RESUMO
Spatial confinement and temporal regulation of signaling by nitric oxide (NO) and reactive oxygen species (ROS) occurs in cancer cells. Signaling mediated by NO and ROS was investigated in two sub clones of the murine melanoma B16F10-Nex2 cell line, Nex10C and Nex8H treated or not with bradykinin (BK). The sub clone Nex10C, similar to primary site cells, has a low capacity for colonizing the lungs, whereas the sub clone Nex8H, similar to metastatic cells, corresponds to a highly invasive melanoma. BK-treated Nex10C cells exhibited a transient increase in NO and an inhibition in basal O2- levels. Inhibition of endogenous NO production by l-NAME resulted in detectable levels of O2-. l-NAME promoted Rac1 activation and enhanced Rac1-PI3K association. l-NAME in the absence of BK resulted in Nex10C cell migration and invasion, suggesting that NO is a negative regulator of O2- mediated cell migration and cell invasion. BK-treated Nex8H cells sustained endogenous NO production through the activation of NOS3. NO activated Rac1 and promoted Rac1-PI3K association. NO stimulated cell migration and cell invasion through a signaling axis involving Ras, Rac1 and PI3K. In conclusion, a role for O2- and NO as positive regulators of Rac1-PI3K signaling associated with cell migration and cell invasion is proposed respectively for Nex10C and Nex8H murine melanoma cells.
Assuntos
Bradicinina , Melanoma , Camundongos , Animais , Bradicinina/farmacologia , Bradicinina/metabolismo , Superóxidos , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Movimento CelularRESUMO
Significance: The epithelial/mesenchymal transition (EMT) is commonly associated with tumor metastasis. Oxidative and nitrosative stress is maintained in cancer cells and is involved in the EMT. Cancer cells are endowed with high levels of enzymatic and nonenzymatic antioxidants, which counteract the effects of oxidative and nitrosative stress. Thiol-based antioxidant systems such as the thioredoxin/thioredoxin reductase (Trx/TrxR) and glutathione/glutaredoxin (GSH/Grx) are continually active in cancer cells, while the thioredoxin-interacting protein (Txnip), the negative regulator of the Trx/TrxR system, is downregulated. Recent Advances: Trx/TrxR and GSH/Grx systems play a major role in maintaining EMT signaling and cancer cell progression. Critical Issues: Enhanced stress conditions stimulated in cancer cells inhibit EMT signaling. The elevated expression levels of the Trx/TrxR and GSH/Grx systems in these cells provide the antioxidant protection necessary to guarantee the occurrence of the EMT. Future Directions: Elevation of the intracellular reactive oxygen species and nitric oxide concentrations in cancer cells has been viewed as a promising strategy for elimination of these cells. The development of inhibitors of GSH synthesis and of the Trx/TrxR system together with genetic-based strategies to enhance Txnip levels may provide the necessary means to achieve this goal. Antioxid. Redox Signal. 36, 1037-1050.
Assuntos
Antioxidantes , Neoplasias , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Glutationa/metabolismo , Humanos , Neoplasias/genética , Oxirredução , Compostos de Sulfidrila , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismoRESUMO
Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.
Assuntos
Adenocarcinoma/enzimologia , Autofagia , Neoplasias do Colo/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Estresse Nitrosativo , Adenocarcinoma/genética , Adenocarcinoma/secundário , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Óxido Nítrico Sintase Tipo II/genética , Isoformas de Proteínas , Transdução de SinaisRESUMO
Nitric Oxide (NO) and Hydrogen Sulfide (H2S) are components of an "interactome", which is defined as a redox system involving the interactions of RSS, RNS and ROS. Chemical interaction by these species is common and is characterized by one and two electron oxidation, nitrosylation, nitration and sulfuration/polysulfidation reactions. NO and H2S are gases that penetrate cell membranes, are synthesized by specific enzymes, are ubiquitous, regulate protein activities through post-translational modifications and participate in cell signaling. The two molecules at high concentrations compared to physiological concentrations may result in cellular damage particularly through their interaction with other reactive species. NO and H2S can interact with each other and form a variety of molecular species which may have constructive or destructive behavior depending on the cell type, the cellular environment (ex. oxygen tension, pH, redox state), where the products are produced and in what concentrations. Cross talk exists between NO and H2S, whereby they can influence the generation and signaling behavior of each other. Given the above mentioned properties of NO and H2S and studies in cancer cells and animal models employing NO and H2S donors that generate higher than physiological concentrations of NO and H2S and are effective in killing cancer cells but not normal cells, lend credence to the possibility of the utility of these donors in an approach to the treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Sulfeto de Hidrogênio/farmacologia , Neoplasias/tratamento farmacológico , S-Nitrosotióis/farmacologia , Animais , Humanos , Neoplasias/metabolismo , Óxido Nítrico/metabolismo , Oxirredução/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.
Assuntos
Anoikis/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Compostos Nitrosos/farmacologia , Quinases da Família src/genética , Animais , Anoikis/genética , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Ativação Enzimática/efeitos dos fármacos , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Óxido Nítrico/química , Doadores de Óxido Nítrico/química , Nitroprussiato/química , Compostos Nitrosos/química , Transdução de Sinais , Células Tumorais Cultivadas , Quinases da Família src/metabolismoRESUMO
Accumulating mutations may drive cells into the acquisition of abnormal phenotypes that are characteristic of cancer cells. Cancer cells feature profound alterations in proliferation programs that result in a new population of cells that overrides normal tissue construction and maintenance programs. To achieve this goal, cancer cells are endowed with up regulated survival signaling pathways. They also must counteract the cytotoxic effects of high levels of nitric oxide (NO) and of reactive oxygen species (ROS), which are by products of cancer cell growth. Accumulating experimental evidence associates cancer cell survival with their capacity to up-regulate antioxidant systems. Elevated expression of the antioxidant protein thioredoxin-1 (Trx1) has been correlated with cancer development. Trx1 has been characterized as a multifunctional protein, playing different roles in different cell compartments. Trx1 migrates to the nucleus in cells exposed to nitrosative/oxidative stress conditions. Trx1 nuclear migration has been related to the activation of transcription factors associated with cell survival and cell proliferation. There is a direct association between the p21Ras-ERK1/2 MAP Kinases survival signaling pathway and Trx1 nuclear migration under nitrosative stress. The expression of the cytoplasmic protein, the thioredoxin-interacting protein (Txnip), determines the change in Trx1 cellular compartmentalization. The anti-apoptotic actions of Trx1 and its denitrosylase activity occur in the cytoplasm and serve as important regulators of cell survival. Within this context, this review focuses on the participation of Trx1 in cells under nitrosative/oxidative stress in survival signaling pathways associated with cancer development.
Assuntos
Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxinas/farmacologia , Humanos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/fisiologiaRESUMO
The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.
Assuntos
Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Óxido Nítrico/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Bradicinina/farmacologia , Células Endoteliais/metabolismo , Humanos , Óxido Nítrico/biossínteseRESUMO
Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.
Assuntos
Indutores da Angiogênese/farmacologia , Bradicinina/farmacologia , Receptores ErbB/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico/biossíntese , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Coelhos , S-Nitrosotióis/metabolismo , Tirosina/metabolismoRESUMO
Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras-ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Estresse Oxidativo/fisiologia , Tiorredoxinas/metabolismo , Análise de Variância , Western Blotting , Catalase/metabolismo , Primers do DNA/genética , Técnica Indireta de Fluorescência para Anticorpo , Vetores Genéticos/genética , Glutationa Peroxidase/metabolismo , Células HeLa , Humanos , Microscopia Confocal , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Imagem com Lapso de TempoRESUMO
AIMS: S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. RESULTS: Ras signaling pathway stimulation by 50 and/or 100 µM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 µM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras. INNOVATION: The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. CONCLUSION: S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane.
Assuntos
Proliferação de Células , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , S-Nitrosoglutationa/farmacologia , Animais , Bradicinina/farmacologia , Células COS , Sinalização do Cálcio , Membrana Celular/enzimologia , Chlorocebus aethiops , Cisteína/análogos & derivados , Cisteína/metabolismo , Ativação Enzimática , Complexo de Golgi/enzimologia , Células HeLa , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Óxido Nítrico/fisiologia , Oxirredução , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Processamento de Proteína Pós-Traducional , S-Nitrosotióis/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
The role of NO in regulating the focal adhesion proteins, Src, FAK, p130 Cas, and PTP-alpha, was investigated. Fibroblasts expressing PTP-alpha (PTP-alpha(WT) cells), fibroblasts "knockout" for PTP-alpha (PTP-alpha(-/-) cells), and "rescued" "knockout" fibroblasts (PTP-alpha A5/3 cells) were stimulated with either S-nitroso-N-acetylpenicillamine (SNAP) or fetal bovine serum (FBS). FBS increased inducible NO synthase in both cell lines. Activation of Src mediated either by SNAP or by FBS occurred independent of dephosphorylation of Tyr527 in PTP-alpha(-/-) cells. Both stimuli promoted dephosphorylation of Tyr527 and activation of Src kinase in PTP-alpha(WT) cells. NO-mediated activation of Src kinase affected the activities of FAK and p130Cas and was dependent on the expression of PTP-alpha. Analogous to tyrosine phosphorylation, SNAP and FBS stimulated differential generation of NO and S-nitrosylation of Src kinase in both cell lines. Incubation with SNAP resulted in higher levels of NO and S-nitrosylation of immunoprecipitated Src in PTP-alpha(-/-) cells (oxidizing redox environment) as compared with the levels of NO and S-nitrosylated Src in PTP-alpha(WT) cells (reducing redox environment). SNAP differentially stimulated cell proliferation of both cell lines is dependent on the intracellular redox environment, Src activity, and PTP-alpha expression. This dependence also is observed with FBS-stimulated cell migration.
Assuntos
Proteína Substrato Associada a Crk/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Óxido Nítrico/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Quinases da Família src/metabolismo , Animais , Bovinos , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteína Substrato Associada a Crk/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Camundongos , Camundongos Knockout , Óxido Nítrico/genética , Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Oxirredução , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , S-Nitroso-N-Acetilpenicilamina/metabolismo , Quinases da Família src/genéticaRESUMO
Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.
Assuntos
S-Nitrosoglutationa/farmacologia , S-Nitrosotióis/metabolismo , Tiorredoxinas/metabolismo , Regulação para Cima/efeitos dos fármacos , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitritos/metabolismo , Fosforilação/efeitos dos fármacos , S-Nitrosoglutationa/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.
Assuntos
Apoptose , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Flavonoides/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , S-Nitrosoglutationa/farmacologiaRESUMO
S-Nitrosylation reactions are considered to be a major mechanism by which NO-related bioactivities are regulated in vivo. In the present study, we show the effects of the low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), on cell cycle progression of rabbit aortic endothelial cells (RAEC). SNAP at low concentrations (0.1mM) stimulated the p21Ras-ERK1/2 MAP kinase signaling pathway. Activation of this signaling pathway was strongly inhibited in cells stably transfected with S-nitrosylation insensitive p21Ras (p21(Ras (C118S))). Furthermore, the SNAP-induced effects on cell cycle progression were eliminated in RAEC expressing N17Ras, a negative dominant mutant of p21Ras. Upon stimulation with SNAP, ERK1/2 MAP kinases become phosphorylated and translocate to the nucleus promoting the phosphorylation of the transcription factor Elk1. Synthesis of Cyclin D1 and stimulation of the cyclin-dependent kinases cdk4 and cdk6 resulted in the phosphorylation of the nuclear protein Rb and its dissociation from the E2F family of transcription factors. Cells then pass the restriction point in the late G1 phase. Cyclins E and A were expressed as the cell cycle progressed through the S phase upon stimulation with SNAP. Further transition in the cell cycle from the G2 to M phase was evidenced by the G2/M peak found in a histogram of the cell-phase distribution in SNAP-treated RAEC. These observations suggest that low molecular weight S-nitrosothiols may promote cell cycle progression possibly through the transnitrosation of p21Ras, and activation of the Ras-ERK1/2 MAP kinases signaling pathway.
Assuntos
Aorta/citologia , Ciclo Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , S-Nitroso-N-Acetilpenicilamina/farmacologia , S-Nitrosotióis/farmacologia , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peso Molecular , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Coelhos , Proteína do Retinoblastoma/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Oxidative modifications of proteins are fundamental biochemical events that regulate cellular signaling, protein expression, and function. The redox status is balanced by reductants in which GSH plays a major role. This study investigated whether or not p21Waf1 expression and TNFalpha biosynthesis in macrophage differentiation/activation were regulated by GSH modulators and whether or not the JNK and ERK pathway were involved. We observed an increase of p21Waf1 expression and TNFalpha biosynthesis in the THP1 monocyte/macrophage cell line treated with PMA. Treatment of THP1 cultures with NAC prior to adding PMA abrogates the expression of p21Waf1 mRNA and decreases the level of TNFalpha whereas GSH depletion by BSO enhances the levels of TNFalpha with minor effects on p21Waf1 expression. To assess whether or not ERK and JNK were involved in the redox mechanism of p21Waf1 and TNFalpha, we used pharmacological inhibitors for JNK and ERK. Both PD98095 and dicoumarol were capable of blocking TNFalpha production but had only a small effect on p21Waf1 expression. We next observed that activation of JNK was significantly inhibited in cells pretreated with NAC with no effect on ERK. Taken together, our findings suggest that the modulation of GSH regulate the magnitude the cell response to PMA in which JNK and ERK have a particular role in redox signaling.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glutationa/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Biomarcadores , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
The free radical nitric oxide is a very effective signal transducer, stimulating the enzyme guanylyl cyclase, the oncoprotein p21Ras, and protein tyrosine phosphorylation. In the present study using rabbit aortic endothelial cells (RAEC), it is demonstrated that the nitric-oxide-generating substances sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and a stable analog of cyclic GMP, 8BrcGMP stimulate p21Ras activity. Tyrosine phosphorylation of cytosolic proteins was stimulated and intracellular production of cGMP was increased, indicating that the NO/cGMP-stimulated tyrosine phosphorylation-dependent signaling pathway is most likely associated with the activation of p21Ras. NO and cGMP-dependent activation of p21Ras result in binding of the oncoprotein to the Ras-binding domain of Raf-1 kinase. Incubation of RAEC with FPT II, a potent and selective inhibitor of p21Ras, prevented NO-dependent tyrosine phosphorylation. ODQ, a potent inhibitor of the soluble form of guanylyl cyclase, inhibited the signal as well. Conversely, the use of KT5823, a cGMP-dependent protein kinase (PKG) blocker, showed no effect on protein tyrosine phosphorylation. To further establish a role for p21Ras on the NO-stimulated tyrosine phosphorylation-signaling pathway, RAEC were constitutively transfected with a dominant negative mutant of p21Ras, N17Ras. NO and cGMP-stimulated tyrosine phosphorylation were prevented in N17Ras-expressing RAEC exposed to NO donors and 8BrcGMP. The above findings indicate that NO and cGMP stimulation of protein tyrosine phosphorylation requires the participation of fully functional p21Ras. ERK1/2 MAP kinases and their subsequent targets, the transcription factors, lie downstream to Ras, Raf-1 kinase, and MEK. Treatment of both RAEC and mock-transfected RAEC with NO resulted in phosphorylation and activation of ERK1/2. On the other hand, NO did not stimulate phosphorylation of ERK1/2 in N17Ras-expressing RAEC. In addition, PD98059, a MEK inhibitor, prevented overall tyrosine phosphorylation and phosphorylation of ERK1/2. Upstream to Ras ERK1/2 MAP kinases target the EGF receptor. Incubation of RAEC or mock-transfected RAEC with NO donors resulted in activation of the EGF receptor autophosphorylation. PD98059 effectively blocked this activation. EGF receptor autophosphorylation was insensitive to NO stimulation in N17Ras-expressing RAEC. It is concluded that NO and cGMP stimulate a signaling pathway involving p21Ras-Raf-1 kinase-MEK-ERK1/2. Activation of this signaling pathway is connected to NO-stimulated overall tyrosine phosphorylation that also involves the transactivation of the EGF receptor mediated by ERK1/2.
Assuntos
Aorta/metabolismo , GMP Cíclico/metabolismo , Endotélio Vascular/citologia , Sistema de Sinalização das MAP Quinases , Óxido Nítrico/metabolismo , Tirosina/metabolismo , Animais , Western Blotting , Carbazóis/farmacologia , Citosol/metabolismo , Endotélio Vascular/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Radicais Livres , Genes Dominantes , Glutationa Transferase/metabolismo , Indóis/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Nitroprussiato/metabolismo , Nitroprussiato/farmacologia , Oxidiazóis/farmacologia , Fosforilação , Testes de Precipitina , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinoxalinas/farmacologia , Coelhos , S-Nitroso-N-Acetilpenicilamina/farmacologia , Transdução de Sinais , TransfecçãoRESUMO
Reversibie phosphorylation of protein tyrosine residues by protein tyrosine kinases (PTK) mediated by polypeptide growth factor receptors (e.g. epidermal growth factor receptor, platelet derived growth factor receptor, insulin receptor) is a signalling process implicated in a variety of processes such as cellular proliferation, differentiation and transformation. On the other hand, many reports converged to the idea that cellular phosphotyrosine levels are regulated by a family of enzymes referred to as protein tyrosine phosphatases (PTP). An essential cysteine residue located in the conserved catalytic domain is responsible for PTP activity. Furthermore, inhibition of PTP activities by oxidative stress inducing agents has been recently documented. It is well known that free radicais and other oxidants at high concentrations are toxic to cells. However, at low concentrations these species may act as modulators/mediators of signal transduction processes in the presence and absence of polypeptide growth factors. Thus, in this article we reviewed our contribution in the general context of the redox modulation of PTK and PTP mediated signal transduction pathways.