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
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
Cancer development is closely related to chronic inflammation, which is associated with identifiable markers of tumor progression, such as uncontrolled cell proliferation, angiogenesis, genomic instability, chemotherapeutic resistance, and metastases. Redox processes mediated by reactive oxygen species (ROS) and nitric oxide (NO) within the inflammatory tumor microenvironment play an essential role in directly influencing intercellular and intracellular signaling. These reactive species originating in the cancer cell or its microenvironment, mediate the epithelial-mesenchymal transition (EMT) and the mesenchymal-epithelial transition (MET). However, intracellular interactions between NO and ROS must be controlled to prevent cell death. Melanoma, breast, and colon cancer cells have developed a mechanism to survive and adapt to oxidative and nitrosative stress. The mechanism involves a spatial-temporal fine adjustment of the intracellular concentrations of NO and ROS, thereby guaranteeing the successful development of cancer cells. Physiological concentrations of NO and supra physiological concentrations of ROS are prevalent in cancer cells at the primary site. The situation reverses in cancer cells undergoing the EMT prior to being released into the blood stream. Intracellular supra physiological concentrations of NO found in circulating cancer cells endow them with anoikis resistance. When the anoikis-resistant cancer cells arrive at a metastatic site they undergo the MET. Endogenous supra physiological concentrations of ROS and physiological NO concentrations are prevalent in these cells. Understanding tumor progression from the perspective of redox signaling permits the characterization of new markers and approaches to therapy. The synthesis and use of compounds with the capacity of modifying intracellular concentrations of NO and ROS may prove effective in disrupting a redox homeostasis operative in cancer cells.
Assuntos
Neoplasias da Mama/fisiopatologia , Neoplasias do Colo/fisiopatologia , Melanoma/fisiopatologia , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Melanoma/tratamento farmacológico , Doadores de Óxido Nítrico/uso terapêutico , Microambiente Tumoral/fisiologiaRESUMO
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
Cancer is a worldwide health problem leading to a high incidence of morbidity and mortality. Malignant transformation can occur by expression of oncogenes, over-expression and deregulated activation of proto-oncogenes, and inactivation of tumor suppressor genes. These cellular actions occur through stimulation of oncogenic signaling pathways. Nitric oxide (NO) can induce genetic changes in cells and its intracellular generation can lead to tumor formation and progression. It can also promote anti-tumor activities. The pro- and anti-tumor activities of NO are dependent on its intracellular concentration, cell compartmentalization, and cell sensitivity. NO affects a number of oncogenic signaling pathways. This review focuses on two oncogenic signaling pathways: NO-EGFR-Src-FAK and NO-Ras-EGFR-ERK1/2 MAP kinases. In these pathways, low to intermediate concentrations of NO/S-nitrosothiols (RSNOs) stimulate oncogenic signaling, while high concentrations of NO/RSNO stimulate anti-oncogenic signaling. Increasing knowledge on pro- and anti-tumorigenic activities of NO and related reactive species such as RSNOs has fostered the research and synthesis of novel NO-based chemotherapeutic agents. RSNOs, effective as NO donors and trans-nitrosylating agents under appropriate conditions, may operate as potential chemotherapeutic agents.
Assuntos
Neoplasias/metabolismo , Óxido Nítrico/metabolismo , Proteína S/metabolismo , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Animais , Humanos , Neoplasias/terapia , FosforilaçãoRESUMO
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
We investigated the role of protein tyrosine phosphatase-alpha (PTPα) expression in the cell death profile of the A431 human carcinoma cell line that was induced by cytotoxic concentrations of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18). Both NO donors promoted extensive cell detachment in A431 parental cells as compared to the detachment observed for A431 cells that ectopically expressed PTPα (A431 (A27B(PTPα)) cells). The NO-induced cell death characteristics for both cell lines were examined. After incubation for 10 hours with 2.0 mM SNP, attached or detached A431 cells underwent apoptosis. Cells were highly positive for Annexin-V, featured increased cleavage of procaspase-8, activation of downstream caspase-3, and activation of poly-ADP-ribose polymerase 1 (PARP-1). In contrast, exposure of A431 (A27B(PTPα)) cells to 2.0 mM SNP produced an increase in the release of lactate dehydrogenase and enhanced incorporation of propidium iodide. In addition, A431 (A27B(PTPα)) cells showed partial inhibition of the activities of caspase-8, caspase-3, and PARP-1 upon detachment and cell death induced by SNP treatment. Results indicate that necrotic cell damage was induced, characterized by cellular swelling and lysis. We conclude from these results that PTPα regulates the A431 tumor cell death profile mediated by NO donors. Expression of PTPα or its absence may determine the occurrence of NO-induced cell death with necrotic or apoptotic features, respectively.
Assuntos
Apoptose , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Compostos Nitrosos/farmacologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Caspases/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Humanos , L-Lactato Desidrogenase/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fosfatidilserinas/análise , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Propídio/análise , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , TransfecçãoRESUMO
Introdução: A gravidez de adolescentes é tema frequente de debates por suas importantes implicações médicas e sociais e por sua crescente incidência. Este estudo pretende verificar se a gravidez na adolescencia é marcador de risco para prematuridade, baixo peso do recém-nascido, baixa vitalidade, parto cesáreo e não-realização de exames pré-natais. Material e métodos: Realizou-se estudo epidemiológico transversal de natureza analítica, utilizando-se dados do Sistema de Informações de Nascidos Vivos, no Distrito Federal, Brasil, de 1995 e 1996. Foram comparados partos de mães adolescentes e adultas, e partos de adolescentes de até 16 anos com os de adolescentes de 17 a 19 anos. Resultados: Recém-nascidos de mães adolescentes tiveram maior proporção de prematuridade, baixo peso ao nascer e baixa vitalidade do que os de mães adultas. As mães adolescentes realizaram menos pré-natal e tiveram menor percentagem de aoprtos cesáreos. Todas essas diferenças foram estatisticamente significativas. Conclusões: A adolescência foi marcador de risco para prematuridade, baixo peso do recém-nascido e baixa vitalidade, em partos realizados no Distrito Federal, nos anos de 1995 e 1996. Os autores enfatizam a necessidade de abordagem especial desse grupo populacional, tanto na prevenção da gravidez, como nos cuidados do pré-natal e do parto