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1.
Cancer Sci ; 111(4): 1156-1164, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31967368

RESUMO

p62 is associated with 2 major cellular defense mechanisms against metabolic and oxidative stress, autophagy and the Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor-E2-related factor 2 (NRF2) system. Recent studies indicate that the p62-KEAP1-NRF2 pathway promotes tumorigenesis and tumor growth mediated by NRF2-dependent antioxidative response. However, whether p62 is involved in bladder cancer (BCa) development remains unknown. Here, we found that p62 is overexpressed in BCa tissue and several BCa cell lines. The knockdown of p62 inhibits BCa cell growth both in vitro and in vivo, with increased intracellular reactive oxygen species level. Mechanically, p62 activates NRF2 signaling by sequestrating KEAP1, which leads to the upregulation of antioxidant genes (Gclc, Gstm5, and Gpx2), thus protecting BCa cells from oxidative stress. Our findings indicate that p62 might be involved in the development of BCa and serve as a potential therapeutic target.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Proteínas de Ligação a RNA/genética , Neoplasias da Bexiga Urinária/genética , Animais , Antioxidantes/metabolismo , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutationa Peroxidase/genética , Glutationa Transferase/genética , Xenoenxertos , Humanos , Camundongos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Ativação Transcricional/genética , Neoplasias da Bexiga Urinária/patologia
2.
Chemosphere ; 240: 124914, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31557642

RESUMO

Arsenic (As) contamination is one of the most daunting environmental problem bothering the whole world. Exploring a suitable bioremediation technique is an urgent need of the hour. The present study focusses on scrutinizing the ectomycorrhizal (ECM) fungus for its potential role in As detoxification and understanding the molecular mechanisms responsible for its tolerance. When exposed to increasing concentrations of external As, the ECM fungus H. cylindrosporum accumulated the metalloid intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for GSH biosynthesis enzymes, γ-glutamylcysteine synthetase (Hcγ-GCS) and glutathione synthetase (HcGS) were highly regulated by As stress. Arsenic coordinately upregulated the expression of both Hcγ-GCS and HcGS genes, thus resulting in increased Hcγ-GCS and HcGS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (Hcγ-GCS and HcGS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating the As induced responses and its detoxification in ECM fungus H. cylindrosporum.


Assuntos
Arsênico/toxicidade , Glutationa/biossíntese , Hebeloma/efeitos dos fármacos , Micorrizas/efeitos dos fármacos , Poluentes do Solo/toxicidade , Antioxidantes/metabolismo , Arsênico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Teste de Complementação Genética , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Hebeloma/genética , Hebeloma/metabolismo , Inativação Metabólica , Mutação , Micorrizas/genética , Micorrizas/metabolismo , Saccharomyces cerevisiae/metabolismo , Poluentes do Solo/metabolismo
3.
Biosci Biotechnol Biochem ; 83(12): 2202-2212, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31409200

RESUMO

In this study, we isolated eight phenylethanoid glycosides from Paraboea martinii for the first time, and evaluated the mechanism underlying their neuroprotective effects against H2O2-induced injury in PC12 cells. The MTS method was utilized to screen the phenylethanoid glycosides for protective ability. Next, qRT-PCR and western blotting analysis were used to detect the transcription levels of HO-1 and GCLC, which are regulated by Nrf2. The inhibitor ZnPP was used to analyze the involvement of Nrf2 in HO-1 expression. Analyses showed that caleolarioside B, paraboside B, and paraboside II also upregulated the expression of HO-1, but showed no obvious effect on GCLC. Pretreatment with ZnPP significantly reduced the neuroprotective effects. Thus, phenylethanoid glycosides isolated from P. martinii protected PC12 cells from H2O2-induced damage by upregulating HO-1. The results provided evidence that P. martinii might be a potential therapeutic agent for the treatment of neurodegenerative diseases.


Assuntos
Glicosídeos/farmacologia , Peróxido de Hidrogênio/toxicidade , Fármacos Neuroprotetores/farmacologia , Feocromocitoma/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células PC12 , Ratos , Transcrição Genética/efeitos dos fármacos
4.
BMC Complement Altern Med ; 19(1): 139, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221142

RESUMO

BACKGROUND: Several studies have found that caffeic acid (CA), a well-known phytochemical, displays important antioxidant and anti-cancer activities. However, no evidence exists on the protective effect and its mechanisms that CA treatment alone has against oxidative stress induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells. METHODS: Hepatoprotective activities such as cell viability, mRNA expression, and report gene assay were measured using HepG2 cell. Three types of genes and proteins related with detoxification in liver were used for measuring the hepatoprotective effects. Statistical analysis was performed using one-way ANOVA test and differences among groups were evaluated by Tukey's studentized range tests. RESULTS: The present study indicate that treatment with CA up-regulates heme oxygenase-1 (HO-1) and glutamate-cysteine ligase (GCL) mRNA and protein expressions in a CA-dose-dependent manner. In addition, translocation of nuclear factor-E2 p45-related factor (Nrf2) from the cytoplasm to the nucleus and phosphorylation of extracellular signal-regulated kinase, ERK and c-Jun N-terminal kinase, JNK which have been shown to be involved in mitogen-activated protein kinases, MAPKs are significantly enhanced by CA treatment. Furthermore, in cell nuclei, CA enhances the 5'-flanking regulatory region of human antioxidant response element (ARE) and activates the ARE binding site. CONCLUSION: Therefore, CA proved to be a stimulant of the expression of detoxification enzymes such as HO-1, GCLC, and GCLM through the ERK/Nrf2 pathway, and it may be an effective chemoprotective agent for protecting liver damage against oxidative damage.


Assuntos
Ácidos Cafeicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , terc-Butil Hidroperóxido/toxicidade , Elementos de Resposta Antioxidante/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/genética , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Hep G2 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/genética , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Redox Biol ; 26: 101231, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31203195

RESUMO

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.


Assuntos
Antioxidantes/metabolismo , Glutamato-Cisteína Ligase/genética , Glutationa/análogos & derivados , Proteínas de Protozoários/genética , Espermidina/análogos & derivados , Tiorredoxinas/genética , Trypanosoma cruzi/efeitos dos fármacos , Amida Sintases/genética , Amida Sintases/metabolismo , Butionina Sulfoximina/farmacologia , Linhagem Celular , Combinação de Medicamentos , Resistência a Medicamentos/genética , Fibroblastos/parasitologia , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/metabolismo , Glutationa/antagonistas & inibidores , Glutationa/biossíntese , Humanos , Peróxido de Hidrogênio/farmacologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Nitroimidazóis/farmacologia , Oxirredução , Estresse Oxidativo , Peroxidases/genética , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Espermidina/antagonistas & inibidores , Espermidina/biossíntese , Tiorredoxinas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética
6.
Biofactors ; 45(4): 563-574, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31131946

RESUMO

Isoflavones are one group of the major flavonoids and possess multiple biological activities due to their antioxidant properties. However, a clear antioxidant mechanism of dietary isoflavones is still remained to be answered. In this study, the effects of isoflavones on the nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway and the underlying molecular mechanisms were investigated. Results showed that isoflavones are potential Nrf2-ARE activators while their activities were structure dependent. Biochanin A (BCA), an O-methylated isoflavone with low direct antioxidant activity, can effectively protect HepG2 cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage via activation of the Nrf2 signaling, and thereby the induction of downstream cytoprotective enzymes including NAD(P)H quinone oxidoreductase-1, heme oxygenasae-1, and glutamate-cysteine ligase catalytic subunit. A molecular docking study revealed that BCA could directly bind into the pocket of Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1), a cytoplasmic suppressor of Nrf2, to facilitate Nrf2 activation. The upstream mitogen-activated protein kinase (MAPK) pathways were also involved in the activation of Nrf2 signaling. These findings indicate that the protective actions of dietary isoflavones against oxidative damage may be at least partly due to their ability to enhance the intracellular antioxidant response system by modulating the Nrf2-ARE signaling pathway.


Assuntos
Elementos de Resposta Antioxidante/efeitos dos fármacos , Antioxidantes/farmacologia , Genisteína/farmacologia , Proteínas Quinases Ativadas por Mitógeno/genética , Fator 2 Relacionado a NF-E2/genética , Espécies Reativas de Oxigênio/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Hep G2 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Oxidantes/antagonistas & inibidores , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , terc-Butil Hidroperóxido/antagonistas & inibidores , terc-Butil Hidroperóxido/farmacologia
7.
Gene ; 703: 120-124, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-30959073

RESUMO

AIMS: Given the participation of oxidative stress in the pathogenesis of diabetic complications, we evaluated, in type 1 diabetes (T1D) individuals, the association between diabetic retinopathy (DR) and functional single nucleotide polymorphisms (SNPs) in regulatory regions of two genes belonging to the antioxidant glutathione (GSH) system: rs17883901 in GCLC and rs713041 in GPX4. METHODS: A cross-sectional case-control study included 288 individuals (61% women, 34[±11] years old, diabetes duration of 22[±9] years, mean [±SD]) sorted according to DR stages: absence of DR (ADR), non-proliferative DR (NPDR) and proliferative DR (PDR). SNPs were genotyped by real-time PCR using fluorescent labelled probes. Logistic regression models with adjustment for confounding covariates were employed. RESULTS: The presence of at least one T-allele of rs17883901 in GCLC was an independent risk factor for PDR (OR 4.13, 95% CI 1.38-13.66, p = 0.014) in a polytomous regression model (PDR versus ADR). The presence of at least one T-allele of rs713041 in GPX4 conferred protection against PDR (OR 0.30, 95% CI 0.11-0.80, p = 0.017) in female T1D individuals. CONCLUSION: The functional SNPs rs17883901 and rs713041 modulate the risk for PDR in the studied population of T1D individuals, widening the spectrum of candidate genes for this complication.


Assuntos
Diabetes Mellitus Tipo 1/complicações , Retinopatia Diabética/genética , Glutamato-Cisteína Ligase/genética , Glutationa Peroxidase/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idade de Início , Estudos de Casos e Controles , Estudos Transversais , Diabetes Mellitus Tipo 1/genética , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
8.
Molecules ; 24(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018491

RESUMO

This study aimed to investigate the antioxidant activity and release behavior of anthocyanin (ANC) loaded within FA-g-MD wall (ANC-FA-g-MD microcapsule) in vitro. The microencapsulation of ANC was prepared by spray drying and displayed a biphasic release profile. The combination of ANC and FA-g-MD (0.0625-1 mg/mL) showed a higher antioxidant activity than that of both individuals. A possible intermolecular interaction between ANC and FA-g-MD was studied by UV-vis spectra. Intracellular reactive oxygen species (ROS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, and protein expression of quinone oxidoreductase 1(NQO1), glutathione reductase (GSR) and γ-glutamate cysteine ligase catalytic subunit (γ-GCLC) were measured through human colon cancer cells (HT-29). After a 24-hour incubation of the HT-29, the combinations (0-60 µg/mL) exhibited a high potential to diminish the ROS level. And the distinct upregulated expressions of GCLC and NQO1 of HT-29 were detected after treatment with combinations compared to those of single ones. These results suggested that the ANC-FA-g-MD microcapsules exerts enhanced antioxidant effect with capability of the modulation of GCLC and NQO1.


Assuntos
Antocianinas/farmacologia , Cápsulas/síntese química , Ácidos Cumáricos/química , Portadores de Fármacos , Depuradores de Radicais Livres/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Polissacarídeos/química , Antocianinas/química , Sobrevivência Celular/efeitos dos fármacos , Composição de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Depuradores de Radicais Livres/química , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Células HT29 , Humanos , Peróxido de Hidrogênio/farmacologia , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Subunidades Proteicas/agonistas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
9.
Biochem J ; 476(7): 1191-1203, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30877193

RESUMO

Plant γ-glutamylcysteine ligase (GCL), catalyzing the first and tightly regulated step of glutathione (GSH) biosynthesis, is redox-activated via formation of an intramolecular disulfide bond. In vitro, redox-activation of recombinant GCL protein causes formation of homo-dimers. Here, we have investigated whether dimerization occurs in vivo and if so whether it contributes to redox-activation. FPLC analysis indicated that recombinant redox-activated WT (wild type) AtGCL dissociates into monomers at concentrations below 10-6 M, i.e. below the endogenous AtGCL concentration in plastids, which was estimated to be in the micromolar range. Thus, dimerization of redox-activated GCL is expected to occur in vivo To determine the possible impact of dimerization on redox-activation, AtGCL mutants were generated in which salt bridges or hydrophobic interactions at the dimer interface were interrupted. WT AtGCL and mutant proteins were analyzed by non-reducing SDS-PAGE to address their redox state and probed by FPLC for dimerization status. Furthermore, their substrate kinetics (K M, V max) were compared. The results indicate that dimer formation is not required for redox-mediated enzyme activation. Also, crystal structure analysis confirmed that dimer formation does not affect binding of GSH as competitive inhibitor. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated.


Assuntos
Proteínas de Arabidopsis/metabolismo , Glutamato-Cisteína Ligase/química , Glutamato-Cisteína Ligase/metabolismo , Glutationa/biossíntese , Sequência de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Domínio Catalítico , Ativação Enzimática , Glutamato-Cisteína Ligase/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Plantas Geneticamente Modificadas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Int J Mol Sci ; 20(3)2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717178

RESUMO

Cadmium (Cd) is harmful for humans and animals, especially for the reproductive system. However, the mechanism of its toxicity has not been elucidated, and how to alleviate its toxicity is very important. This study aimed to explore the role and mechanism of action of sulforaphane (SFN) in protecting mouse Leydigs (TM3) cells from cadmium (Cd)-induced damage. The half-maximal inhibitory concentration (IC50) of Cd and the safe doses of SFN were determined using a methyl thiazolyl tetrazolium (MTT) assay. The testosterone secretion from TM3 cells was measured using the enzyme-linked immunosorbent assay. The intracellular oxidative stress was evaluated using corresponding kits. The cell apoptosis was detected using flow cytometry. The mRNA expression of genes associated with NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling was detected using reverse transcription⁻polymerase chain reaction, including Nrf2, heme oxygenase I (HO-1), glutathione peroxidase (GSH-Px), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS). The protein expression of Nrf2, GSH-Px, HO-1, γ-GCS, and NQO1 was detected using Western blot analysis. The results showed that the IC50 of Cd to TM3 cells was 51.4 µmol/L. SFN reduced the release of lactate dehydrogenase from Cd-exposed cells. Cd + SFN 2.5 treatment significantly elevated testosterone concentration compared with the Cd group (p < 0.05). SFN significantly increased total superoxide dismutase (T-SOD) and GSH-Px activity and GSH content in Cd-treated cells (p < 0.05; p < 0.01), inhibited the production of malondialdehyde or reactive oxygen species caused by Cd (p < 0.05; p < 0.01), and reduced the apoptotic rate of Cd-induced TM3 cells (p < 0.01). SFN upregulated the mRNA expression of Nrf2, GSH-Px, HO-1, NQO1, and γ-GCS in Cd-treated cells, indicating the protective effect of SFN against Cd-induced oxidative stress or cell apoptosis by activating the Nrf2/ARE signaling pathway.


Assuntos
Antioxidantes/farmacologia , Cloreto de Cádmio/antagonistas & inibidores , Isotiocianatos/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Elementos de Resposta Antioxidante/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Testosterona/biossíntese
11.
Oxid Med Cell Longev ; 2019: 7945983, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30805084

RESUMO

Background: Aurothioglucose- (ATG-) mediated inhibition of thioredoxin reductase-1 (TXNRD1) improves alveolarization in experimental murine bronchopulmonary dysplasia (BPD). Glutathione (GSH) mediates susceptibility to neonatal and adult oxidative lung injury. We have previously shown that ATG attenuates hyperoxic lung injury and enhances glutathione- (GSH-) dependent antioxidant defenses in adult mice. Hypothesis: The present studies evaluated the effects of TXNRD1 inhibition on GSH-dependent antioxidant defenses in newborn mice in vivo and lung epithelia in vitro. Methods: Newborn mice received intraperitoneal ATG or saline prior to room air or 85% hyperoxia exposure. Glutamate-cysteine ligase (GCL) catalytic (Gclc) and modifier (Gclm) mRNA levels, total GSH levels, total GSH peroxidase (GPx) activity, and Gpx2 expression were determined in lung homogenates. In vitro, murine transformed club cells (mtCCs) were treated with the TXNRD1 inhibitor auranofin (AFN) or vehicle in the presence or absence of the GCL inhibitor buthionine sulfoximine (BSO). Results: In vivo, ATG enhanced hyperoxia-induced increases in Gclc mRNA levels, total GSH contents, and GPx activity. In vitro, AFN increased Gclm mRNA levels, intracellular and extracellular GSH levels, and GPx activity. BSO prevented AFN-induced increases in GSH levels. Conclusions: Our data are consistent with a model in which TXNRD1 inhibition augments hyperoxia-induced GSH-dependent antioxidant responses in neonatal mice. Discrepancies between in vivo and in vitro results highlight the need for methodologies that permit accurate assessments of the GSH system at the single-cell level.


Assuntos
Antioxidantes/metabolismo , Displasia Broncopulmonar/enzimologia , Displasia Broncopulmonar/patologia , Glutationa/metabolismo , Tiorredoxina Redutase 1/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Aurotioglucose , Displasia Broncopulmonar/genética , Células Epiteliais/metabolismo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa Peroxidase/metabolismo , Hiperóxia/genética , Hiperóxia/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tiorredoxina Redutase 1/metabolismo
12.
Chem Biol Interact ; 303: 1-6, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30794799

RESUMO

Glutathione (GSH), the most abundant cellular non-protein thiol, plays a pivotal role in hepatic defense mechanisms against oxidative damage. Despite a strong association between disrupted GSH homeostasis and liver diseases of various etiologies, it was shown that GSH-deficient glutamate-cysteine ligase modifier subunit (Gclm)-null mice are protected against fatty liver development induced by a variety of dietary and environmental insults. The biochemical mechanisms underpinning this protective phenotype have not been clearly defined. The purpose of the current study was to characterize the intrinsic metabolic signature in the livers from GSH deficient Gclm-null mice. Global profiling of hepatic polar metabolites revealed a spectrum of changes in amino acids and metabolites derived from fatty acids, glucose and nucleic acids due to the loss of GCLM. Overall, the observed low GSH-driven metabolic changes represent metabolic adaptations, including elevations in glutamate, aspartate, acetyl-CoA and gluconate, which are beneficial for the maintenance of cellular redox and metabolic homeostasis.


Assuntos
Glutationa/deficiência , Fígado/metabolismo , Animais , Modelos Animais de Doenças , Fígado Gorduroso , Glutamato-Cisteína Ligase/deficiência , Glutamato-Cisteína Ligase/genética , Homeostase , Metabolômica , Camundongos , Camundongos Knockout , Oxirredução
13.
Plant Mol Biol ; 99(1-2): 149-159, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30617455

RESUMO

KEY MESSAGE: The WRKY transcription factor WRKY12 negatively regulates Cd tolerance in Arabidopsis via the glutathione-dependent phytochelatin synthesis pathway by directly targeting GSH1 and indirectly repressing phytochelatin synthesis-related gene expression. Cadmium (Cd) is a widespread pollutant toxic to plants. The glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway plays key roles in Cd detoxification. However, its regulatory mechanism remains largely unknown. Here, we showed a previously unknown function of the WRKY transcription factor WRKY12 in the regulation of Cd tolerance by repressing the expression of PC synthesis-related genes. The expression of WRKY12 was inhibited by Cd stress. Enhanced Cd tolerance was observed in the WRKY12 loss-of-function mutants, whereas increased Cd sensitivity was found in the WRKY12-overexpressing plants. Overexpression and loss-of-function of WRKY12 were associated respectively with increased and decreased Cd accumulation by repressing or releasing the expression of the genes involved in the PC synthesis pathway. Transient expression assay showed that WRKY12 repressed the expression of GSH1, GSH2, PCS1, and PCS2. Further analysis indicated that WRKY12 could directly bind to the W-box of the promoter in GSH1 but not in GSH2, PCS1, and PCS2 in vivo. Together, our results suggest that WRKY12 directly targets GSH1 and indirectly represses PC synthesis-related gene expression to negatively regulate Cd accumulation and tolerance in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cádmio/metabolismo , Regulação da Expressão Gênica de Plantas , Glutamato-Cisteína Ligase/metabolismo , Fitoquelatinas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutationa/metabolismo , Inativação Metabólica , Mutação com Perda de Função , Regiões Promotoras Genéticas/genética , Estresse Fisiológico , Fatores de Transcrição/genética
14.
Food Chem Toxicol ; 125: 305-312, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30654098

RESUMO

Coffee is a highly consumed beverage with many putative beneficial health effects, however these often come from observational studies. In the current work, a lightly roasted coffee extract that has previously been reported to exhibit potent antioxidant properties was administered for two weeks in rats to examine the potential improvement of blood and tissue redox status. The dose was equivalent to a moderate human daily consumption. According to our results, coffee exerted beneficial effects in all tissues mainly by increasing reduced glutathione (GSH) levels. Interestingly, the brain was the most significantly affected tissue, while the gastrointestinal tract, the main metabolic organs and the quadriceps were also benefited. In addition, protein and lipid oxidation was reduced in several tissues. The observed increase in GSH was attributed to increased levels of the rate-limiting enzyme in its biosynthesis pathway, namely γ-glutamylcysteine ligase both in the protein and gene levels. Overall, moderate coffee consumption showed beneficial short term effects in rat tissues by stimulating parts of the endogenous antioxidant mechanisms.


Assuntos
Antioxidantes/farmacologia , Café/química , Glutationa/metabolismo , Extratos Vegetais/farmacologia , Animais , Coffea/química , Glutamato-Cisteína Ligase/genética , Masculino , Oxirredução , RNA Mensageiro/genética , Ratos Wistar , Superóxido Dismutase/genética , Regulação para Cima/efeitos dos fármacos
15.
Cancer Res ; 79(1): 125-132, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30401714

RESUMO

Glutathione (GSH)/GSH reductase (GSR) and thioredoxin/thioredoxin reductase (TXNRD) are two major compensating thiol-dependent antioxidant pathways that maintain protein dithiol/disulfide balance. We hypothesized that functional deficiency in one of these systems would render cells dependent on compensation by the other system for survival, providing a mechanism-based synthetic lethality approach for treatment of cancers. The human GSR gene is located on chromosome 8p12, a region frequently lost in human cancers. GSR deletion was detected in about 6% of lung adenocarcinomas in The Cancer Genome Atlas database. To test whether loss of GSR sensitizes cancer cells to TXNRD inhibition, we knocked out or knocked down the GSR gene in human lung cancer cells and evaluated their response to the TXNRD inhibitor auranofin. GSR deficiency sensitized lung cancer cells to this agent. Analysis of a panel of 129 non-small cell lung cancer (NSCLC) cell lines revealed that auranofin sensitivity correlated with the expression levels of the GSR, glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H quinone dehydrogenase 1 (NQO1) genes. In NSCLC patient-derived xenografts with reduced expression of GSR and/or GCLC, growth was significantly suppressed by treatment with auranofin. Together, these results provide a proof of concept that cancers with compromised expression of enzymes required for GSH homeostasis or with chromosome 8p deletions that include the GSR gene may be targeted by a synthetic lethality strategy with inhibitors of TXNRD. SIGNIFICANCE: These findings demonstrate that lung cancers with compromised expression of enzymes required for glutathione homeostasis, including reduced GSR gene expression, may be targeted by thioredoxin/thioredoxin reductase inhibitors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Glutamato-Cisteína Ligase/metabolismo , Glutationa Redutase/metabolismo , Glutationa/metabolismo , Mutações Sintéticas Letais , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Animais , Antirreumáticos/farmacologia , Auranofina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutationa Redutase/genética , Homeostase , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Environ Microbiol Rep ; 11(2): 53-61, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30411517

RESUMO

Ectomycorrhizal fungi play an important role in protecting their host plant from metal(loid) stresses by synthesizing various thiol rich compounds like metallothioneins and glutathione. We investigated the effect of cadmium (Cd) and arsenic (As) stress with a specific interest on glutathione (GSH) in the ectomycorrhizal fungus Laccaria bicolor. The total GSH levels inside the cell were significantly increased with increase in external metal(loid) stress. An analysis of the transcript levels of genes responsible for GSH synthesis, γ-glutamylcysteine synthetase (Lbγ-GCS) and glutathione synthetase (LbGS), using qPCR revealed that expression of both genes increased as a function of external metal(loid) concentration. The enzyme activity of both Lbγ-GCS and LbGS were increased with increase in external Cd and As concentration. Further, the functional role of Lbγ-GCS and LbGS genes in response to Cd and As stress was studied using their respective yeast mutant strains gsh1 Δ and gsh2 Δ . The mutant strains successfully expressed the two genes resulting in wild-type phenotype restoration of Cd and As tolerance. From these results, it was concluded that GSH act as a core component in the mycorrhizal defence system under Cd and As stress for metal(loid) homeostasis and detoxification.


Assuntos
Arsênico/metabolismo , Cádmio/metabolismo , Glutationa/metabolismo , Homeostase/fisiologia , Laccaria/metabolismo , Micorrizas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/biossíntese , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Inativação Metabólica , Laccaria/enzimologia , Laccaria/crescimento & desenvolvimento , Micorrizas/enzimologia , Micorrizas/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Estresse Fisiológico
17.
Free Radic Biol Med ; 130: 256-266, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391672

RESUMO

Huntington's disease (HD) is a heritable neurodegenerative disorder, and has been characterized as an increase of oxidative stress in brain regions. In our previous results, we showed fibroblast growth factor 9 (FGF9) provides neuroprotective functions to suppress cell death in HD striatal cells dominantly through ERK signalling. However, whether the working mechanism of FGF9 is related to anti-oxidative stress in HD is still unknown. In this study, STHdhQ7/Q7 (Q7) and STHdhQ111/Q111 (Q111) striatal knock-in cell lines were used to examine the neuroprotective effects of FGF9 against oxidative stress in HD. Results show that FGF9 alleviates oxidative stress induced by starvation in Q7 and Q111 cells. The treatment of FGF9 not only induces upregulation and activation of nuclear factor erythroid 2-like 2 (Nrf2), a critical transcription factor for anti-oxidative stress, but also further upregulates its downstream targets, such as superoxide dismutase 2, gamma-glutamylcysteine synthetase and glutathione reductase. Furthermore, blockage of the Nrf2 pathway abolishes the anti-oxidative functions of FGF9, and inhibition of ERK signalling reduces the activation of the FGF9-Nrf2 pathway, resulting in higher level of oxidative stress in HD cells. These results support the neuroprotective effects of FGF9 against oxidative stress through the ERK-Nrf2 pathway, and imply one of potential strategies for therapy of HD.


Assuntos
Antioxidantes/farmacologia , Encéfalo/metabolismo , Fator 9 de Crescimento de Fibroblastos/genética , Doença de Huntington/tratamento farmacológico , Fator 2 Relacionado a NF-E2/genética , Animais , Encéfalo/patologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Glutationa Redutase/genética , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/genética , Córtex Visual/metabolismo , Córtex Visual/patologia
18.
J Med Chem ; 62(2): 811-830, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30540174

RESUMO

We previously developed a novel series of vinyl sulfones as nuclear factor erythroid 2-related factor 2 (Nrf2) activators with therapeutic potential for Parkinson's disease (PD). However, the previously developed lead compound (1) exhibited undesirable druglike properties. Here, we optimized vinyl sulfones by introducing nitrogen heterocycles to improve druglike properties. Among the synthesized compounds, 17e was the most promising drug candidate with good druglike properties. Compound 17e showed superior effects on Nrf2 activation in cell-based assays compared to compound 1 (17e: half-maximal effective concentration (EC50) = 346 nM; 1: EC50 = 530 nM). Compound 17e was further confirmed to induce expression of Nrf2-dependent antioxidant enzymes at both mRNA and protein levels. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of PD, 17e significantly attenuated loss of tyrosine hydroxylase-immunopositive dopaminergic neurons, suppressed microglial activation, and alleviated PD-associated motor dysfunction. Thus, 17e is a novel Nrf2 activator with excellent druglike properties and represents a potential therapeutic candidate for PD.


Assuntos
Fator 2 Relacionado a NF-E2/agonistas , Fármacos Neuroprotetores/química , Sulfonas/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Sulfonas/metabolismo , Sulfonas/farmacologia , Sulfonas/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Compostos de Vinila/química
19.
Free Radic Biol Med ; 131: 376-381, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578920

RESUMO

Chronic kidney disease (CKD) is a worldwide public health problem with an estimated prevalence of 8.2%. This study reports glutathione deficiency, excess oxidative stress, and altered vitamin D metabolism in the kidney of mice fed a high-fat diet (HFD). The levels of GCLC and GCLM gene expression were significantly downregulated and the protein carbonylation level, a hallmark of oxidative damage, was significantly increased in the kidney of HFD-fed mice. While the levels of VD-regulatory genes 1-alpha-hydroxylase (CYP27B1), VDR, and RXRα were significantly downregulated in the kidney of mice fed a HFD, those of 24-hydroxylase (CYP24A1) were significantly elevated. In vitro, GSH deficiency per se causes excess oxidative damage (protein carbonylation), and significantly decreases the levels of VD-regulatory genes (CYP27B1, VDR, and RXRα), but increases levels of CYP24A1 in human renal proximal tubule epithelial cells (RPTEC), similar to findings in the kidney of HFD-fed diabetic mice. L-cysteine supplementation restores GSH and prevents oxidative damage in RPTEC. These studies suggest a potential role of GSH precursor in reducing excess oxidative stress and renal injury that commonly accompanies obesity/diabetes.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Diabetes Mellitus Experimental/enzimologia , Glutationa/deficiência , Receptores de Calcitriol/genética , Insuficiência Renal Crônica/enzimologia , Vitamina D3 24-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Cisteína/farmacologia , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica/efeitos adversos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Cultura Primária de Células , Carbonilação Proteica , Receptores de Calcitriol/metabolismo , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Transdução de Sinais , Vitamina D3 24-Hidroxilase/metabolismo
20.
Appl Microbiol Biotechnol ; 103(3): 1243-1254, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30448906

RESUMO

Glutathione has diverse physiological functions, and therefore, the demand for it has increased recently. Currently, industrial mass production of glutathione is performed from D-glucose via fermentation by the budding yeast Saccharomyces cerevisiae. However, use of D-glucose often competes with demands for various other industries, leading to high production costs. To affordably produce glutathione, we aimed to produce high amounts of glutathione from D-glucose and D-xylose, which are the main constituents of lignocellulosic biomass pre-treated with acids. Genetically engineered S. cerevisiae strains that can produce high amounts of glutathione and assimilate D-xylose were constructed and cultured in media containing D-xylose. Among these recombinant strains, a S. cerevisiae GCI (XR/XDH/XK) strain over-expressing γ-glutamylcysteine synthetase, glutathione synthetase, D-xylose reductase, xylitol dehydrogenase, and xylulokinase genes successfully consumed D-xylose in the medium and produced the highest amount of glutathione. When strains were grown in media containing D-glucose and D-xylose, the GCI (XR/XDH/XK) strain showed 4.6-fold higher volumetric glutathione production (mg/L-broth), 2.2-fold higher glutathione content (%), and 2.1-fold higher cell growth (g-cell/L-broth) than the vector control strain of YPH499 (Vector). Furthermore, when recombinant S. cerevisiae strains were grown in medium containing fermentation inhibitory materials, the GCI (XR/XDH/XK) strain produced 5.8- and higher volumetric glutathione, 2.6-fold higher intracellular glutathione, and 2.9-fold higher cell growth than the vector control YPH499 (Vector) strain. The gradual sugar consumption by recombinant S. cerevisiae strains in medium containing D-glucose and D-xylose leads to high yields of glutathione. These results indicate the potential for glutathione production from lignocellulosic materials.


Assuntos
Reatores Biológicos/microbiologia , Engenharia Genética/métodos , Glutationa/biossíntese , Lignina/metabolismo , Saccharomyces cerevisiae/metabolismo , D-Xilulose Redutase/genética , Glucose/metabolismo , Glutamato-Cisteína Ligase/genética , Glutationa Sintase/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Xilose/metabolismo
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