scholarly journals iNOS Associates With Poor Survival in Melanoma: A Role for Nitric Oxide in the PI3K-AKT Pathway Stimulation and PTEN S-Nitrosylation

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhen Ding ◽  
Dai Ogata ◽  
Jason Roszik ◽  
Yong Qin ◽  
Sun-Hee Kim ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Nitrosative Stress ◽  
Akt Pathway ◽  
Melanoma Tumor ◽  
Inos Protein ◽  
Akt Activation ◽  
Inos Protein Expression ◽  
Patient Prognosis

We previously showed that inducible nitric oxide synthase (iNOS) protein expression in melanoma tumor cells is associated with poor patient prognosis. Here, we analyzed the association between iNOS and the oncogenic PI3K-AKT pathway. TCGA data show that iNOS and phospho-Akt Ser473 expression were associated significantly only in the subset of tumors with genetically intact PTEN. Employing a stage III melanoma TMA, we showed that iNOS protein presence is significantly associated with shorter survival only in tumors with PTEN protein expression. These findings led to our hypothesis that the iNOS product, nitric oxide (NO), suppresses the function of PTEN and stimulates PI3K-Akt activation. Melanoma cells in response to NO exposure in vitro exhibited enhanced AKT kinase activity and substrate phosphorylation, as well as attenuated PTEN phosphatase activity. Biochemical analysis showed that NO exposure resulted in a post-translationally modified S-Nitrosylation (SNO) PTEN, which was also found in cells expressing iNOS. Our findings provide evidence that NO-rich cancers may exhibit AKT activation due to post-translational inactivation of PTEN. This unique activation of oncogenic pathway under nitrosative stress may contribute to the pathogenesis of iNOS in melanoma. Significance: Our study shows that iNOS expression is associated with increased PI3K-AKT signaling and worse clinical outcomes in melanoma patients with wt (intact) PTEN. Mutated PTEN is already inactivated. We also demonstrate that NO activates the PI3K-AKT pathway by suppressing PTEN suppressor function concurrent with the formation of PTEN-SNO. This discovery provides insight into the consequences of inflammatory NO produced in human melanoma and microenvironmental cells. It suggests that NO–driven modification provides a marker of PTEN inactivation, and represents a plausible mechanism of tumor suppressor inactivation in iNOS expressing subset of cancers.

scholarly journals l-Arginine Availability Regulates Inducible Nitric Oxide Synthase-Dependent Host Defense against Helicobacter pylori

2007 ◽  
Vol 75 (9) ◽  
pp. 4305-4315 ◽  
Author(s):  
Rupesh Chaturvedi ◽  
Mohammad Asim ◽  
Nuruddeen D. Lewis ◽  
Holly M. Scott Algood ◽  
Timothy L. Cover ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Helicobacter Pylori ◽  
Protein Expression ◽  
Translation Initiation Factor ◽  
No Production ◽  
Dependent Manner ◽  
Inos Protein ◽  
Inos Protein Expression ◽  
H Pylori ◽  
Inducible Nitric Oxide

ABSTRACT Helicobacter pylori infection of the stomach causes an active immune response that includes stimulation of inducible nitric oxide (NO) synthase (iNOS) expression. Although NO can kill H. pylori, the bacterium persists indefinitely, suggesting that NO production is inadequate. We determined if the NO derived from iNOS in macrophages was dependent on the availability of its substrate, l-arginine (l-Arg). Production of NO by H. pylori-stimulated RAW 264.7 cells was dependent on the l-Arg concentration in the culture medium, and the 50% effective dose for l-Arg was 220 μM, which is above reported plasma l-Arg levels. While iNOS mRNA induction was l-Arg independent, iNOS protein increased in an l-Arg-dependent manner that did not involve changes in iNOS protein degradation. l-Lysine, an inhibitor of l-Arg uptake, attenuated H. pylori-stimulated iNOS protein expression, translation, NO levels, and killing of H. pylori. While l-Arg starvation suppressed global protein translation, at concentrations of l-Arg at which iNOS protein was only minimally expressed in response to H. pylori, global translation was fully restored and eukaryotic translation initiation factor α was dephosphorylated. H. pylori lacking the gene rocF, which codes for a bacterial arginase, induced higher levels of NO production by increasing iNOS protein levels. When murine gastric macrophages were activated with H. pylori, supraphysiologic levels of l-Arg were required to permit iNOS protein expression and NO production. These findings indicate that l-Arg is rate limiting for iNOS translation and suggest that the levels of l-Arg that occur in vivo do not permit sufficient NO generation by the host to kill H. pylori.

Differences in in vitro interactions between macrophages with pathogenic and environmental strains of Prototheca

2020 ◽  
Vol 15 (6) ◽  
pp. 427-436
Author(s):  
Jin Yang ◽  
Junhao Zhu ◽  
Timothy Kudinha ◽  
Fanrong Kong ◽  
Qiang-qiang Zhang
Keyword(s):  
Protein Expression ◽  
Rt Pcr ◽  
Inos Protein ◽  
Colony Forming Unit ◽  
Inos Protein Expression ◽  
Cytokine Levels ◽  
Different Strains ◽  
Different Levels ◽  
In Vitro Interactions

Aim: We investigated the interactions between macrophage and different strains of Prototheca. Materials & method: J774A.1 macrophages were infected with clinical isolates of Prototheca ciferrii 18125 and P. ciferrii 50779 and environmental isolate of  P. ciferrii N71. Phagocytosis activities were compared by colony-forming unit assays at 3, 6 and 9 h after infection. Cytokine levels were detected by RT-PCR and ELISA. iNOS protein expression was examined by western blotting. Results: All P. ciferrii strains were phagocytized by macrophages but induced different levels of cytokines in macrophages. Moreover, infected by  P. ciferrii N71 upregulated much higher iNOS protein expression in J774A.1 than that infected by the clinical strains. Conclusion: Clinical and environmental P. ciferrii strains show differences in their interactions with macrophages, which may be attributed to their virulence.

scholarly journals Effects of antisense oligonucleotide to iNOS on hemodynamic and vascular changes induced by LPS

1998 ◽  
Vol 275 (3) ◽  
pp. H1078-H1083 ◽  
Author(s):  
Azizul M. Hoque ◽  
Andreas Papapetropoulos ◽  
Richard C. Venema ◽  
John D. Catravas ◽  
Leslie C. Fuchs
Keyword(s):  
Protein Expression ◽  
Lung Lavage ◽  
Mesenteric Arteries ◽  
Intraluminal Pressure ◽  
Inos Mrna ◽  
Inos Protein ◽  
Inos Protein Expression ◽  
Male Wistar Rats

Lipopolysaccharide (LPS) causes impaired vascular contractility proposed to be mediated by induction of nitric oxide synthase (iNOS). Antisense (AS) oligonucleotide inhibits the translation of target mRNA into functional proteins. We hypothesize that in vivo pretreatment with AS oligonucleotide targeted to iNOS mRNA can prevent LPS-induced hyporeactivity to norepinephrine (NE). Three groups of conscious male Wistar rats received one of the following: saline, AS, or mismatch (MM) oligonucleotide at 0.4 mg/kg iv at 12 and 24 h before LPS (5 mg/kg iv). The fourth group received saline only. Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded before and 6 h after LPS or saline administration. Aorta, lung lavage, and lung tissue were collected for determination of iNOS protein expression and NOS activity. Small mesenteric arteries (≈250 μm) were isolated, denuded of endothelium, and maintained at a constant intraluminal pressure of 40 mmHg for study in vitro. LPS produced significant tachycardia that was not altered by AS or MM oligonucleotide. AS, but not MM oligonucleotide, reduced the accumulation of cGMP, the increase in conversion ofl-[3H]arginine tol-[3H]citrulline, and iNOS protein expression in tissue from LPS-treated rats. Small mesenteric arterial contraction to NE was significantly impaired in vessels from LPS-treated rats and was restored by AS, but not MM, oligonucleotide. In a rat model of septic shock, AS oligonucleotide to iNOS mRNA inhibits NOS activity and iNOS protein expression and prevents the vascular hyporeactivity to NE, which may contribute to hypotension in shock.

scholarly journals Indomethacin augments lipopolysaccharide-induced expression of inflammatory molecules in the mouse brain

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10391
Author(s):  
Mona Yasin Mohamed ◽  
Willias Masocha
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Mrna Expression ◽  
Calcium Binding ◽  
Necrosis Factor Alpha ◽  
Inos Protein ◽  
Inos Protein Expression ◽  
Anti Inflammatory ◽  
The Brain ◽  
Inducible Nitric Oxide

Indomethacin and other non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain and fever including during infections. However, some studies suggest that NSAIDs protect against neuroinflammation, while some find no effects or worsening of neuroinflammation. We evaluated the effect of indomethacin alone on in combination with minocycline, a drug that inhibits neuroinflammation, on the expression of transcripts of neuroinflammatory molecules-induced by lipopolysaccharide (LPS) in the brain of mice. Inoculation of male BALB/c mice with LPS induced the expression of the microglia marker ionized calcium binding adaptor molecule protein, mRNA expression of the genes for cytokines interleukin-1beta (Il1b) and tumor necrosis factor-alpha (Tnf) and inducible nitric oxide synthase gene (Nos2), but not Il10, in the brain. Treatment with indomethacin had no significant effect on the cytokines or Nos2 mRNA expression in naïve animals. However, pretreatment with indomethacin increased LPS-induced Nos2 mRNA and inducible nitric oxide (iNOS) protein expression, but had no significant effect on LPS-induced mRNA expression of the cytokines. Minocycline reduced LPS-induced Il1b and Tnf, but not Nos2, mRNA expression. Treatment with indomethacin plus minocycline had no effect on LPS-induced Il1b, Tnf and Nos2 mRNA expression. In conclusion these results show that indomethacin significantly augments LPS-induced Nos2 mRNA and iNOS protein expression in the brain. In the presence of indomethacin, minocycline could not inhibit LPS-induced pro-inflammatory cytokine expression. Thus, indomethacin could exacerbate neuroinflammation by increasing the expression of iNOS and also block the anti-inflammatory effects of minocycline.

iNOS activity is essential for endothelial stress gene expression protecting against oxidative damage

2003 ◽  
Vol 95 (5) ◽  
pp. 1937-1946 ◽  
Author(s):  
Karsten Hemmrich ◽  
Christoph V. Suschek ◽  
Guido Lerzynski ◽  
Victoria Kolb-Bachofen
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Stress Response ◽  
Endothelial Function ◽  
Protective Role ◽  
Inos Expression ◽  
Heme Oxygenase 1 ◽  
Inos Protein ◽  
Inos Protein Expression ◽  
Exogenous Substrate

In endothelial cells, the expression of the inducible nitric oxide synthase (iNOS) and the resulting high-output nitric oxide synthesis have often been assumed as detrimental to endothelial function, but recent publications have demonstrated a protective role resulting from iNOS espression and activity. To address this question, we used antisense-mediated iNOS knockdown during proinflammatory cytokine challenge in primary endothelial cell cultures and studied endothelial function by monitoring the expression of stress defense genes. Using antisense oligonucleotides, we achieved a block of iNOS protein formation, accompanied by a strong decrease in the expression of the protective stress response genes bcl-2, vascular endothelial growth factor, and heme oxygenase-1 (HO-1). Additionally, cells were also maintained in the presence of limited exogenous substrate concentrations during cytokine challenge, thereby mimicking a situation of low serum arginine level during inflammation. Under these conditions, cytokine addition results in full iNOS protein expression with minimal nitric oxide formation, concomitant with a significant reduction in stress response gene expression and susceptibility to cell death induced by reactive oxygen species. Taken together, our data suggest that cytokine-induced endogenous iNOS expression and activity have key functions in increasing endothelial survival and maintaining function. Thus suppression of iNOS expression or limited substrate supply, as has been reported to occur in atherosclerosis patients, appears to significantly contribute to endothelial dysfunction and death during oxidative stress.

scholarly journals Differential regulation of metabolism by nitric oxide andS-nitrosothiols in endothelial cells

2011 ◽  
Vol 301 (3) ◽  
pp. H803-H812 ◽  
Author(s):  
Anne R. Diers ◽  
Katarzyna A. Broniowska ◽  
Victor M. Darley-Usmar ◽  
Neil Hogg
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Oxidative Phosphorylation ◽  
Metabolic Pathways ◽  
Nitrosative Stress ◽  
Surrogate Marker ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
No Donor

S-nitrosation of thiols in key proteins in cell signaling pathways is thought to be an important contributor to nitric oxide (NO)-dependent control of vascular (patho)physiology. Multiple metabolic enzymes are targets of both NO and S-nitrosation, including those involved in glycolysis and oxidative phosphorylation. Thus it is important to understand how these metabolic pathways are integrated by NO-dependent mechanisms. Here, we compared the effects of NO and S-nitrosation on both glycolysis and oxidative phosphorylation in bovine aortic endothelial cells using extracellular flux technology to determine common and unique points of regulation. The compound S-nitroso-l-cysteine (l-CysNO) was used to initiate intracellular S-nitrosation since it is transported into cells and results in stable S-nitrosation in vitro. Its effects were compared with the NO donor DetaNONOate (DetaNO). DetaNO treatment caused only a decrease in the reserve respiratory capacity; however, l-CysNO impaired both this parameter and basal respiration in a concentration-dependent manner. In addition, DetaNO stimulated extracellular acidification rate (ECAR), a surrogate marker of glycolysis, whereas l-CysNO stimulated ECAR at low concentrations and inhibited it at higher concentrations. Moreover, a temporal relationship between NO- and S-nitrosation-mediated effects on metabolism was identified, whereby NO caused a rapid impairment in mitochondrial function, which was eventually overwhelmed by S-nitrosation-dependent processes. Taken together, these results suggest that severe pharmacological nitrosative stress may differentially regulate metabolic pathways through both intracellular S-nitrosation and NO-dependent mechanisms. Moreover, these data provide insight into the role of NO and related compounds in vascular (patho)physiology.

Combined lack of estrogen receptors a and ? affects vascular iNOS protein expression

2003 ◽  
Vol 313 (1) ◽  
pp. 63-70 ◽  
Author(s):  
M. Liang ◽  
E. Ekblad ◽  
M.-L. Lydrup ◽  
B.-O. Nilsson
Keyword(s):  
Protein Expression ◽  
Estrogen Receptors ◽  
Inos Protein ◽  
Inos Protein Expression

scholarly journals Vagus Nerve Stimulation Ameliorates Renal Ischemia-Reperfusion Injury through Inhibiting NF-κB Activation and iNOS Protein Expression

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Meng Wang ◽  
Jielin Deng ◽  
Huanzhu Lai ◽  
Yanqiu Lai ◽  
Guannan Meng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Expression ◽  
Nerve Stimulation ◽  
Vagus Nerve Stimulation ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Inos Expression ◽  
Inos Protein ◽  
Inos Protein Expression ◽  
Renal Ischemia Reperfusion Injury

Objective. In renal ischemia/reperfusion injury (RIRI), nuclear factor κB (NF-κB) initiates the expression of multiple genes involved in inflammatory disease. Inhibition of NF-κB-mediated inducible nitric oxide synthase (iNOS) expression can ameliorate RIRI. Vagus nerve stimulation (VNS) protects against various organs I/R injury. The present study was designed to elucidate the protective effect of VNS on RIRI and its influence on iNOS protein expression. Methods. Eighteen male Sprague-Dawley rats were randomly allocated into the sham group, the I/R group, and the VNS+I/R group, 6 rats per group. An RIRI model was induced by a right nephrectomy and blockade of the left renal pedicle vessels for 45 min. After 6 h of reperfusion, the blood samples and renal samples were collected. The VNS treatment was performed throughout the I/R process in the VNS+I/R group using specific parameters (20 Hz, 0.1 ms in duration, square waves) known to produce a small but reliable bradycardia. Blood was used for evaluation of renal function and inflammatory state. Renal injury was evaluated via TUNEL staining. Renal samples were harvested to evaluate renal oxidative stress, NF-κB p65 levels, and iNOS protein expression. Results. The VNS treatment reduces serum creatinine (Cr) and blood urea nitrogen (BUN) levels. Simultaneously, the levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1-beta (IL-1β) were significantly increased in the I/R group, but VNS treatment markedly ameliorated this inflammatory response. Furthermore, the VNS ameliorated oxidant stress and renal injury, indicated by a decrease in 3-nitrotyrosine (3-NT) formation and MDA and MPO levels and an increase in the SOD level compared to that in the I/R group. Finally, the VNS also significantly decreases NF-κB p65, iNOS, and nitrite/nitrate levels compared to that in the I/R group. Conclusion. Our findings indicate that NF-κB activation increased iNOS expression and promoted RIRI and that VNS treatment attenuated RIRI by inhibiting iNOS expression, oxidative stress, and inflammation via NF-κB inactivation.

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