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1.
Bioorg Med Chem ; 28(9): 115440, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32205046

RESUMO

A chip-based screening system for IκB kinase ß (IKKß) has been developed by physically immobilizing the substrate IκBα on a glass matrix using a calixarene linker. Phosphorylation of IκBα by IKKß and ATP was quantitated using a fluorescently labeled antibody. Using this efficient assay system a chemical library of 2000 bioactive compounds was screened against IKKß and four were identified as good inhibitors, namely, aurintricarboxylic acid, diosmin, ellagic acid, and hematein. None of them have been reported to be an inhibitor of IKKß although they were implicated in various NFκB-mediated biological processes. Our enzyme-based assay showed that IC50 of the four inhibitors is comparable with that of IKK-16, a previously known strong inhibitor. Molecular docking simulation shows that the hydrophobic moiety of an inhibitor interacts with the four hydrophobic residues (Leu21, Val29, Val152, and Ile165) of the active site. The MM-PBSA calculation suggests that these hydrophobic interactions appear to be the predominant contributor to the binding free energy. As IKKß is ubiquitously expressed in various cell types and executes many biological functions, the enzyme and cell specificity of the four inhibitors need to be rigorously tested before accepted as a drug candidate.


Assuntos
Quinase I-kappa B/antagonistas & inibidores , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Quinase I-kappa B/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Termodinâmica
2.
J Biol Chem ; 290(1): 467-77, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25391655

RESUMO

Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE(-/-) mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and Gi-mediated phospholipase C/Ca(2+)/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC50 of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature.


Assuntos
Indutores da Angiogênese/farmacologia , Ginsenosídeos/farmacologia , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Receptor IGF Tipo 1/agonistas , Vasodilatação/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
3.
J Liposome Res ; 26(4): 336-44, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26982006

RESUMO

Bilayers prepared from sorbitan fatty acid esters (Span) have been frequently used for delivery of drugs including flavonoids. We applied molecular dynamics simulation to characterize the structure of a sorbitan monostearate (Span 60) bilayer in complex with three representative flavones, a subclass of flavonoids. At a low concentration, unsubstituted flavone, the most hydrophobic member, was able to flip over and cross the bilayer with a large diffusion coefficient. At a high concentration, it was accumulated at the bilayer center resulting in a phase separation. The leaflets of the bilayer were pushed in the opposite directions increasing the membrane thickness. Order parameter of the stearate chain of Span 60 was not affected significantly by unsubstituted flavone. In contrast, chrysin with hydroxylated ring A was lined up with the acyl chains of Span 60 with its hydroxyl group facing the membrane surface. Neither flipping nor transbilayer movement were allowed. Diffusion coefficient was only 15-25% of that of unsubstituted flavone and order parameter decreased with the concentration of chrysin. Luteolin, the most hydroxylated member, interacted mainly with the headgroup of Span 60 and assumed many different orientations without crossing the bilayer. Unlike chrysin and unsubstituted flavone the bilayer integrity was disrupted at 50 mol% luteolin. These behaviors and structures of flavones in a Span 60 bilayer can be accounted for by their hydrophobicity and sites of hydroxylation.


Assuntos
Flavonas/química , Hexoses/química , Bicamadas Lipídicas/química , Lipossomos/química , Simulação de Dinâmica Molecular , Difusão , Interações Hidrofóbicas e Hidrofílicas , Hidroxilação , Luteolina/química , Estrutura Molecular
4.
Biochem Biophys Res Commun ; 377(2): 612-616, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-18929538

RESUMO

Estimation of structural perturbation induced by S-nitrosation is important to understand the mode of cellular signal transduction mediated by nitric oxide. Crystal structures of S-nitrosated proteins have been solved only for a few cases, however, so that molecular dynamics simulation may provide an alternative tool for probing structural perturbation. In this study AMBER-99 force field parameters for S-nitrosocysteine were developed and applied to molecular dynamics simulations of S-nitrosated thioredoxin. Geometry optimization at the level of HF/6-31G * was followed by a restrained electrostatic potential charge-fitting to obtain the atomic charges of S-nitrosocysteine. Force constants for bonds and angles were obtained from generalized AMBER force field. Torsional force constants for CC-SN and CS-NO were determined by fitting the torsional profiles obtained from geometry optimization with those from molecular mechanical energy minimization. Finally molecular dynamics simulations were performed with theses parameters on oxidized and reduced thioredoxin with and without S-nitrosocysteine. In all cases the root-mean-square deviations of alpha-carbons yielded well-behaved trajectories. The CC-SH dihedral angle which fluctuated severely during the simulation became quiet upon S-nitrosation. In conclusion the force field parameters developed in this study for S-nitrosocysteine appear to be suitable for molecular dynamics simulations of S-nitrosated proteins.


Assuntos
Simulação por Computador , Cisteína/análogos & derivados , Modelos Químicos , Nitratos/química , S-Nitrosotióis/química , Tiorredoxinas/química , Cisteína/química , Nitrosação , Conformação Proteica , Processamento de Proteína Pós-Traducional
5.
Oncotarget ; 8(7): 11763-11777, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28052029

RESUMO

The tetrapeptide Arg-Leu-Tyr-Glu (RLYE) is known to inhibit vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis in vitro. Herein, we examined its underlying mechanism and antitumor activity associated with vascular remodeling. RLYE inhibited VEGF-A-induced angiogenesis in a mouse model and suppressed VEGF-A-induced angiogenic signal cascades in human endothelial cells. However, RLYE showed no inhibitory effect on VEGF-A-induced proliferation and migration of multiple myeloma cells expressing VEGF receptor (VEGFR)-1, but not VEGFR-2. In addition, RLYE showed no inhibitory effect on angiogenic activities induced by VEGF-B, basic fibroblast growth factor, epithermal growth factor, sphingosine-1-phosphate, and placental growth factor. RLYE bound specifically to VEGFR-2 at the VEGF-A binding site, thereby blocking VEGF-A-VEGFR-2 binding and VEGF-A-induced VEGFR-2 internalization. The RLYE peptide inhibited tumor growth and metastasis via suppression of tumor angiogenesis in tumor-bearing mice. Moreover, RLYE showed a synergistic effect of the cytotoxic agent irinotecan on tumor cell apoptosis and tumor progression via tumor vessel normalization due to stabilization of VE-cadherin-mediated adherens junction, improvement of pericyte coverage, and inhibition of vascular leakage in tumors. Our results suggest that RLYE can be used as an antiangiogenic and tumor blood vessel remodeling agent for inhibition of tumor growth and metastasis by antagonizing VEGFR-2, with the synergistic anti-cancer effect via enhancement of drug delivery and therapeutic efficacy.


Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias do Colo/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Oligopeptídeos/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Permeabilidade Capilar/efeitos dos fármacos , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/metabolismo , Progressão da Doença , Células HCT116 , Humanos , Masculino , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Ratos Sprague-Dawley , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
6.
Biochimie ; 88(1): 53-8, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16040185

RESUMO

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.


Assuntos
Citocromos c/química , Citocromos c/metabolismo , Ácido Peroxinitroso/química , Animais , Cianetos/metabolismo , Estabilidade Enzimática , Fluoresceínas/metabolismo , Oxirredução , Peroxidases/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Superóxidos/metabolismo , Tirosina/análogos & derivados , Tirosina/síntese química
7.
Mol Cells ; 21(1): 161-5, 2006 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-16511360

RESUMO

Dichlorodihydrofluorescein (DCFH(2)) is a widely used probe for intracellular H(2)O(2). However, H(2)O(2) can oxidize DCFH(2) only in the presence of a catalyst, whose identity in cells has not been clearly defined. We compared the peroxidase activity of Cu,Zn-superoxide dismutase (CuZnSOD), cytochrome c, horseradish peroxidase (HRP), Cu(2+), and Fe(3+) under various condi-tions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of DCFH(2) oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by Cu(2+) and Fe(3+). Oxidation of DCFH(2) by H(2)O(2) in the presence of a cell lys-ate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of H(2)O(2)-treated cells showed enhanced DCF fluorescence in the presence of bicarbonate and attenuated fluorescence for the cells pre-incubated with KCN. Moreover, DCF fluorescence was intensified in CuZnSOD-transfected HaCaT and RAW 264.7 cells. We propose that CuZnSOD is a potential intracellular catalyst for the H(2)O(2)-dependent oxidation of DCFH(2).


Assuntos
Cobre/metabolismo , Fluoresceínas/metabolismo , Peróxido de Hidrogênio/farmacologia , Superóxido Dismutase/metabolismo , Zinco/metabolismo , Animais , Catálise , Bovinos , Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Queratinócitos/citologia , Queratinócitos/enzimologia , Macrófagos/citologia , Macrófagos/enzimologia , Camundongos , Oxirredução/efeitos dos fármacos , Peroxidase/metabolismo
8.
Eur J Pharmacol ; 551(1-3): 143-51, 2006 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-17027748

RESUMO

We here investigated the functional effect of 4-O-methylgallic acid (4-OMGA), a major metabolite of gallic acid abundant in red wine, on vascular inflammation and its action mechanism. 4-OMGA inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-alpha (TNF-alpha), resulting in the suppression of leukocyte adhesion to HUVECs. In addition, 4-OMGA inhibited the promoter activities of ICAM-1 and VCAM-1 and the activity of nuclear factor-kappaB (NF-kappaB) without affecting cytosolic IkappaB kinase (IKK) activation, inhibitor of kappaB (IkappaB) phosphorylation and degradation, and nuclear translocation of NF-kappaB. This compound did not alter nitric oxide (NO) generation, but inhibited reactive oxygen species (ROS) production in TNF-alpha-stimulated HUVECs, suggesting that NO and ROS are not involved in 4-OMGA-mediated inhibition of NF-kappaB activity. Moreover, 4-OMGA directly blocked the binding activity of NF-kappaB to its consensus DNA oligonucleotide, when pre-incubated with the nuclear extract from TNF-alpha-stimulated HUVECs, but not with the oligonucleotide alone. This inhibition was completely abolished by the addition of dithiothreitol. 4-OMGA exhibits an anti-inflammatory property by interfering with the formation of the NF-kappaB-DNA complex in the nuclei through direct and redox-sensitive interactions and may play an important role in the prevention of inflammatory responses such as the atherosclerotic process.


Assuntos
Anti-Inflamatórios/farmacologia , Moléculas de Adesão Celular/metabolismo , DNA/metabolismo , Células Endoteliais/efeitos dos fármacos , Ácido Gálico/análogos & derivados , NF-kappa B/antagonistas & inibidores , Anti-Inflamatórios/uso terapêutico , Aterosclerose/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/metabolismo , Ácido Gálico/farmacologia , Ácido Gálico/uso terapêutico , Genes Reporter , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/efeitos dos fármacos , Luciferases , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
9.
Cancer Lett ; 381(2): 314-22, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27543359

RESUMO

Transforming growth factor-ß1 (TGF-ß1) promotes tumor metastasis by inducing an epithelial-to-mesenchymal transition (EMT) in cancer cells. In this study, we investigated the effects of BIX02189 and XMD8-92, pharmacologic inhibitors of the MEK5 [mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK)5] signaling pathway, on the EMT and migration of cancer cells induced by TGF-ß1. In human A549 lung cancer cells, TGF-ß1-induced EMT, cell motility, and expression of matrix metalloproteinase-2 were completely inhibited by BIX02189, but not by XMD8-92 or small interference RNAs specific to MEK5 and ERK5. Interestingly, BIX02189 strongly blocked the activation of TGF-ß1 signaling components, and this inhibitory effect was not reproduced by MEK5 inhibition. Molecular docking simulation and kinase assays revealed that BIX02189 binds directly to the ATP-binding site of the TGF-ß receptor type I (TßRI) and suppresses its kinase activity. Finally, the anti-metastatic effect of BIX02189 was validated in a TßRI-derived A549 xenograft mouse model. Collectively, these findings newly characterize BIX02189 as a potent inhibitor of TßRI that can block the tumor metastatic activity of TGF-ß1.


Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fator de Crescimento Transformador beta1/farmacologia , Células A549 , Trifosfato de Adenosina/metabolismo , Compostos de Anilina/metabolismo , Animais , Antineoplásicos/metabolismo , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/secundário , Relação Dose-Resposta a Droga , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Indóis/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MAP Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase 5/genética , MAP Quinase Quinase 5/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Invasividade Neoplásica , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Biochim Biophys Acta ; 1623(1): 1-5, 2003 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-12957710

RESUMO

Cytochrome c peroxidase (CcP) uses hydrogen peroxide as an electron acceptor to oxidize cytochrome c (Cc) in the mitochondrial intermembrane space. A null allele of yeast CCP1 gene encoding CcP was created by one-step gene disruption method in a diploid yeast strain. Haploid yeast cells with the disrupted CCP1 gene were viable and able to grow in a medium containing lactic acid or glycerol as an energy source, indicating that CcP is not essential for both cell viability and respiration. However, CCP1-disrupted cells were more sensitive to H2O2 than wild-type cells. We also constructed a CCP1-lacZ fused gene and integrated this gene into yeast chromosomal DNA to monitor the expression of CCP1 gene. We found that expression of CCP1 gene increases under respiratory culture conditions and by treatments with H2O2. These results hint that the biological function of CcP is to reduce H2O2 generated during aerobic respiratory process. Moreover, expression of CCP1 gene increased by treatments with peroxynitrite, indicating that CcP may act as a peroxynitrite scavenger.


Assuntos
Citocromo-c Peroxidase/genética , Citocromo-c Peroxidase/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Respiração Celular/fisiologia , Sobrevivência Celular/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Glicerol/metabolismo , Peróxido de Hidrogênio/farmacologia , Ácido Láctico/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ácido Peroxinitroso/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/efeitos dos fármacos , Especificidade da Espécie
11.
Biochimie ; 85(10): 947-52, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14644549

RESUMO

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.


Assuntos
Antioxidantes , Catalase/antagonistas & inibidores , Glutationa Peroxidase/antagonistas & inibidores , Ozônio/farmacologia , Superóxido Dismutase/antagonistas & inibidores , Animais , Bovinos , Inibidores Enzimáticos/farmacologia , Cavalos , Técnicas In Vitro
12.
Mol Cells ; 17(2): 347-52, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-15179053

RESUMO

We examined the damage to mitochondrial electron transport caused by photosensitization of a pheophorbide a derivative, DH-I-180-3, shown recently to induce necrosis of lung carcinoma cells with low dark toxicity. Confocal microscopy showed that DH-I-180-3 co-localized with dihydrorhodamine-123 suggesting that it mainly accumulates in mitochondria. The photosensitizer alone in the dark did not affect mitochondrial electron transport. Illumination of isolated mitochondria in the presence of DH-I-180-3 resulted in inhibition of both NADH- and succinate-dependent respiration. Measurement of the activity of each component of the electron transport chain revealed that Complex I and III were very susceptible to the treatment whereas Complex IV was resistant. We conclude that the photosensitizer is localized in mitochondria and, upon illumination, produces reactive oxygen species that inactivate Complexes I and III.


Assuntos
Clorofila/análogos & derivados , Clorofila/química , Transporte de Elétrons/fisiologia , Luz , Mitocôndrias/metabolismo , Radiossensibilizantes/química , Animais , Benzoquinonas/metabolismo , Bovinos , Clorofila/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , NAD/metabolismo , Radiossensibilizantes/metabolismo , Ácido Succínico/metabolismo
13.
Chem Phys Lipids ; 181: 83-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24721595

RESUMO

Certain non-ionic surfactants form lamellar vesicles called niosomes. Being elastic and deformable, niosomes have been used as an efficient vehicle for transdermal drug delivery. However, dynamic properties of niosomes have not been studied extensively. In this study we used electron spin resonance (ESR) technique to measure the membrane fluidity of niosomes. In parallel with phospholipid liposomes, the ESR spectra of 5- and 16-doxyl stearate in niosomes of sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80) showed that motion of the spin label was more restricted at the region near the headgroup than near the bilayer center. Cholesterol increased fluidity of Span 60 niosomes whereas it decreased fluidity of Span 80 niosomes. Dicetyl phosphate added at 10 mol% concentration as a stabilizer had a minimal effect on the membrane fluidity throughout the bilayer. We also used ESR technique to monitor the hydration-induced transformation of Span 60 proniosome gel to niosome and showed that the niosome prepared by hydration of proniosome gel was identical to the niosome obtained from a thin film hydration method. Finally the ESR spectra of Span niosomes were compared with those of polysorbate (Tween) niosomes and polyethoxy fatty ether (Brij) niosomes. Tween niosomes had a bulky headgroup and were much less rigid than Span niosomes. This effect of headgroup size on fluidity was also manifest in Brij niosomes where fluidity increased with the number of ethoxy units in the polyethoxy headgroup.


Assuntos
Lipossomos/química , Tensoativos/química , Colesterol/química , Espectroscopia de Ressonância de Spin Eletrônica , Estradiol/química , Fluidez de Membrana , Organofosfatos/química , Polissorbatos/química
14.
Chem Phys Lipids ; 175-176: 79-83, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23994553

RESUMO

When oleic acid and oleate coexist in comparable amounts they form unilamellar vesicles called ufasomes in aqueous phase. Intrinsic pH sensitivity of ufasomes makes it an attractive vehicle for drug delivery. Physical properties of ufasomes have been studied by using spectroscopic techniques but an atomistic model for a ufasome has not been proposed. In this study molecular dynamics simulation was performed on oleic acid/oleate bilayers with the oleate concentration varying from 40 to 70 mol%. All the bilayers reached an equilibrium and stayed stable during a 40 ns simulation. Area per lipid increased with mol% of oleate probably due to charge repulsion between anionic oleate molecules. Oleate was pulled out toward the aqueous phase so that the carboxyl groups of oleic acid and oleate were separated by 0.392 nm in the bilayer of oleic acid/oleate 1:1. Water concentration at the depth of carboxyl group of oleate was five times as high as that of oleic acid. Number of hydrogen bonds between oleic acid and oleate was small in contrast to a proposal that it is an important factor for the bilayer stability. However there was an extensive array of hydrogen bonds between the lipids and water molecules. Acyl chain order was within a normal range for a lipid bilayer but lateral diffusion was an order of magnitude faster in oleic acid/oleate bilayer than in dioleoylphosphatidylcholine bilayer. Cholesterol increased the bilayer thickness and order parameter and decreased the rate of lateral diffusion.


Assuntos
Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Ácido Oleico/química , Colesterol/química , Difusão , Lipossomas Unilamelares/química , Água/química
15.
Biochimie ; 93(2): 168-74, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20888885

RESUMO

In this study, we demonstrate that raloxifene, a selective estrogen receptor modulator, is a potent inducer of the anti-inflammatory enzyme heme oxygenase-1 (HO-1). In RAW264.7 macrophages, raloxifene induced HO-1 mRNA and protein expression. Pretreatment of ICI182780, an estrogen receptor (ER) antagonist or knock-down of endogenous ERα or ERß gene by RNA interference failed to reverse raloxifene-mediated HO-1 induction, indicating an estrogen receptor-independent mechanism. Interestingly, the raloxifene-induced HO-1 expression was suppressed by reactive oxygen species (ROS) scavengers, including glutathione, TEMPO, Me(2)SO, 1,10-phenanthroline, or allopurinol. In addition, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe(2+)/Cu(2+) ions enhanced the positive effect of raloxifene on HO-1 expression. Consistent with these findings, raloxifene induced production of intracellular ROS and increased xanthine oxidase activity in vitro. Additional experiments revealed the involvement of mitogen-activated protein kinase (MAPK) kinase6 and p38 MAPK in the up-regulation of HO-1 by raloxifene and identified p38 MAPK as a downstream effector of ROS. Furthermore, the ROS-p38 MAPK cascade targeted the transcription factor cAMP-responsive element-binding protein (CREB). Finally, the functional significance of HO-1 induction was revealed by raloxifene-mediated inhibition of inducible nitric oxide synthase expression and nitric oxide production, a response reversed by the inhibition of HO-1 protein synthesis or blockade of p38 MAPK or xanthine oxidase activity. Therefore, identification of ROS-p38 MAPK-CREB-linked cascade as cellular relays in raloxifene-mediated HO-1 expression defines the signaling events that could participate in raloxifene-mediated anti-inflammatory response.


Assuntos
Heme Oxigenase-1/metabolismo , Lipopolissacarídeos/farmacologia , Óxido Nítrico/biossíntese , Cloridrato de Raloxifeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Peróxido de Hidrogênio/metabolismo , Ferro/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Xantina/metabolismo , Xantina Oxidase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Eur J Pharmacol ; 612(1-3): 131-42, 2009 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-19356729

RESUMO

The role of celastrol, a triterpene extracted from the Chinese "Thunder of God Vine," in allergic inflammation was investigated. Celastrol decreased the secretion of beta-hexosaminidase, decreased the release of histamine, decreased the expression of Th2 cytokines and decreased calcium influx and cell adhesion in antigen-stimulated RBL2H3 cells. Exposure to celastrol decreased the phosphorylation of extracellular regulated kinase (ERK) and the ERK kinase activity was decreased in RBL2H3 cells. A molecular dynamics simulation showed binding of celastrol to a large pocket in ERK2, which serves as the ATP-binding site. Exposure to celastrol inhibited the interaction between immunoglobulin Fc epsilon receptor I (FcepsilonRIgamma) and ERK and inhibited interaction between FcepsilonRIgamma and protein kinase C delta (PKCdelta). Antigen stimulation induced an interaction between Rac1 and ERK as well as an interaction between Rac1 and PKCdelta. Inhibition of ERK decreased Rac1 activity and inhibition of Rac1 decreased ERK activity in antigen-stimulated RBL2H3 cells. Celastrol regulated the expression of epithelial-mesenchymal transition (EMT)-related proteins through inhibition of PKCalpha, PKCdelta, and Rac1 in antigen-stimulated RBL2H3 cells. Exposure to celatrol inhibited PKCdelta activity in antigen-stimulated RBL2H3 cells. Celastrol exerted a negative effect on FcepsilonRIbeta signaling by inhibiting the interaction between heat shock protein 90 (hsp90) and proteins, such as, FcepsilonRIbeta, Akt and PKCalpha. Celastrol exerted a negative effect on in vivo atopic dermatitis induced by 2, 4-dinitrofluorobenzene (DNFB), which requires ERK. Celastrol also showed an inhibitory effect on skin inflammation induced by phorbol myristate acetate (PMA) in Balb/c mice. In summary, celastrol binds to ERK and inhibits FcepsilonRI signaling to exert an anti-inflammatory effect.


Assuntos
Antialérgicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Receptores de IgE/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Liberação de Histamina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Triterpenos Pentacíclicos , Ratos , Transdução de Sinais/fisiologia , Organismos Livres de Patógenos Específicos , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores
18.
Eur J Pharmacol ; 602(2-3): 422-31, 2009 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-19027002

RESUMO

We investigated the effect of desmethylanhydroicaritin (DMAI), a major compound of the Chinese herbal medicine Epimedium, on inflammatory gene expression and the NF-kappaB signaling pathway. We found that DMAI suppressed the expression of NF-kappaB-responsive genes, such as inducible nitric oxide synthase, cyclooxygenase-2, interleukin-1beta, and tumor necrosis factor-alpha, in lipopolysaccharide (LPS)-stimulated macrophages and endotoxemic mice as well as protected mice against LPS-induced lethality. DMAI inhibited NF-kappaB activation through the inhibition of IkappaB kinase (IKK) activation, IkappaB phosphorylation and degradation, and NF-kappaB nuclear translocation in LPS-stimulated macrophages. This compound inhibited in vitro and in vivo LPS-induced phosphatidylinositol 3-kinase (PI3K) activation, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) oxidation, and Akt phosphorylation, which are upstream modulators of IKK activation. Moreover, treatment with DMAI was not observed to affect the interaction between the Toll-like receptor 4, MyD88, and TRAF6 as well as mitogen-activated protein kinase activation. DMAI also suppressed intracellular H(2)O(2) accumulation, hydroxyl radical production, and glutathione oxidation without affecting superoxide generation and accumulation by NADPH oxidase. Moreover, DMAI inhibited redox-sensitive activation of the PI3K/PTEN/Akt pathway and NF-kappaB activation in macrophages treated with H(2)O(2). These results indicate that DMAI negatively regulates canonical NF-kappaB-regulated inflammatory gene expression by functioning as an inhibitor of the NF-kappaB pathway through the suppression of redox-based PI3K activation and PTEN inactivation and therefore can be considered as a potential drug for inflammatory diseases.


Assuntos
Flavonas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/genética , NF-kappa B/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular , Endotoxemia/induzido quimicamente , Endotoxemia/tratamento farmacológico , Endotoxemia/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Flavonas/uso terapêutico , Peróxido de Hidrogênio/metabolismo , Inflamação/tratamento farmacológico , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , NF-kappa B/metabolismo , Oxirredução/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
19.
Biochem Biophys Res Commun ; 362(2): 532-7, 2007 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-17716625

RESUMO

Thioredoxin-1 (Trx) becomes inactive when cysteine-73 forms a mixed disulfide with glutathione. This reversible S-glutathiolation may serve as a regulatory mechanism. However, molecular basis for the glutathiolation-induced inhibition has not been established due to the lack of its structure. Molecular dynamics (MD) simulations were performed with Gromacs to obtain structural information on glutathiolated Trx. Glutathiolation did not cause a large change in overall shape of Trx although small local changes in the secondary structures were evident. The glutathione moiety was much more flexible than the peptide and spanned a large conformational space. It remained very close to the active site for a large part of the simulation time. Therefore inhibition of Trx by glutathiolation appears to be due to steric hindrance imposed by the covalently attached glutathione.


Assuntos
Simulação por Computador , Cisteína/química , Glutationa/química , Modelos Moleculares , Tiorredoxinas/química , Sítios de Ligação , Cisteína/metabolismo , Glutationa/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tiorredoxinas/metabolismo
20.
Biochem Biophys Res Commun ; 335(2): 300-8, 2005 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-16081047

RESUMO

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has anti-bacterial, anti-inflammatory, and anti-tumor-promoting activities. Here, we describe its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF. Moreover, i.p. administration, without reaching tumor cytotoxic blood levels, to mice receiving i.v. injection of B16F10 melanoma cells, reduced the number of lung metastasis, with preservation of apparently healthy behavior. Taken together, these results demonstrate that [6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumors and other angiogenesis-dependent diseases.


Assuntos
Álcoois Graxos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica , Zingiber officinale/química , Animais , Aorta/metabolismo , Aorta/patologia , Western Blotting , Catecóis , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Colágeno/química , Córnea/metabolismo , Ciclina D1/metabolismo , DNA/química , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fase G1 , Humanos , Técnicas In Vitro , Laminina/química , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Químicos , Mutagênicos , Células NIH 3T3 , Metástase Neoplásica , Transplante de Neoplasias , Extratos Vegetais , Proteoglicanas/química , Ratos , Ratos Sprague-Dawley , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
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