated with antigen processing (TAP), which may change the peptide binding affinity or the TAP expression impacting the efficiency of peptide transport in the secretory pathway, and the presentation of peptides to cytotoxic T lymphocytes. This study aimed to evaluate the role of the TAP1 and TAP2 polymorphisms, TAP1, and TAP2 genes expressions, and protein levels in cervical cells presenting different degrees of pre-cancerous lesions in 296 immunocompetent women infected or not by HPV. TAP SNPs were genotyped by Sanger sequencing, and gene expression by real-time PCR. Aneuploidy was determined by DNA index using flow cytometry. TAP-1 and TAP-2 tissue expressions were evaluated by immunohistochemistry. The Asp697Gly SNP of TAP1 presented a risk for cellular aneuploidy (P=0.0244). HPV+ women had higher TAP-2 mRNA (P=0.0212) and protein (P<0.0001) levels. The TAP2D and TAP2E haplotypes were associated with the risk for aneuploidy and pre-cancerous lesions. In conclusion, nucleotide variability at the peptide binding region of peptide transporter genes, particularly of the TAP2 gene, may influence the HPV-peptide transportation from the cytosol to the endoplasmic reticulum, increasing the susceptibility to the development of high-grade cervical lesions.
Asunto(s)
Neoplasias , Infecciones por Papillomavirus , Humanos , Femenino , Presentación de Antígeno , Virus del Papiloma Humano , Infecciones por Papillomavirus/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Transporte de Membrana/genética , Polimorfismo de Nucleótido Simple , Péptidos/genéticaRESUMEN
The multidrug-resistance (MDR) phenotype is typically observed in patients with refractory epilepsy (RE) whose seizures are not controlled despite receiving several combinations of more than two antiseizure medications (ASMs) directed against different ion channels or neurotransmitter receptors. Since the use of bromide in 1860, more than 20 ASMs have been developed; however, historically ~30% of cases of RE with MDR phenotype remains unchanged. Irrespective of metabolic biotransformation, the biodistribution of ASMs and their metabolites depends on the functional expression of some ATP-binding cassette transporters (ABC-t) in different organs, such as the blood-brain barrier (BBB), bowel, liver, and kidney, among others. ABC-t, such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP-1), and breast cancer-resistance protein (BCRP), are mainly expressed in excretory organs and play a critical role in the pharmacokinetics (PK) of all drugs. The transporter hypothesis can explain pharmacoresistance to a broad spectrum of ASMs, even when administered simultaneously. Since ABC-t expression can be induced by hypoxia, inflammation, or seizures, a high frequency of uncontrolled seizures increases the risk of RE. These stimuli can induce ABC-t expression in excretory organs and in previously non-expressing (electrically responsive) cells, such as neurons or cardiomyocytes. In this regard, an alternative mechanism to the classical pumping function of P-gp indicates that P-gp activity can also produce a significant reduction in resting membrane potential (ΔΨ0 = -60 to -10 mV). P-gp expression in neurons and cardiomyocytes can produce membrane depolarization and participate in epileptogenesis, heart failure, and sudden unexpected death in epilepsy. On this basis, ABC-t play a peripheral role in controlling the PK of ASMs and their access to the brain and act at a central level, favoring neuronal depolarization by mechanisms independent of ion channels or neurotransmitters that current ASMs cannot control.
Asunto(s)
Epilepsia , Proteínas de Neoplasias , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/uso terapéutico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/uso terapéutico , Epilepsia/tratamiento farmacológico , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/uso terapéutico , Convulsiones/tratamiento farmacológico , Distribución TisularRESUMEN
Several proteins are involved in cholesterol homeostasis, as scavenger receptor class B type I and ATP-binding cassette (ABC) transporters including ABCA1, ABCG1, ABCG5, and ABCG8. This study aimed to determine the effects of single nucleotide variants (SNVs) rs2275543 (ABCA1), rs1893590 (ABCG1), rs6720173 (ABCG5), rs6544718 (ABCG8), and rs5888 (SCARB1) on plasma lipids, lipoproteins, and adiposity markers in an asymptomatic population and its sex-specific effects. Volunteers (n = 590) were selected and plasma lipids, lipoproteins, and adiposity markers (waist-to-hip and waist-to-height ratios, lipid accumulation product and body adiposity index) were measured. Genomic DNA was isolated from peripheral blood cells according to the method adapted from Gross-Bellard. SNVs were detected in the TaqMan® OpenArray® Real-Time polymerase chain reaction platform and data analyses were performed using the TaqMan® Genotyper Software. The rs2275543*C point to an increase of high-density lipoprotein size in females while in males very-low-density lipoprotein, cholesterol, and triglycerides were statistically lower (P value < 0.05). The rs1893590*C was statistically associated with lower apolipoprotein A-I levels and higher activities of paraoxonase-1 and cholesteryl ester transfer protein (P value < 0.05). The rs6720173 was statistically associated with an increase in cholesterol and low-density lipoprotein cholesterol in males; moreover, rs6544718*T reduced adiposity markers in females (P value < 0.05). Regarding the rs5888, a decreased adiposity marker in the total population and in females occurred (P value < 0.05). Multivariate analysis of variance showed that SNVs could influence components of high-density lipoprotein metabolism, mainly through ABCG1 (P value < 0.05). The ABCA1 and ABCG5 variants showed sex-specific effects on lipids and lipoproteins, while SCARB1 and ABCG8 variants might influence adiposity markers in females. Our data indicate a possible role of ABCG1 on HDL metabolism.
Asunto(s)
Adiposidad , Lipoproteínas , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adiposidad/genética , Colesterol/metabolismo , Femenino , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Lipoproteínas HDL/genética , Masculino , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismoRESUMEN
The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Eliminación de Gen , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xenobióticos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Antifúngicos/metabolismo , Antifúngicos/farmacología , Transporte Biológico , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Proteínas de Transporte de Monosacáridos/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Xenobióticos/farmacologíaRESUMEN
Cell adhesion to stromal support and the associated intracellular signaling are central to drug resistance, therefore blocking both has been effective in increasing drug sensitization in leukemia. The stromal Ser/Thr protein kinase C (PKC) has been found to be important for conferring protection to leukemic cells. We aimed at elucidating the intracellular signals connected to cell adhesion and to stromal PKC. We found that NF-κB and Akt were up-regulated in mesenchymal stem cells (MSC) after binding of B-cell acute lymphoblastic leukemia (B-ALL) cells. Nevertheless, Akt inhibition did not induce B-ALL cell detachment. In spite of a clear activation of the NF-κB signaling pathway after B-ALL cell binding (up-regulation NF-κB1/2, and down-regulation of the IKBε and IKBα inhibitors) and an important reduction in cell adhesion after NF-κB inhibition, sensitization to the drug treatment was not observed. This was opposite to the PKC inhibitors Enzastaurin and HKPS, a novel chimeric peptide inhibitor, that were able to increase sensitization to dexamethasone, methotrexate, and vincristine. PLCγ1, Erk1/2, and CREB appear to be related to PKC signaling and PKC effect on drug sensitization since they were contra-regulated by HKPS when compared to dexamethasone-treated cells. Additionally, PKC inhibition by HKPS, but not by Enzastaurin, in MSC reduced the activity of three ABC transporters in leukemic cells treated with dexamethasone, a new indirect mechanism to increase sensitization to drug treatment in B-ALL cells. Our results show the validity of targeting the functional characteristic acquired and modulated during cell-to-cell interactions occurring in the leukemic niche.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Células Precursoras de Linfocitos B/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Adhesión Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , FN-kappa B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Células Tumorales CultivadasRESUMEN
Trypanosoma cruzi is the etiologic agent for Chagas disease, which affects 6-7 million people worldwide. The biological diversity of the parasite reflects on inefficiency of benznidazole, which is a first choice chemotherapy, on chronic patients. ABC transporters that extrude xenobiotics, metabolites, and mediators are overexpressed in resistant cells and contribute to chemotherapy failure. An ABCC-like transport was identified in the Y strain and extrudes thiol-conjugated compounds. As thiols represent a line of defense towards reactive species, we aimed to verify whether ABCC-like transport could participate in the regulation of responses to stressor stimuli. In order to achieve this, ABCC-like activity was measured by flow cytometry using fluorescent substrates. The present study reveals the participation of glutathione and ceramides on ABCC-like transport, which are both implicated in stress. Hemin modulated the ABCC-like efflux which suggests that this protein might be involved in cellular detoxification. Additionally, all strains evaluated exhibited ABCC-like activity, while no ABCB1-like activity was detected. Results suggest that ABCC-like efflux is not associated with natural resistance to benznidazole, since sensitive strains showed higher activity than the resistant ones. Although benznidazole is not a direct substrate, ABCC-like efflux increased after prolonged drug exposure and this indicates that the ABCC-like efflux mediated protection against cell stress depends on the glutathione biosynthesis pathway.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Enfermedad de Chagas/tratamiento farmacológico , Glutatión/metabolismo , Nitroimidazoles/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Transporte Biológico , Enfermedad de Chagas/parasitología , Resistencia a Medicamentos , Estrés Oxidativo/fisiología , Tripanocidas/farmacología , Trypanosoma cruzi/metabolismoRESUMEN
Oxidative stress (OS) is a key factor in the development of gastrointestinal disorders, in which the intestinal barrier is altered. However, the Multidrug resistance-associated protein 2 (Mrp2) status, an essential component of the intestinal transcellular barrier exhibiting pharmaco-toxicological relevance by limiting the orally ingested toxicants and drugs absorption, has not been investigated. We here evaluated the short-term effect of OS on Mrp2 by treatment of isolated rat intestinal sacs with tert-butyl hydroperoxide (TBH) for 30 min. OS induction by TBH (250 and 500 µM) was confirmed by increased lipid peroxidation end products, decreased reduced glutathione (GSH) content and altered antioxidant enzyme activities. Under this condition, assessment of Mrp2 distribution between brush border (BBM) and intracellular (IM) membrane fractions, showed that Mrp2 protein decreased in BBM and increased in IM, consistent with an internalization process. This was associated with decreased efflux activity and, consequently, impaired barrier function. Subsequent incubation with N-Acetyl-L-Cysteine (NAC, 1 mM) reestablished GSH content and reverted concomitantly the alteration in Mrp2 localization and function induced by TBH. Cotreatment with a specific inhibitor of classic calcium-dependent Protein Kinase C (cPKC) implicated this kinase in TBH-effects. In conclusion, we demonstrated a negative posttranslational regulation of rat intestinal Mrp2 after short-term exposition to OS, a process likely mediated by cPKC and dependent on intracellular GSH content. The concomitant impairment of the Mrp2 barrier function may have implications in xenobiotic absorption and toxicity in a variety of human diseases linked to OS, with notable consequences on the toxicity/safety of therapeutic agents.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Microvellosidades/metabolismo , Estrés Oxidativo/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Animales , Relación Dosis-Respuesta a Droga , Mucosa Intestinal/efectos de los fármacos , Yeyuno/efectos de los fármacos , Masculino , Microvellosidades/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Wistar , terc-Butilhidroperóxido/toxicidadRESUMEN
Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Placenta/ultraestructura , Placenta/virología , Complicaciones Infecciosas del Embarazo , Infección por el Virus Zika/genética , Infección por el Virus Zika/virología , Virus Zika/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apoptosis , Biomarcadores , Femenino , Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad , Huésped Inmunocomprometido , Inmunohistoquímica , Masculino , Ratones , Embarazo , Infección por el Virus Zika/patologíaRESUMEN
The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the "arm-like" domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the "arm-like" domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the "arm-like" domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Hidrólisis , FosforilaciónRESUMEN
Prenylated flavonoids are an important class of naturally occurring flavonoids with important biological activity, but their low abundance in nature limits their application in medicines. Here, we showed the hemisynthesis and the determination of various biological activities of seven prenylated flavonoids, named 7-13, with an emphasis on antimicrobial ones. Compounds 9, 11, and 12 showed inhibitory activity against human pathogenic fungi. Compounds 11, 12 (flavanones) and 13 (isoflavone) were the most active against clinical isolated Staphylococcus aureus MRSA, showing that structural requirements as prenylation at position C-6 or C-8 and OH at positions C-5, 7, and 4' are key to the antibacterial activity. The combination of 11 or 12 with commercial antibiotics synergistically enhanced the antibacterial activity of vancomycin, ciprofloxacin, and methicillin in a factor of 10 to 100 times against drug-resistant bacteria. Compound 11 combined with ciprofloxacin was able to decrease the levels of ROS generated by ciprofloxacin. According to docking results of S enantiomer of 11 with ATP-binding cassette transporter showed the most favorable binding energy; however, more studies are needed to support this result.
Asunto(s)
Antibacterianos/farmacología , Flavonoides/farmacología , Prenilación/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Simulación por Computador , Flavanonas/farmacología , Hongos/efectos de los fármacos , Humanos , Isoflavonas/farmacología , Ratones , Pruebas de Sensibilidad Microbiana/métodos , Especies Reactivas de Oxígeno/metabolismo , Staphylococcus aureus/efectos de los fármacosRESUMEN
The ability of Listeria monocytogenes grow on ready-to-eat food is a major concern in food safety. Natural antimicrobials, such as nisin, can be used to control this pathogen, but the increasing reports of nisin tolerance and resistance make necessary novel approaches to increase its effectiveness, such as encapsulation. The goal of this study was to investigate how L. monocytogenes ATCC7644 regulates and shapes its proteome in response to sublethal doses of nisin and nisin-loaded phosphatidylcholine liposomes (lipo-nisin), compared to untreated cells growing under optimal conditions. Total proteins were extracted from L. monocytogenes cells treated for 1 h with free and lipo-nisin. As result, of 803 proteins that were initially identified, 64 and 53 proteins were differentially upregulated and downregulated respectively, in the treatments with nisin and lipo-nisin. Changes of Listeria proteome in response to treatments containing nisin were mainly related to ATP-binding cassette (ABC) transporter systems, transmembrane proteins, RNA-binding proteins and diverse stress response proteins. Some of the proteins uniquely detected in samples treated with free nisin were the membrane proteins SecD, Lmo1539 and the YfhO enzyme, which are related to translocation of L. monocytogenes virulence factors, activation of the LiaR-mediated stress defense and glycosylation of wall teichoic acid, respectively. The L. monocytogenes treated with liposome encapsulated nisin showed no expression of some stress response factors as compared with the free nisin, suggesting a reduction of stress mediated response and production of nisin-resistance factors by exposure to encapsulated nisin.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Listeria monocytogenes/efectos de los fármacos , Nisina/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Composición de Medicamentos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Nisina/química , ProteómicaRESUMEN
INTRODUCTION: The solute carrier (SLC) and the ATP-binding cassette (ABC) transporter superfamilies play essential roles in the disposition of small molecules (endogenous metabolites, uremic toxins, drugs) in the blood, kidney, liver, intestine, and other organs. In chronic kidney disease (CKD), the loss of renal function is associated with altered function of remote organs. As renal function declines, many molecules accumulate in the plasma. Many studies now support the view that ABC and SLC transporters as well as drug metabolizing enzymes (DMEs) in renal and non-renal tissues are directly or indirectly affected by the presence of various types of uremic toxins, including those derived from the gut microbiome; this can lead to aberrant inter-organ communication. AREAS COVERED: Here, the expression, localization and/or function of various SLC and ABC transporters as well as DMEs in the kidney and other organs are discussed in the context of CKD and systemic pathophysiology. EXPERT OPINION: According to the Remote Sensing and Signaling Theory (RSST), a transporter and DME-centric network that optimizes local and systemic metabolism maintains homeostasis in the steady state and resets homeostasis following perturbations due to renal dysfunction. The implications of this view for pharmacotherapy of CKD are also discussed.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Proteínas Transportadoras de Solutos/metabolismo , Animales , Enzimas/metabolismo , Microbioma Gastrointestinal , Humanos , Insuficiencia Renal Crónica/tratamiento farmacológicoRESUMEN
The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Membrana Celular/enzimología , Proteolisis , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Transportadoras de Casetes de Unión a ATP/metabolismo , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Mercury is a widespread pollutant. Mercuric ions uptake into tubular cells is supported by the Organic anion transporter 1 (Oat1) and 3 (Oat3) and its elimination into urine is through the Multidrug resistance-associated protein 2 (Mrp2). We investigated the effect of recombinant human erythropoietin (Epo) on renal function and on renal expression of Oat1, Oat3, and Mrp2 in a model of mercuric chloride (HgCl2)-induced renal damage. Four experimental groups of adult male Wistar rats were used: Control, Epo, HgCl2, and Epo + HgCl2. Epo (3000 IU/kg, b.w., i.p.) was administered 24 h before HgCl2 (4 mg/kg, b.w., i.p.). Experiments were performed 18 h after the HgCl2 dose. Parameters of renal function and structure were evaluated. The protein expression of Oat1, Oat3 and Mrp2 in renal tissue was assessed by immunoblotting techniques. Mercury levels were determined by cold vapor atomic absorption spectrometry. Pretreatment with Epo ameliorated the HgCl2-induced tubular injury as assessed by histopathology and urinary biomarkers. Immunoblotting showed that pretreatment with Epo regulated the renal expression of mercury transporters in a way to decrease mercury content in the kidney. Epo pretreatment ameliorates HgCl2-induced renal tubular injury by modulation of mercury transporters expression in the kidneys.