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1.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073365

RESUMO

Ferroptosis is a new type of programmed cell death, which occurs with iron dependence. Previous studies have showed that ferroptosis plays an important regulatory role in the occurrence and development of tumors. Colon cancer is one of the major morbidities and causes of mortality in the world. This study used RNA-seq and colon cancer clinical data to explore the relationship between ferroptosis-related genes and colon cancer. Based on the fifteen prognostic ferroptosis-related genes, two molecular subgroups of colon cancer were identified. Surprisingly, we also found cluster2 was characterized by lower mutation burden and expression of checkpoint genes, better survival, and higher expression of NOX1. Moreover, cluster2 has fewer BRAF mutations. We also found the expression of NOX1 is related to the status of BRAF. Finally, using 15 ferroptosis-related genes from The Cancer Genome Atlas cohort, we constructed a prognosis model, and this model may be used to predict the prognosis of patients in clinics.


Assuntos
Neoplasias do Colo/metabolismo , Bases de Dados de Ácidos Nucleicos , Ferroptose , Regulação Neoplásica da Expressão Gênica , Modelos Biológicos , NADPH Oxidase 1/biossíntese , Proteínas Proto-Oncogênicas B-raf/biossíntese , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Humanos , NADPH Oxidase 1/genética , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , RNA-Seq
2.
J Neurosci ; 41(12): 2780-2794, 2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33563722

RESUMO

Repetitive behavior is a widely observed neuropsychiatric symptom. Abnormal dopaminergic signaling in the striatum is one of the factors associated with behavioral repetition; however, the molecular mechanisms underlying the induction of repetitive behavior remain unclear. Here, we demonstrated that the NOX1 isoform of the superoxide-producing enzyme NADPH oxidase regulated repetitive behavior in mice by facilitating excitatory synaptic inputs in the central striatum (CS). In male C57Bl/6J mice, repeated stimulation of D2 receptors induced abnormal behavioral repetition and perseverative behavior. Nox1 deficiency or acute pharmacological inhibition of NOX1 significantly shortened repeated D2 receptor stimulation-induced repetitive behavior without affecting motor responses to a single D2 receptor stimulation. Among brain regions, Nox1 showed enriched expression in the striatum, and repeated dopamine D2 receptor stimulation further increased Nox1 expression levels in the CS, but not in the dorsal striatum. Electrophysiological analyses revealed that repeated D2 receptor stimulation facilitated excitatory inputs in the CS indirect pathway medium spiny neurons (iMSNs), and this effect was suppressed by the genetic deletion or pharmacological inhibition of NOX1. Nox1 deficiency potentiated protein tyrosine phosphatase activity and attenuated the accumulation of activated Src kinase, which is required for the synaptic potentiation in CS iMSNs. Inhibition of NOX1 or ß-arrestin in the CS was sufficient to ameliorate repetitive behavior. Striatal-specific Nox1 knockdown also ameliorated repetitive and perseverative behavior. Collectively, these results indicate that NOX1 acts as an enhancer of synaptic facilitation in CS iMSNs and plays a key role in the molecular link between abnormal dopamine signaling and behavioral repetition and perseveration.SIGNIFICANCE STATEMENT Behavioral repetition is a form of compulsivity, which is one of the core symptoms of psychiatric disorders, such as obsessive-compulsive disorder. Perseveration is also a hallmark of such disorders. Both clinical and animal studies suggest important roles of abnormal dopaminergic signaling and striatal hyperactivity in compulsivity; however, the precise molecular link between them remains unclear. Here, we demonstrated the contribution of NOX1 to behavioral repetition induced by repeated stimulation of D2 receptors. Repeated stimulation of D2 receptors upregulated Nox1 mRNA in a striatal subregion-specific manner. The upregulated NOX1 promoted striatal synaptic facilitation in iMSNs by enhancing phosphorylation signaling. These results provide a novel mechanism for D2 receptor-mediated excitatory synaptic facilitation and indicate the therapeutic potential of NOX1 inhibition in compulsivity.


Assuntos
Comportamento Compulsivo/metabolismo , Locomoção/fisiologia , NADPH Oxidase 1/biossíntese , NADPH Oxidases/biossíntese , Receptores de Dopamina D2/biossíntese , Sinapses/metabolismo , Animais , Células Cultivadas , Comportamento Compulsivo/induzido quimicamente , Comportamento Compulsivo/psicologia , Agonistas de Dopamina/farmacologia , Agonistas de Dopamina/toxicidade , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 1/antagonistas & inibidores , NADPH Oxidases/antagonistas & inibidores , Pirazolonas/farmacologia , Piridonas/farmacologia , Receptores de Dopamina D2/agonistas , Sinapses/efeitos dos fármacos
3.
Cell Death Dis ; 11(6): 435, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513988

RESUMO

Many studies have shown that long-noncoding RNA (lncRNA) is associated with cardiovascular disease, but its molecular mechanism is still unclear. In this study, we explored the role of lncRNA ANRIL in ox-LDL-induced phenotypic transition of human aortic smooth muscle cells (HASMC). The results of quantitative fluorescence PCR showed that the expression of ANRIL in patients with coronary atherosclerotic heart disease (CAD) was significantly higher than that in normal subjects. RNA-FISH detection showed that the ANRIL expression increased in HASMC treated by ox-LDL. Ox-LDL could upregulate the expression of ANRIL and ROS and promote the phenotypic transition of HASMC. After downregulation of ANRIL by siRNA, ROS level decreased and HASMC phenotypic transition alleviated. ANRIL could act as a molecular scaffold to promote the binding of WDR5 and HDAC3 to form WDR5 and HDAC3 complexes, they regulated target genes such as NOX1 expression by histone modification, upregulated ROS level and promote HASMC phenotype transition. Therefore, we found a new epigenetic regulatory mechanism for phenotype transition of VSMC, ANRIL was a treatment target of occlusive vascular diseases.


Assuntos
Doença da Artéria Coronariana/metabolismo , Histona Desacetilases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Músculo Liso Vascular/metabolismo , RNA Longo não Codificante/metabolismo , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Histona Desacetilases/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/patologia , NADPH Oxidase 1/biossíntese , NADPH Oxidase 1/genética , Fenótipo , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Transfecção
4.
PLoS One ; 15(5): e0233208, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428030

RESUMO

To facilitate functional investigation of the role of NADPH oxidase 1 (NOX1) and associated reactive oxygen species in cancer cell signaling, we report herein the development and characterization of a novel mouse monoclonal antibody that specifically recognizes the C-terminal region of the NOX1 protein. The antibody was validated in stable NOX1 overexpression and knockout systems, and demonstrates wide applicability for Western blot analysis, confocal microscopy, flow cytometry, and immunohistochemistry. We employed our NOX1 antibody to characterize NOX1 expression in a panel of 30 human colorectal cancer cell lines, and correlated protein expression with NOX1 mRNA expression and superoxide production in a subset of these cells. Although a significant correlation between oncogenic RAS status and NOX1 mRNA levels could not be demonstrated in colon cancer cell lines, RAS mutational status did correlate with NOX1 expression in human colon cancer surgical specimens. Immunohistochemical analysis of a comprehensive set of tissue microarrays comprising over 1,200 formalin-fixed, paraffin-embedded tissue cores from human epithelial tumors and inflammatory disease confirmed that NOX1 is overexpressed in human colon and small intestinal adenocarcinomas, as well as adenomatous polyps, compared to adjacent, uninvolved intestinal mucosae. In contradistinction to prior studies, we did not find evidence of NOX1 overexpression at the protein level in tumors versus histologically normal tissues in prostate, lung, ovarian, or breast carcinomas. This study constitutes the most comprehensive histopathological characterization of NOX1 to date in cellular models of colon cancer and in normal and malignant human tissues using a thoroughly evaluated monoclonal antibody. It also further establishes NOX1 as a clinically relevant therapeutic target in colorectal and small intestinal cancer.


Assuntos
Adenocarcinoma/enzimologia , Neoplasias do Colo/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Intestino Delgado/enzimologia , NADPH Oxidase 1/biossíntese , Proteínas de Neoplasias/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/patologia , Células CACO-2 , Neoplasias do Colo/genética , Células HT29 , Humanos , Intestino Delgado/patologia , Modelos Biológicos , NADPH Oxidase 1/genética , Proteínas de Neoplasias/genética
5.
Oncol Rep ; 42(4): 1475-1486, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31364740

RESUMO

Gallbladder cancer (GBC) is a lethal aggressive malignant neoplasm of the biliary tract. Potential prognostic markers and therapeutic targets for this disease are urgently required. Cancer­associated fibroblasts (CAFs) play a key role in tumorigenesis and the development of cancer. Nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) expression has been reported to be involved in tumorigenesis and useful for tumor prognosis. However, NOX1 expression in the stroma of GBCs, particularly gallbladder cancer­associated fibroblasts (GCAFs), and its prognostic significance in GBC patients remains unclear. In the present study, NOX1 expression in the stroma of human gallbladder lesions in vivo was investigated, as well as in GCAFs and co­cultures of GBC­SD+GCAFs in vitro, and their correlation with clinicopathological parameters and the prognosis of GBC patients were evaluated. The results revealed that NOX1 expression was significantly upregulated in the stroma of GBCs compared with precancerous and benign lesions of the gallbladder; NOX1 expression was localized to gallbladder stromal fibroblasts expressing α­smooth muscle actin and fibroblast secreted protein­1. Furthermore, these observations were confirmed by the fact that NOX1 expression was upregulated in GCAFs as determined by Affymetrix gene profile chip analysis and reverse transcription­quantitative PCR. In addition, overexpression was observed in formed spheroids of GBC­SD+GCAF co­cultures by immunohistochemistry and western blotting in vitro. Thus, it was verified that NOX1 expression was upregulated in GCAFs. Furthermore, upregulated stromal NOX1 expression was correlated with aggressive characteristics such as differentiation degree (P=0.042), venous invasion (P=0.041), resection methods (P=0.002), and a lower survival rate (P=0.025, log­rank test) of patients with GBC. Stromal NOX1 expression (P=0.047) was an independent prognostic factor for the overall survival rate of patients with GBC. GBC patients with upregulated NOX1 expression in GCAFs had a poorer prognosis. These results revealed that stromal NOX1 may be a novel biomarker and/or target, and may contribute to the discovery of new tumor markers and potential targeted therapeutics for human GBCs.


Assuntos
Fibroblastos Associados a Câncer/enzimologia , Neoplasias da Vesícula Biliar/enzimologia , NADPH Oxidase 1/biossíntese , Actinas/biossíntese , Idoso , Proteínas de Ligação ao Cálcio/biossíntese , Fibroblastos Associados a Câncer/patologia , Feminino , Neoplasias da Vesícula Biliar/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteína A4 de Ligação a Cálcio da Família S100 , Regulação para Cima
6.
J Neurotrauma ; 35(17): 2077-2090, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29648986

RESUMO

Blast-induced traumatic brain injury (bTBI) is a leading cause of morbidity in soldiers on the battlefield and in training sites with long-term neurological and psychological pathologies. Previous studies from our laboratory demonstrated activation of oxidative stress pathways after blast injury, but their distribution among different brain regions and their impact on the pathogenesis of bTBI have not been explored. The present study examined the protein expression of two isoforms: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and 2 (NOX1, NOX2), corresponding superoxide production, a downstream event of NOX activation, and the extent of lipid peroxidation adducts of 4-hydroxynonenal (4HNE) to a range of proteins. Brain injury was evaluated 4 h after the shock-wave exposure, and immunofluorescence signal quantification was performed in different brain regions. Expression of NOX isoforms displayed a differential increase in various brain regions: in hippocampus and thalamus, there was the highest increase of NOX1, whereas in the frontal cortex, there was the highest increase of NOX2 expression. Cell-specific analysis of changes in NOX expression with respect to corresponding controls revealed that blast resulted in a higher increase of NOX1 and NOX 2 levels in neurons compared with astrocytes and microglia. Blast exposure also resulted in increased superoxide levels in different brain regions, and such changes were reflected in 4HNE protein adduct formation. Collectively, this study demonstrates that primary blast TBI induces upregulation of NADPH oxidase isoforms in different regions of the brain parenchyma and that neurons appear to be at higher risk for oxidative damage compared with other neural cells.


Assuntos
Traumatismos por Explosões/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , NADPH Oxidases/biossíntese , Animais , Astrócitos/metabolismo , Química Encefálica , Cerebelo/metabolismo , Hipocampo/metabolismo , Isoenzimas , Peroxidação de Lipídeos , Masculino , NADPH Oxidase 1/biossíntese , NADPH Oxidase 1/genética , NADPH Oxidase 2/biossíntese , NADPH Oxidase 2/genética , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Tálamo/metabolismo
7.
Clin Exp Ophthalmol ; 46(6): 652-660, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29360265

RESUMO

BACKGROUND: Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. METHODS: Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 µM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. RESULTS: Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. CONCLUSIONS: Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.


Assuntos
Retinopatia Diabética/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , NADPH Oxidase 1/genética , Estresse Oxidativo , Vasos Retinianos/patologia , Proliferação de Células , Células Cultivadas , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/patologia , Humanos , NADPH Oxidase 1/biossíntese , RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Vasos Retinianos/metabolismo
8.
Brain Behav Immun ; 59: 190-199, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27614125

RESUMO

Traumatic brain injury (TBI), even at mild levels, can activate matrix metalloproteinases (MMPs) and the induction of neuroinflammation that can result in blood brain barrier breakdown and neurodegeneration. MMP2 has a significant role in neuroinflammation and neurodegeneration by modulating the chemokine CXCL12α (stromal cell derived factor SDF-1α) signaling pathway and the induction of apoptosis. SDF-1α is responsible for cell proliferation and differentiation throughout the nervous system and is also implicated in various neurodegenerative illnesses. We hypothesized that TBI leads to MMP2 activation and cleavage of the N-terminal 4 amino acid residues of CXCL12α with generation of the highly neurotoxic fragment SDF-1(5-67). Using an in vitro stretch-injury model of rat neuronal cultures and the in vivo fluid percussion injury (FPI) model in rats, we found that oxidative stress has a significant role in the activation of MMP2. This is initiated by the induction of free radical generating enzyme NADPH oxidase 1 (NOX1). Induction of NOX1 correlated well with the signatures of oxidative stress marker, 4HNE in the injured neuronal cultures and cerebral cortex of rats. Further, using MMP2 siRNA and pharmacological MMP2 inhibitor, ARP100, we established the neurodegenerative role of MMP2 in cleaving SDF-1α to a neurotoxic fragment SDF-1(5-67). By immunofluorescence, western blotting and TUNEL experiments, we show the cleaved form of SDF leads to apoptotic cell death in neurons. This work identifies a new potential therapeutic target to reduce the complications of brain damage in TBI.


Assuntos
Lesões Encefálicas Traumáticas/enzimologia , Quimiocina CXCL12/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Degeneração Neural/enzimologia , Degeneração Neural/genética , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas Traumáticas/genética , Caspase 3/biossíntese , Caspase 3/genética , Sobrevivência Celular/genética , Células Cultivadas , Quimiocina CXCL12/genética , Ativação Enzimática , Técnicas de Silenciamento de Genes , Metaloproteinase 2 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/farmacologia , NADPH Oxidase 1/biossíntese , NADPH Oxidase 1/genética , Neurônios/efeitos dos fármacos , Estresse Oxidativo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley
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