RESUMEN
In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O']-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (Ë93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4ß7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101's affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.
Asunto(s)
Etilenos/uso terapéutico , Integrinas/antagonistas & inhibidores , Leishmania donovani/enzimología , Leishmaniasis Visceral/tratamiento farmacológico , NADH NADPH Oxidorreductasas/efectos de los fármacos , Animales , Células Cultivadas , Cricetinae , Modelos Animales de Enfermedad , Etilenos/farmacología , Femenino , Interacciones Huésped-Parásitos/efectos de los fármacos , Integrinas/efectos de los fármacos , Leishmania donovani/efectos de los fármacos , Leishmania donovani/metabolismo , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/patología , Masculino , Ratones , Ratones Endogámicos BALB C , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Excess catecholamine levels are suggested to be cardiotoxic and to underlie stress-induced heart failure. The cardiotoxic effects of norepinephrine and epinephrine are well recognized. However, although cardiac and circulating dopamine levels are also increased in stress cardiomyopathy patients, knowledge regarding putative toxic effects of excess dopamine levels on cardiomyocytes is scarce. We now studied the effects of elevated dopamine levels in H9c2 cardiomyoblasts. H9c2 cells were cultured and treated with dopamine (200 µM) for 6, 24, and 48 h. Subsequently, the effects on lipid accumulation, cell viability, flippase activity, reactive oxygen species (ROS) production, subcellular NADPH oxidase (NOX) protein expression, and ATP/ADP and GTP/GDP levels were analyzed. Dopamine did not result in cytotoxic effects after 6 h. However, after 24 and 48 h dopamine treatment induced a significant increase in lipid accumulation, nitrotyrosine levels, indicative of ROS production, and cell death. In addition, dopamine significantly reduced flippase activity and ATP/GTP levels, coinciding with phosphatidylserine exposure on the outer plasma membrane. Furthermore, dopamine induced a transient increase in cytoplasmic and (peri)nucleus NOX1 and NOX4 expression after 24 h that subsided after 48 h. Moreover, while dopamine induced a similar transient increase in cytoplasmic NOX2 and p47phox expression, in the (peri)nucleus this increased expression persisted for 48 h where it colocalized with ROS. Exposure of H9c2 cells to elevated dopamine levels induced lipid accumulation, oxidative stress, and a proinflammatory status of the plasma membrane. This can, in part, explain the inflammatory response in patients with stress-induced heart failure.
Asunto(s)
Dopaminérgicos/farmacología , Dopamina/farmacología , Inflamación/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Mioblastos Cardíacos/efectos de los fármacos , NADPH Oxidasas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular , Citometría de Flujo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Microscopía Fluorescente , Mioblastos Cardíacos/metabolismo , Mioblastos Cardíacos/ultraestructura , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/metabolismo , Peroxidasa/efectos de los fármacos , Peroxidasa/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Tirosina/análogos & derivados , Tirosina/efectos de los fármacos , Tirosina/metabolismoRESUMEN
The synthesis of hitherto unreported S-alkyl/aryl benzothiazole-2-carbothioate is reported from thiols, oxalyl chloride, and 2-aminothiophenols using 10 mol % n-tetrabutylammonium iodide (TBAI) as catalyst in acetonitrile through multicomponent reaction (MCR) strategy. The present protocol favored formation of benzothiazoles and thioesters via simultaneous formation of C-N and C-S bonds in good yields with a wide range of substrates. A few of the synthesized derivatives were evaluated for their antimicrobial activity against the protozoan parasite Leishmania donovani, a causative agent of visceral leishmaniasis (VL). Further, these compounds displayed no toxicity toward macrophage RAW 264.7 cells and are therefore nontoxic and effective antileishmanial leads. In silico docking studies were performed to understand the possible binding site interaction with trypanothione reductase (TryR).
Asunto(s)
Antiprotozoarios/síntesis química , Antiprotozoarios/farmacología , Benzotiazoles/síntesis química , Benzotiazoles/farmacología , Leishmania donovani/efectos de los fármacos , Macrófagos/efectos de los fármacos , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/efectos de los fármacos , Compuestos de Sulfhidrilo/química , Antiprotozoarios/química , Benzotiazoles/química , Sitios de Unión , Leishmania donovani/enzimología , Leishmaniasis Visceral/enzimología , NADH NADPH Oxidorreductasas/metabolismo , Ornitina Descarboxilasa/metabolismoRESUMEN
OBJECTIVE: NOX-1 and NOX-4 are key enzymes responsible for reactive oxygen species (ROS) generation in vascular smooth muscle cells (VSMC). The RNA-binding protein Hu antigen R (HuR) is implicated in posttranscriptional regulation of gene expression; however, its role regulating NOX is unknown. We investigated transcriptional and posttranscriptional mechanisms underlying angiotensin II (AngII) and IL-1ß regulation of NOX-1 and NOX-4 in VSMC and their implications in cell migration. METHODS: Rat and human VSMC were stimulated with AngII (0.1âµmol/l) and/or IL-1ß (10âng/ml). NOX-1 and NOX-4 mRNA and protein levels, NOX-1 and NOX-4 promoter and 3'UTR activities, NADPH oxidase activity, ROS production, and cell migration were studied. RESULTS: IL-1ß increased NOX-1 expression, NADPH oxidase activity and ROS production, and decreased NOX-4 expression and H2O2 production in VSMC. AngII potentiated the IL-1ß-mediated induction of NOX-1 expression, NADPH oxidase activity, ROS production, and cell migration. However, AngII did not influence IL-1ß-induced NOX-4 downregulation. AngIIâ+âIL-1ß interfered with the decay of NOX-1 mRNA and promoted HuR binding to NOX-1 mRNA. Moreover, HuR blockade reduced NOX-1 mRNA stability and AngIIâ+âIL-1ß-induced NOX-1 mRNA levels. IL-1ß decreased NOX-4 expression through a transcriptional mechanism that involved response elements situated in the proximal promoter. AngII and/or IL-1ß-induced cell migration were prevented by NOX-1 and HuR blockade and were augmented by NOX-4 overexpression. CONCLUSION: In VSMC HuR-mediated mRNA stabilization is partially responsible for AngIIâ+âIL-1ß-dependent NOX-1 expression, whereas transcriptional mechanisms are involved in decreased NOX-4 expression induced by IL-1ß. NOX4 and HuR regulation of NOX-1 contributes to VSMC migration, important in vascular inflammation and remodeling.
Asunto(s)
Angiotensina II/farmacología , Proteína 1 Similar a ELAV/metabolismo , Interleucina-1beta/farmacología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Regulación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 4 , NADPH Oxidasas/efectos de los fármacos , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Inflammation and the excess production of reactive oxygen species (ROS) contribute significantly to the pathogenesis of ischemic retinopathies such as diabetic retinopathy and retinopathy of prematurity. We hypothesized that GKT137831, a dual inhibitor of NADPH oxidases (NOX) 1 and NOX4, reduces inflammation in the ischemic retina by dampening the pro-inflammatory phenotype of retinal immune cells as well as macroglial Müller cells and neurons. METHODS: Ischemic retinopathy was induced in Sprague-Dawley rats by exposure to 80 % O2 cycled with 21 % O2 for 3 h per day from postnatal day (P) 0 to P11, followed by room air (P12 to P18). GKT137831 was administered P12 to P18 (60 mg/kg, subcutaneous) and comparisons were to room air controls. Retinal inflammation was examined by measuring leukocyte adherence to the retinal vasculature, ionized calcium-binding adaptor protein-1-positive microglia/macrophages, and the mRNA and protein levels of key inflammatory factors involved in retinal disease. Damage to Müller cells was evaluated by quantitating glial fibrillary acidic protein-positive cells and vascular leakage with an albumin ELISA. To verify the anti-inflammatory actions of GKT137831 on glia and neurons involved in ischemic retinopathy, primary cultures of rat retinal microglia, Müller cells, and ganglion cells were exposed to the in vitro counterpart of ischemia, hypoxia (0.5 %), and treated with GKT137831 for up to 72 h. ROS levels were evaluated with dihydroethidium and the protein and gene expression of inflammatory factors with quantitative PCR, ELISA, and a protein cytokine array. RESULTS: In the ischemic retina, GKT137831 reduced the increased leukocyte adherence to the vasculature, the pro-inflammatory phenotype of microglia and macroglia, the increased gene and protein expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and leukocyte adhesion molecules as well as vascular leakage. In all cultured cell types, GKT137831 reduced the hypoxia-induced increase in ROS levels and protein expression of various inflammatory mediators. CONCLUSIONS: NOX1/4 enzyme inhibition with GKT137831 has potent anti-inflammatory effects in the retina, indicating its potential as a treatment for a variety of vision-threatening retinopathies.
Asunto(s)
NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADPH Oxidasas/antagonistas & inhibidores , Neuroglía/efectos de los fármacos , Pirazoles/farmacología , Pirazoles/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Retinitis/prevención & control , Animales , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Hipoxia/complicaciones , Técnicas In Vitro , Molécula 1 de Adhesión Intercelular/metabolismo , Isquemia/etiología , Isquemia/patología , Isquemia/prevención & control , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADPH Oxidasa 1 , NADPH Oxidasa 4 , NADPH Oxidasas/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patología , Pirazolonas , Piridonas , Ratas , Especies Reactivas de Oxígeno/metabolismo , Retinitis/metabolismo , Retinitis/patología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pulmonary vascular remodeling, characterized by disordered proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a pathognomonic feature of pulmonary arterial hypertension. Thus, pharmacologic strategy targeting on anti-proliferation and anti-migration of PASMCs may have therapeutic implications for PAH. Here we investigated the effects and underlying mechanisms of B-type natriuretic peptide (BNP) on angiotensin II (Ang II)-induced proliferation and migration of PASMCs. Proliferation and migration of PASMCs cultured from Wistar rats were induced by Ang II, with or without BNP treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca(2+) overload, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and the cGMP/PKG pathway were examined. We found that BNP inhibited Ang II-induced PASMCs proliferation and migration dose dependently. BNP could also arrest the cell cycle progression in the G0/G1-phase. In addition, BNP attenuated intracellular calcium overload caused by Ang II. Moreover, Ang II-induced ROS production was mitigated by BNP, with associated down-regulation of NAD(P)H oxidase 1 (Nox1) and reduced mitochondrial ROS production. Finally, Ang II-activated MAPKs and Akt were also counteracted by BNP. Of note, all these effects of BNP were abolished by a PKG inhibitor (Rp-8-Br-PET-cGMPS). In conclusion, BNP inhibits Ang II-induced PASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production by Nox1 and mitochondria, and down-regulation of MAPK and Akt signal transduction, through the cGMP/PKG pathway. Therefore, this study implicates that BNP may have a therapeutic role in pulmonary vascular remodeling.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Natriuréticos/farmacología , Péptido Natriurético Encefálico/farmacología , Arteria Pulmonar/citología , Angiotensina II/farmacología , Animales , Calcio/metabolismo , Células Cultivadas , Regulación hacia Abajo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Arteria Pulmonar/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno , Vasoconstrictores/farmacologíaRESUMEN
OBJECTIVE: To investigate the possible effect of artesunate (ART) on schistosome thioredoxin glutathione reductase (TGR) and cytochrome c peroxidase (CcP) in Schistosoma mansoni-infected mice. METHODS: A total of 200 laboratory bred male Swiss albino mice were divided into 4 groups (50 mice in each group). Group I: infected untreated group (Control group) received a vehicle of 1% sodium carbonyl methylcellulose (CMC-Na); Group II: infected then treated with artesunate; Group III: infected then treated with praziquantel, and group IV: infected then treated with artesunate then praziquantel. Adult S. mansoni worms were collected by Animal Perfusion Method, tissue egg counted, TGR, and CcP mRNA Expression were estimated of in S. mansoni adult worms by semi-quantitative rt-PCR. RESULTS: Semi-quantitative rt-PCR values revealed that treatment with artesunate caused significant decrease in expression of schistosome TGR and CcP in comparison to the untreated group. In contrast, the treatment with praziquantel did not cause significant change in expression of these genes. The results showed more reduction in total worm and female worm count in combined ART-PZQ treated group than in monotherapy treated groups by either ART or PZQ. Moreover, complete disappearance (100%) of tissue eggs was recorded in ART-PZQ treated group with a respective reduction rate of 95.9% and 68.4% in ART- and PZQ-treated groups. CONCLUSION: The current study elucidated for the first time that anti-schistosomal mechanisms of artesunate is mediated via reduction in expression of schistosome TGR and CcP. Linking these findings, addition of artesunate to praziquantel could achieve complete cure outcome in treatment of schistosomiasis.
Asunto(s)
Artemisininas/farmacología , Citocromo-c Peroxidasa/efectos de los fármacos , Complejos Multienzimáticos/efectos de los fármacos , NADH NADPH Oxidorreductasas/efectos de los fármacos , Schistosoma/efectos de los fármacos , Animales , Artesunato , Citocromo-c Peroxidasa/genética , Masculino , Ratones , Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Schistosoma/enzimologíaRESUMEN
Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P < 0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Hifa/efectos de los fármacos , Lipopolisacáridos/farmacología , Pseudomonas aeruginosa/fisiología , Adenosina Trifosfatasas/efectos de los fármacos , Candida albicans/genética , Candida albicans/fisiología , AMP Cíclico/análisis , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas Fúngicas/efectos de los fármacos , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glicina Hidroximetiltransferasa/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Hidrolasas/efectos de los fármacos , Hifa/genética , Klebsiella pneumoniae/fisiología , Glicoproteínas de Membrana/efectos de los fármacos , Interacciones Microbianas , NADH NADPH Oxidorreductasas/efectos de los fármacos , Fosfoglicerato Quinasa/efectos de los fármacos , Proteoma/genética , Deshidrogenasas del Alcohol de Azúcar/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate-glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.
Asunto(s)
Avicennia/enzimología , Cloroplastos/enzimología , NADH NADPH Oxidorreductasas/genética , Tolerancia a la Sal/fisiología , Cloruro de Sodio/farmacología , Activación Transcripcional/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , NADH NADPH Oxidorreductasas/efectos de los fármacos , Plantas Modificadas Genéticamente , Plantas Tolerantes a la SalRESUMEN
The bioassay-guided fractionation of the EtOH extract of the leaves of Galenia africana led to the isolation of three known flavonoids, (2S)-5,7,2'-trihydroxyflavanone (1), (E)-3,2',4'-trihydroxychalcone (2), and (E)-2',4'-dihydroxychalcone (3), and the new (E)-3,2',4'-trihydroxy-3'-methoxychalcone (4). Compounds 1 and 3 exhibited moderate antituberculosis activity. During synergistic studies, a combination of compound 4 and an existing antituberculosis drug, isoniazid, reduced their original MICs 4-fold, resulting in a fractional inhibitory concentration of 0.50. The most pronounced effect was demonstrated by compound 1 and isoniazid reducing their MICs 16-fold and resulting in an FIC of 0.12. Both EtOH extract and isolated compounds failed to exhibit any NADPH oxidase activity at 800.0 muM concentrations, indicating that mycothiol disulfide reductase is not the target for their antituberculosis activity.
Asunto(s)
Antituberculosos/aislamiento & purificación , Antituberculosos/farmacología , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Magnoliopsida/química , Plantas Medicinales/química , Antituberculosos/química , Flavonoides/química , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADPH Oxidasas/efectos de los fármacos , Hojas de la Planta/química , Sudáfrica , EstereoisomerismoRESUMEN
Phenoxodiol is an experimental anticancer drug under development as a chemosensitizer intended to reverse multidrug resistance mechanisms in ovarian and prostate cancer cells to most standard cytotoxics. The putative molecular target of phenoxodiol is a cell-surface, tumor-specific NADH oxidase, ENOX2 (tNOX), with phenoxodiol having no apparent effect on the constitutive form of this enzyme ENOX1 (CNOX). Using ENOX2 as the target, this study was conducted to explore the temporal relationship between phenoxodiol and paclitaxel or cisplatin in achieving chemosensitization in HeLa cells which are relatively resistant to both paclitaxel and cisplatin. Sequential addition of phenoxodiol and paclitaxel or phenoxodiol and cisplatin showed greater inhibition of HeLa cell ENOX1 activity and growth compared to adding the drugs simultaneously or individually. In parallel, a similar chemosensitizing response of phenoxodiol for cisplatin was observed. ENOX1 was not affected and trans-platinum had no effect. With spent media from phenoxodiol-treated cells sensitivity was enhanced to both paclitaxel and cisplatin if the cells were first pretreated with phenoxodiol. Similar results were obtained with ENOX2-enriched preparations stripped from the surfaces of phenoxodiol-treated cells. In keeping with a speculative prion model, it seems as though the ENOX2 "remembers" the phenoxodiol and "teaches" other ENOX2 molecules to respond to paclitaxel and cisplatin as if phenoxodiol were still present.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Isoflavonas/farmacología , NADH NADPH Oxidorreductasas , Paclitaxel/farmacología , Antineoplásicos/farmacología , Membrana Celular/metabolismo , Medios de Cultivo Condicionados/farmacología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Microsomas/metabolismo , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , Paclitaxel/metabolismoRESUMEN
Our work has identified an aging-related ECTO-NOX activity (arNOX), a hydroquinone oxidase which is cell surface located and generates superoxide. This activity increases with increasing age beginning >30 y. Because of its cell surface location and ability to generate superoxide, the arNOX proteins may serve to propagate an aging cascade both to adjacent cells and to oxidize circulating lipoproteins as significant factors determining atherogenic risk. The generation of superoxide by arNOX proteins is inhibited by Coenzyme Q10 as one basis for an anti-aging benefit of CoQ10 supplementation in human subjects. In a preliminary pilot study, 25 female subjects between 45 and 55 y of age were recruited at Stanford University from the Palo Alto, CA area. Informed consent was obtained. Ten of the subjects received Coenzyme Q10 supplementation of 180 (3 x 60 mg) per day for 28 days. Serum, saliva and perspiration levels of arNOX were determined at 7, 14 and 28 days of CoQ10 supplementation and compared to the initial baseline value. Activity correlated with subject age up to a maximum between age 50 and 55 years of age for saliva and perspiration as well and then declined. With all three sources, the arNOX activity extrapolated to zero at about age 30. Response to Coenzyme Q10 also increased with age being least between ages 45 and 50 and greatest between ages 60 and 65. With all three biofluids, arNOX activity was reduced between 25 and 30% by a 3 x 60 mg daily dose Coenzyme Q10 supplementation. Inhibition was the result of Coenzyme Q10 presence.
Asunto(s)
NADH NADPH Oxidorreductasas/metabolismo , Ubiquinona/análogos & derivados , Adulto , Anciano , Envejecimiento , Femenino , Humanos , Masculino , Persona de Mediana Edad , NADH NADPH Oxidorreductasas/efectos de los fármacos , Proyectos Piloto , Saliva/efectos de los fármacos , Saliva/enzimología , Sudor/efectos de los fármacos , Sudor/enzimología , Ubiquinona/administración & dosificaciónRESUMEN
arNOX is a coenzyme Q10-inhibited, aging-related ECTO-NOX protein of the cell surface also present in sera. It is capable of superoxide generation measured as superoxide dismutase-inhibited reduction of ferricytochrome c and is a potential contributor to atherogenic risk. Here, we report an arNOX activity of saliva of older individuals also inhibited by coenzyme Q10. The activity first appears after age 30 to a near maximum at about age 55. Those surviving beyond age 55 usually have reduced arNOX activities. Our studies demonstrate significant (25 to 30%) reduction of arNOX levels with coenzyme Q10 supplementation of 60 mg (2 x 30 mg) per day for 28 days. Activity correlated with age. Response to coenzyme Q10 increased with age being greatest between ages 60 and 65. Saliva arNOX levels varied in a regular pattern throughout the day so it was important that samples be collected at approximately the same time each day for comparative purposes. The coenzyme Q10 response was reversible and within 12 h after the last intake of coenzyme Q10, the salivary arNOX levels returned to base line. The findings suggest that salivary arNOX provides a convenient and non-invasive method to monitor arNOX levels in clinical coenzyme Q10 intervention trials with the response levels paralleling those seen with serum and cellular arNOX.
Asunto(s)
NADH NADPH Oxidorreductasas/metabolismo , Saliva/enzimología , Ubiquinona/análogos & derivados , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/efectos de los fármacos , Superóxidos/metabolismo , Ubiquinona/farmacologíaRESUMEN
Lithium has been used widely both as a clinical agent to treat manic depressive disorders and as a substance targeted to the regulation of the circadian cycle. In this study, we show that lithium at physiological concentrations of less than 1microM uniquely induces an ECTO-NOX activity previously inactive from plant (soybean), murine (3T3 cells) and human (HUVEC and HeLa cells) sources and resets the period of the constitutive CNOX. The average period length of the new oscillation set induced by the presence of lithium of 23.5min was slightly less than the period length in the absence of lithium (24min). The constitutive period was retained in the presence of lithium but the period length was increased on an average by 4% to about 25min. Targeting circadian rhythm abnormalities may be a particularly useful strategy in management of bipolar disorder and related illnesses since circadian cycles appear to be an inherent function conserved through evolution in all organisms and consistently implicated in the pathophysiology of bipolar disorder.
Asunto(s)
Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Ritmo Circadiano/efectos de los fármacos , Litio/farmacología , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , Animales , Antimaníacos/farmacología , Antimaníacos/uso terapéutico , Relojes Biológicos/efectos de los fármacos , Relojes Biológicos/fisiología , Trastorno Bipolar/tratamiento farmacológico , Trastorno Bipolar/metabolismo , Trastorno Bipolar/fisiopatología , Ritmo Circadiano/fisiología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Compuestos de Litio/farmacología , Compuestos de Litio/uso terapéutico , Ratones , Células 3T3 NIH , Plantas/efectos de los fármacos , Plantas/metabolismo , Factores de TiempoRESUMEN
Recently, we showed that the MarR-type repressor YkvE (MhqR) regulates multiple dioxygenases/glyoxalases, oxidoreductases and the azoreductase encoding yvaB (azoR2) gene in response to thiol-specific stress conditions, such as diamide, catechol and 2-methylhydroquinone (MHQ). Here we report on the regulation of the yocJ (azoR1) gene encoding another azoreductase by the novel DUF24/MarR-type repressor, YodB after exposure to thiol-reactive compounds. DNA binding activity of YodB is directly inhibited by thiol-reactive compounds in vitro. Mass spectrometry identified YodB-Cys-S-adducts that are formed upon exposure of YodB to MHQ and catechol in vitro. This confirms that catechol and MHQ are auto-oxidized to toxic ortho- and para-benzoquinones which act like diamide as thiol-reactive electrophiles. Mutational analyses further showed that the conserved Cys6 residue of YodB is required for optimal repression in vivo and in vitro while substitution of all three Cys residues of YodB affects induction of azoR1 transcription. Finally, phenotype analyses revealed that both azoreductases, AzoR1 and AzoR2 confer resistance to catechol, MHQ, 1,4-benzoquinone and diamide. Thus, both azoreductases that are controlled by different regulatory mechanisms have common functions in quinone and azo-compound reduction to protect cells against the thiol reactivity of electrophiles.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , NADH NADPH Oxidorreductasas/genética , Quinonas/metabolismo , Proteínas Represoras/genética , Compuestos de Sulfhidrilo/metabolismo , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Catecoles/metabolismo , Cisteína/metabolismo , Huella de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Diamida/metabolismo , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Hidroquinonas/metabolismo , Espectrometría de Masas , Modelos Moleculares , NADH NADPH Oxidorreductasas/efectos de los fármacos , Nitrorreductasas , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Regiones Promotoras Genéticas , Proteómica , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
A nutritional characteristic of trypanosomatid protozoa is that in vitro they need a haem-compound as a growth factor, which is supplied as haemoglobin, haematin or haemin. Because haemin and related porphyrins are an important source of oxidative stress in biological systems, the effect of haemin on growth, protein content and the antioxidant defence system in Trypanosoma cruzi was evaluated. We have observed that, in epimastigotes grown under different haemin concentrations in the culture medium (0-30 mg/l), 5 mg/l was the haemin concentration yielding optimum growth. Above 15 mg/l there was a clear decrease in growth rate, producing the epimastigote to amastigote transformation. Such morphological change was observed together with a marked injury of the enzymatic machinery of the parasite, leading to diminished protein synthesis as well as lower activity of the antioxidant enzymes (superoxide dismutase, ascorbate peroxidase and trypanothione reductase), reduced total thiol content and a marked increase in the HaemOx-1 activity and expression. The current work demonstrates that there is a correlation between higher haemin concentrations in the culture medium and oxidative damage in the cells. Under these conditions induction of HaemOx-1 would indicate the important role of this enzyme as an antioxidant defence response in Trypanosoma cruzi.
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Hemina/farmacología , Estrés Oxidativo/fisiología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/fisiología , Animales , Ascorbato Peroxidasas , Medios de Cultivo , Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/análisis , Hemo-Oxigenasa 1/biosíntesis , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Peroxidasas/efectos de los fármacos , Peroxidasas/metabolismo , Proteínas/análisis , Proteínas/efectos de los fármacos , Compuestos de Sulfhidrilo/metabolismo , Superóxido Dismutasa/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Trypanosoma cruzi/citología , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.
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Acanthamoeba/efectos de los fármacos , Antibacterianos/farmacología , Antifúngicos/farmacología , Antipsicóticos/farmacología , NADH NADPH Oxidorreductasas/efectos de los fármacos , Naegleria fowleri/efectos de los fármacos , Acanthamoeba/enzimología , Animales , Cromatografía Líquida de Alta Presión , Glutatión Reductasa/efectos de los fármacos , Glutatión Reductasa/metabolismo , Humanos , NADH NADPH Oxidorreductasas/metabolismo , Naegleria fowleri/enzimologíaRESUMEN
Numerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process.
Asunto(s)
Anaerobiosis/fisiología , Proteínas Bacterianas/metabolismo , Ácidos Grasos/biosíntesis , NADH NADPH Oxidorreductasas/metabolismo , Streptococcus agalactiae/fisiología , Streptococcus agalactiae/patogenicidad , Acetatos/farmacología , Adaptación Biológica , Animales , Antioxidantes/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/genética , Proliferación Celular , Activación Enzimática , Ácidos Grasos/química , Ácidos Grasos/farmacología , Mutación , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADH NADPH Oxidorreductasas/genética , Ácido Oléico/farmacología , Polisorbatos/farmacología , Ratas , Ratas Sprague-Dawley , Virulencia/fisiologíaRESUMEN
Tumor-associated NOX (tNOX) is a novel cell surface ECTO-NOX protein that represents a promising target for selective antitumor therapy. Studies have confirmed the unique presence of tNOX on the cell surface of invasive human cancers and in the sera of cancer patients. Furthermore, as there is a resolute difference between tNOX and the drug-resistant constitutive NOX isoform constitutive NOX, it represents an attractive target for drug, vaccine and diagnostic strategies for cancer. Interestingly, several products currently utilized or under development for cancer display tNOX inhibition as one of their underlying mechanisms, these include non- steroidal antiinflammatory drugs, (-)-epigallocatechin gallate, phenoxodiol and doxorubicin hydrochloride (Adria- mycin). This spotlight article will also highlight tNOX inhibitors under preclinical development and new lines of research to target tNOX for cancer therapeutics.
Asunto(s)
Antineoplásicos/uso terapéutico , NADH NADPH Oxidorreductasas/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Sistemas de Liberación de Medicamentos , Humanos , NADH NADPH Oxidorreductasas/genéticaRESUMEN
Lactosylceramide (LacCer) is a member of the glycosphingolipid family which has been recently recognized as a signaling intermediate in the regulation of cell proliferation and cell adhesion. In this paper, we present our studies pointing to a potential role of LacCer in inducing apoptosis. In our studies we employed a human osteosarcoma cell line MG-63 (wild type, WT) and a neutral sphingomyelinase (N-SMase) deficient cell line CC derived from MG-63 (mutant) cells. We observed that WT cells were highly sensitive to tumor necrosis factor-alpha (TNF-alpha), ceramide and LacCer-induced apoptosis. In contrast, the mutant cells were insensitive to TNF-alpha-induced apoptosis as they did not generate ceramide and LacCer. However, the exogenous supply of ceramide and/or LacCer rendered the mutant cells apoptotic. Interestingly, preincubation of cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase, abrogated ceramide-induced apoptosis but not LacCer-induced apoptosis in both WT cells and the mutant cells. Moreover, TNF-alpha and LacCer-induced apoptosis required the generation of reactive oxygen species (ROS) in WT cells. However, since mutant cells did not produce significant amounts of LacCer and ROS in response to TNF-alpha treatment they are insensitive to TNF-alpha-induced apoptosis. In summary, our studies suggest that TNF-alpha-induced N-SMase activation and production of ceramide is required to activate the apoptosis pathway in human osteosarcoma cells. But it is not sufficient to induce apoptosis. Rather, the conversion of ceramide to LacCer and ROS generation are critical for apoptosis.