Macrophages, IL-10, and nitric oxide increase, induced by hyperglycemic conditions, impact the development of murine melanoma B16F10-Nex2.

Epidemiological studies show a strong correlation between diabetes and the increased risk of developing different cancers, including melanoma. In the present study, we investigated the impact of a streptozotocin (STZ)-induced hyperglycemic environment on B16F10-Nex2 murine melanoma development. Hyperglycemic male C57Bl/6 mice showed increased subcutaneous tumor development, partially inhibited by metformin. Tumors showed increased infiltrating macrophages, and augmented IL-10 and nitric oxide (NO) concentrations. In vivo neutralization of IL-10, NO synthase inhibition, and depletion of macrophages reduced tumor development. STZ-treated TLR4 KO animals showed delayed tumor development; the transfer of hyperglycemic C57Bl/6 macrophages to TLR4 KO reversed this effect. Increased concentrations of IL-10 present in tumor homogenates of hyperglycemic mice induced a higher number of pre-angiogenic structures in vitro, and B16F10-Nex2 cells incubated with different glucose concentrations in vitro produced increased levels of IL-10. In summary, our findings show that a hyperglycemic environment stimulates murine melanoma B16F10-Nex2 primary tumor growth, and this effect is dependent on tumor cell stimulation, increased numbers of macrophages, and augmented IL-10 and NO concentrations. These findings show the involvement of tumor cells and other components of the tumor microenvironment in the development of subcutaneous melanoma under hyperglycemic conditions, defining novel targets for melanoma control in diabetic patients.

Bradykinin promotes murine melanoma cell migration and invasion through endogenous production of superoxide and nitric oxide.

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.

Knockdown of the inducible nitric oxide synthase (NOS2) splicing variant S3 promotes autophagic cell death from nitrosative stress in SW480 human colon cancer cells.

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.

Nitric oxide stimulates a PKC-Src-Akt signaling axis which increases human immunodeficiency virus type 1 replication in human T lymphocytes.

Human immunodeficiency virus (HIV) infections are typically accompanied by high levels of secreted inflammatory cytokines and generation of high levels of reactive oxygen species (ROS). To elucidate how HIV-1 alters the cellular redox environment during viral replication, we used human HIV-1 infected CD4+T lymphocytes and uninfected cells as controls. ROS and nitric oxide (NO) generation, antioxidant enzyme activity, protein phosphorylation, and viral and proviral loads were measured at different times (2-36 h post-infection) in the presence and absence of the NO donor S-nitroso-N-acetylpenicillamine (SNAP). HIV-1 infection increased ROS generation and decreased intracellular NO content. Upon infection, we observed increases in copper/zinc superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities, and a marked decrease in glutathione (GSH) concentration. Exposure of HIV-1 infected CD4+T lymphocytes to SNAP resulted in an increasingly oxidizing intracellular environment, associated with tyrosine nitration and SOD1 inhibition. In addition, SNAP treatment promoted phosphorylation and activation of the host's signaling proteins, PKC, Src kinase and Akt. Inhibition of PKC leads to inhibition of Src kinase strongly suggesting that PKC is the upstream element in this signaling cascade. Changes in the intracellular redox environment after SNAP treatment had an effect on HIV-1 replication as reflected by increases in proviral and viral loads. In the absence or presence of SNAP, we observed a decrease in viral load in infected CD4+T lymphocytes pre-incubated with the PKC inhibitor GF109203X. In conclusion, oxidative/nitrosative stress conditions derived from exposure of HIV-1-infected CD4+T lymphocytes to an exogenous NO source trigger a signaling cascade involving PKC, Src kinase and Akt. Activation of this signaling cascade appears to be critical to the establishment of HIV-1 infection.

Nitric oxide and interactions with reactive oxygen species in the development of melanoma, breast, and colon cancer: A redox signaling perspective.

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.

Heparan sulfate proteoglycan deficiency up-regulates the intracellular production of nitric oxide in Chinese hamster ovary cell lines.

We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O2- ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.

Basal neutrophil function in human aging: Implications in endothelial cell adhesion.

Much attention has been drawn to the pro-inflammatory condition that accompanies aging. This study compared parameters from non-stimulated neutrophils, obtained from young (18-30 years old [y.o.]) and elderly (65-80 y.o.) human volunteers. Measured as an inflammatory marker, plasmatic concentration of hs-CRP was found higher in elderly individuals. Non-stimulated neutrophil production of ROS and NO was, respectively, 38 and 29% higher for the aged group. From the adhesion molecules evaluated, only CD11b expression was elevated in neutrophils from the aged group, whereas no differences were found for CD11a, CD18, or CD62. A 69% higher non-stimulated in vitro neutrophil/endothelial cell adhesion was observed for neutrophils isolated from elderly donors. Our results suggest that with aging, neutrophils may be constitutively producing more reactive species in closer proximity to endothelial cells of vessel walls, which may both contribute to vascular damage and reflect a neutrophil intracellular disrupted redox balance, altering neutrophil function in aging.

Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

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.

Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis.

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.

Heparin-integrin interaction in endothelial cells: downstream signaling and heparan sulfate expression.

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.

Effects of ethanol on CYP2E1 levels and related oxidative stress using a standard balanced diet.

Expression of cytochrome P4502E1 (CYP2E1) is very much influenced by nutritional factors, especially carbohydrate consumption, and various results concerning the expression of CYP2E1 were obtained with a low-carbohydrate diet. This study describes the effects of ethanol treatment on CYP2E1 levels and its relationship with oxidative stress using a balanced standard diet to avoid low or high carbohydrate consumption. Rats were fed for 1, 2, 3, or 4 weeks a commercial diet plus an ethanol-sucrose solution. The results have shown that ethanol administration was associated with CYP2E1 induction and stabilization without related oxidative stress. Our findings suggest that experimental models with a low-carbohydrate/high-fat diet produce some undesirable CYP2E1 changes that are not present when a balanced standard diet is given.

Native LDL-cholesterol mediated monocyte adhesion molecule overexpression is blocked by simvastatin.

AIM OF THE STUDY: This study sought to evaluate the effect of nLDL concentrations on monocyte adhesion molecule expression in hypercholesterolemic patients with stable coronary artery disease (CAD) and to determine whether lipid-lowering therapy with simvastatin would change this effect. METHODS: Blood samples from patients with hypercholesterolemia (mean LDL 152 mg/dL) and CAD (HC, n = 23) were collected before and after a 12-week treatment with 40 mg of simvastatin. Healthy individuals (mean LDL 111 mg/dL) were used as controls (CT, n = 15). Isolated nLDL, at a fixed concentration of 100 mg/dL, was added to monocyte suspensions obtained before and after the simvastatin treatment. Monocyte activation was determined by changes in cellular adhesion molecule expression. RESULTS: In response to nLDL, CD11b and CD14 adhesion molecule expression was higher in HC patients than in CT patients before treatment (174.2 +/- 8.4 vs 102.2 +/- 6.3, P < 0.03 and 140.4 +/- 5.0 vs 90.4 +/- 6.7, P < 0.04). After simvastatin treatment, CD11b expression decreased to 116.9 +/- 12.5 (P < 0.03) and CD14 expression to 107.5 +/- 6.2 (P < 0.04). Alternatively, L-selectin expression was lower in HC patients than in CT patients before therapy (46.0 +/- 3.5 vs 62.1 +/- 5.5, P < 0.04), and it increased markedly after lipid reduction to 58.7 +/- 5.0 (P < 0.04 vs baseline). After simvastatin treatment, LDL was reduced to mean 101.5 mg/dL. CONCLUSIONS: These data demonstrate that monocytes from HC patients are more prone to marked nLDL-mediated changes of adhesion molecule expression than monocytes from controls. Simvastatin is capable of inhibiting such nLDL effects. This proinflammatory response to nLDL may have a role in the early onset of atherosclerosis.

Silencing cytokeratin 18 gene inhibits intracellular replication of Trypanosoma cruzi in HeLa cells but not binding and invasion of trypanosomes.

BACKGROUND: As an obligatory intracellular parasite, Trypanosoma cruzi, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18) is among the host molecules that have been suggested as a mediator of important events during T. cruzi-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi)-mediated down regulation of the CK18 gene could interfere with the parasite life cycle in vitro. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence. RESULTS: CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different T. cruzi strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18. CONCLUSION: The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of T. cruzi in HeLa cells, but not trypanosome binding and invasion.

Protein tyrosine phosphorylation and protein tyrosine nitration in redox signaling.

Reversible phosphorylation of protein tyrosine residues by polypeptide growth factor-receptor protein tyrosine kinases is implicated in the control of fundamental cellular processes including the cell cycle, cell adhesion, and cell survival, as well as cell proliferation and differentiation. During the last decade, it has become apparent that receptor protein tyrosine kinases and the signaling pathways they activate belong to a large signaling network. Such a network can be regulated by various extracellular cues, which include cell adhesion, agonists of G protein-coupled receptors, and oxidants. It is well documented that signaling initiated by receptor protein tyrosine kinases is directly dependent on the intracellular production of oxidants, including reactive oxygen and nitrogen species. Accumulated evidence indicates that the intracellular redox environment plays a major role in the mechanisms underlying the actions of growth factors. Oxidation of cysteine thiols and nitration of tyrosine residues on signaling proteins are described as posttranslational modifications that regulate, positively or negatively, protein tyrosine phosphorylation (PTP). Early observations described the inhibition of PTP activities by oxidants, resulting in increased levels of proteins phosphorylated on tyrosine. Therefore, a redox circuitry involving the increasing production of intracellular oxidants associated with growth-factor stimulation/cell adhesion, oxidative reversible inhibition of protein tyrosine phosphatases, and the activation of protein tyrosine kinases can be delineated.

The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis.

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.

Serum amyloid A induces CCL20 secretion in mononuclear cells through MAPK (p38 and ERK1/2) signaling pathways.

Although the serum levels of SAA had been reported to be upregulated during inflammatory/infectious process, the role of this acute-phase protein has not been completely elucidated. In previous studies, we demonstrated that SAA stimulated the production of TNF-alpha, IL-1beta, IL-8, NO, and ROS by neutrophils and/or mononuclear cells. Herein we demonstrate that SAA induces the expression and release of CCL20 from cultured human blood mononuclear cells. We also focus on the signaling pathways triggered by SAA. In THP-1 cells SAA promotes phosphorylation of p38 and ERK1/2. Furthermore, the addition of SB203580 (p38 inhibitor) and PD98059 (ERK 1/2 inhibitor) inhibits the expression and release of CCL20 in mononuclear cells treated with SAA. Our results point to SAA as an important link of innate to adaptive immunity, once it might act on the recruitment of mononuclear cells.

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases.

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.

The low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine, promotes cell cycle progression in rabbit aortic endothelial cells.

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.

Src kinase activation by nitric oxide promotes resistance to anoikis in tumour cell lines.

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.