Intracellular NO Levels Research Articles

Effect of the NADPH oxidase inhibitor apocynin on ischemia-reperfusion hippocampus injury in rat brain

Blockage along with sudden restoration of blood following ischemia, results in several cascading events, such as a massive ROS production which plays an important role in the pathophysiology of ischemia. NADPH oxidase complex in mitochondria complex is believed to be the major source for ROS production. The present study explores the therapeutic potential of apocynin, an NADPH oxidase inhibitor in attenuating the ROS production, and the resultant neuroinflammation and mitochondrial injury during cerebral ischemia in rats. Bilateral common carotid artery occlusion (BCCAO) model was chosen for the study where intracellular ROS and NO levels as well as the NADPH oxidase activity were found to be increased significantly post 7th day of ischemic injury. Enhanced glial activation was observed and an upregulated expression of GFAP and Iba-1 in hippocampus along with that of the transcription factor NFκB and inflammatory markers iNOS, IL-1α, IL-1β and TNF-α.The activity of mitochondrial electron transport chain (ETC) complexes I, II, IV and V were significantly decreased following ischemia. Consequently, there was a decrease in mitochondrial membrane potential (MMP) while an increased release of cytochrome c and upregulated apoptotic markers Bax, caspase-3 and 9 initiated the programmed neuronal death which was also reflected by the marked increase in TUNEL positive cells in the hippocampal region. The physiological functional alterations have been observed following ischemic injury i.e memory and motor deficits. The apocynin supplementation significantly reduced the NADPH oxidase activity and resulted in declined ROS production which in-turn prevented the glial activation and downregulated the inflammatory and pro-apoptotic markers. Apocynin also restored the MMP (Δψm) and mitochondrial enzymes via inhibition of ROS vicious and relationship between NADPH oxidase and mitochondrial complexes. Apocynin treatment was also successfully reduced the behavioural deficits in ischemic animals. In conclusion, inhibiting the NADPH oxidase complex presumably attenuated the mitochondrial injury, neuroinflammation and apoptosis following ischemic injury in rat brain.

A STUDY OF THE ANTI-INFLAMMATORY EFFECTS OF THE ETHYL ACETATE FRACTION OF THE METHANOL EXTRACT OF FORSYTHIAE FRUCTUS.

Background:The dried fruit of Forsythia suspensa (Thunb.) Vahl. (Oleaceae) are better known by their herbal name Forsythiae Fructus, and have a bitter taste, slightly pungent smell, and cold habit. FF has been widely used to treat symptoms associated with the lung, heart, and small intestine. Recently, bioactive compounds isolated from hydrophobic solvent fractions of FF have been reported to have anti-oxidant, anti-bacterial, and anti-cancer effects. Traditionally, almost all herbal medicines are water extracts, and thus, extraction methods should be developed to optimize the practical efficacies of herbal medicines.Materials and Methods:In this study, the anti-inflammatory effects of the ethyl acetate fraction of the methanol extract of FF (FFE) were assessed by measuring NO and PGE2 production by and intracellular ROS and protein levels of iNOS and COX-2 in RAW 264.7 cells.Results:FFE inhibited COX-2 expression in LPS-stimulated RAW 264.7 cells.Conclusion:In summary, FFE effectively reduced intracellular ROS and NO levels and inhibited PGE2 production by down-regulating COX-2 levels.Abbreviations: FF, of Forsythiae Fructus; NO, nitric oxide; iNOS, inducible NO synthase; COX-2, cyclooxygenase-2; ROS, reactive oxygen species; PGE2, prostaglandin E2.

Nitrogen oxide cycle regulates nitric oxide levels and bacterial cell signaling.

Nitric oxide (NO) signaling controls various metabolic pathways in bacteria and higher eukaryotes. Cellular enzymes synthesize and detoxify NO; however, a mechanism that controls its cellular homeostasis has not been identified. Here, we found a nitrogen oxide cycle involving nitrate reductase (Nar) and the NO dioxygenase flavohemoglobin (Fhb), that facilitate inter-conversion of nitrate, nitrite, and NO in the actinobacterium Streptomyces coelicolor. This cycle regulates cellular NO levels, bacterial antibiotic production, and morphological differentiation. NO down-regulates Nar and up-regulates Fhb gene expression via the NO-dependent transcriptional factors DevSR and NsrR, respectively, which are involved in the auto-regulation mechanism of intracellular NO levels. Nitrite generated by the NO cycles induces gene expression in neighboring cells, indicating an additional role of the cycle as a producer of a transmittable inter-cellular communication molecule.

Effect of schisantherin A inhibit liver sinusoid endothelial cell function and action against liver fibrosis relating to angiogenesis

To investigate the effect of schisantherin A on liver sinusoid endothelial cell function and angiogenesis. Different dosages (0-40 μmol•L⁻¹) of schisantherin A were incubated 24 h with SK-HEP-1 cells, and the toxicity of SK-HEP-1 cells was assayed by MTT method. The proliferation of SK-HEP-1 cells were induced by the vascular endothelial growth factor (VEGF), with receptor tyrosine kinase inhibitor sorafenib as the control, at the same time, set up the control group, 2, 20 μmol•L⁻¹ schisantherin A were incubated with SK-HEP-1 cells, cell proliferation was analyzed by EdU DNA cell proliferation kit. Fluorescence probe method was used to assay the intracellular NO levels and NOS activity. Tube formation was observed using cell migration and a matrigel tube formation assay. Rat aortic ring assay was performed to observe the sprouting vessels from aortic ring. The fluorescence vessels, the number of functional blood vessels, and intersegmental vessel changes of transgenic zebrafish were also observed. Compared with control group, the proliferation of SK-HEP-1 cells induced by VEGF increased and and the level of NO and NOS activity induced; compared with model group, 2, 20 μmol•L⁻¹ schisantherin A and sorafenib inhibited the proliferation of SK-Hep-1 cells induced by VEGF, and reduced the level of NO and NOS activity. At the dosage of 20 μmol•L⁻¹, schisantherin A attenuated the migration and tube formation of SK-HEP-1 cells induced by VEGF, and also inhibition the formation of rat aortic rings and intersegmental vessel changes of transgenic zebrafish, and significantly reduce the number of vessels in zebrafish. Schisantherin A has potential effects on function of endothelial cell proliferation and angiogenesis.

Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells

Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN–LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

Prolonged Fasting Improves Endothelial Progenitor Cell-Mediated Ischemic Angiogenesis in Mice

Background/Aims: Prolonged fasting (PF) was shown to be of great potency to promote optimal health and reduce the risk of many chronic diseases. This study sought to determine the effect of PF on the endothelial progenitor cell (EPC)-mediated angiogenesis in the ischemic brain and cerebral ischemic injury in mice. Methods: Mice were subjected to PF or periodic PF after cerebral ischemia, and histological analysis and behavioral tests were performed. Mouse EPCs were isolated and examined, and the effects of EPC transplantation on cerebral ischemic injury were investigated in mice. Results: It was found that PF significantly increased the EPC functions and angiogenesis in the ischemic brain, and attenuated the cerebral ischemic injury in mice that was previously subjected to cerebral ischemia. Periodic PF might reduce cortical atrophy and improve long-term neurobehavioral outcomes after cerebral ischemia in mice. The eNOS and MnSOD expression and intracellular NO level were increased, and TSP-2 expression and intracellular O₂^- level were reduced in EPCs from PF-treated mice compared to control. In addition, transplanted EPCs might home into ischemic brain, and the EPCs from PF-treated mice had a stronger ability to promote angiogenesis in ischemic brain and reduce cerebral ischemic injury compared to the EPCs from control mice. The EPC-conditioned media from PF-treated mice exerted a stronger effect on cerebral ischemic injury reduction compared to that from control mice. Conclusion: Prolonged fasting promoted EPC-mediated ischemic angiogenesis and improved long-term stroke outcomes in mice. It is implied that prolonged fasting might potentially be an option to treat ischemic vascular diseases.

Naoxintong Protects Primary Neurons from Oxygen-Glucose Deprivation/Reoxygenation Induced Injury through PI3K-Akt Signaling Pathway.

Naoxintong capsule (NXT), developed from Buyang Huanwu Decoction, has shown the neuroprotective effects in cerebrovascular diseases, but the neuroprotection mechanisms of NXT on ischemia/reperfusion injured neurons have not yet been well known. In this study, we established the oxygen-glucose deprivation/reoxygenation (OGD/R) induced neurons injury model and treat the neurons with cerebrospinal fluid containing NXT (BNC) to investigate the effects of NXT on OGD/R induced neurons injury and potential mechanisms. BNC improved neuron viability and decreased apoptotic rate induced by OGD/R. BNC attenuated OGD/R induced cytosolic and mitochondrial Ca2+ overload, ROS generation, intracellular NO levels and nNOS mRNA increase, and cytochrome-c release when compared with OGD/R group. BNC significantly inhibited both mPTP opening and ΔΨm depolarization. BNC increased Bcl-2 expression and decreased Bax expression, upregulated the Bcl-2/Bax ratio, downregulated caspase-3 mRNA and caspase-9 mRNA expression, and decreased cleaved caspase-3 expression and caspase-3 activity. BNC increased phosphorylation of Akt following OGD/R, while LY294002 attenuated BNC induced increase of phosphorylated Akt expression. Our study demonstrated that NXT protected primary neurons from OGD/R induced injury by inhibiting calcium overload and ROS generation, protecting mitochondria, and inhibiting mitochondrial apoptotic pathway which was mediated partially by PI3K-Akt signaling pathway activation.

Nitric oxide signaling and its role in oxidative stress response in Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, we found that the putative NO dioxygenase SPAC869.02c (named Yhb1) and the S-nitrosoglutathione reductase Fmd2 cooperatively reduced intracellular NO levels as NO-detoxification enzymes. Although both mRNA and protein levels were increased with exogenous NO, their expression patterns were different during growth phases. While treatment with an NO synthase inhibitor in the log phase abrogated both NO production and Yhb1 expression, induction of Fmd2 in the stationary phase was correlated with elevated mitochondrial respiratory chain (MRC) activity, confirmed by the fact that inhibition of MRC complex III led to a decrease in Fmd2 and NO levels. Moreover, NO was localized in the mitochondria in the stationary phase, suggesting that there are two distinctive types of NO signaling in S. pombe. For mitochondria, pretreatment with an NO donor rescued cell growth by repressing generation of reactive oxygen species (ROS) under oxidative stress. DNA microarray analysis revealed that exogenous NO contributes to tolerance to hydrogen peroxide (H2O2) by (i) inhibition of Fe3+ to Fe2+ conversion, (ii) upregulation of the H2O2-detoxifying enzymes, and (iii) downregulation of the MRC genes, suggesting that NO plays a pivotal role in the negative feedback system to regulate ROS levels in S. pombe.

NOAD, a novel nitric oxide donor, induces G2/M phase arrest and apoptosis in human hepatocellular carcinoma Bel-7402 cells

O2-(2,4-dinitro-5-{[2-(12-en-28-b-d-galactopyranosyl-oleanolate-3-yl)-oxy-2-oxoethyl]amino}phenyl)1-(N-hydroxyethylmethylamino)diazen-1-ium-1,2-diolate (NOAD), a novel NO-releasing derivative of oleanolic acid (OA), is an active cytotoxic component. In this study, NOAD induced a rise in intracellular NO levels and showed cytotoxic effects which were prevented by hemoglobin (NO scavenger). Meanwhile, NOAD induced G2/M phase cell cycle arrest in a concentration-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that NOAD did not change the steady-state levels of cyclin A, cyclin B, cyclin E, Cdk2 and Cdk4, but decreased the protein levels of Cdk1 and Cdc25C. Meanwhile, the levels of phosphorylation of Cdc25C and Cdk1 were significantly increased by NOAD in a concentration-dependent manner. Moreover, NOAD modulated the phosphorylation of protein kinases Chk2. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21WAF1/CIP1 and p27kip1 were increased in a concentration-dependent manner. In addition, NOAD also caused a marked increase in the apoptotic cells, as characterized by fragmented nuclei, sub G1 formation, the level of 8-OHDG increase and poly (ADP-ribose) polymerase (PARP) cleavage, which was associated with activation of caspase-3, caspase-8 and caspase-9. Up-regulation of Bax and down-regulation of Bcl-2 were also observed in Bel-7402 cells treated with NOAD. These data suggest that NOAD produces anti-tumor effect via induction of G2/M cell cycle arrest and apoptosis.

Melatonin in sperm biology: breaking paradigms.

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRβ. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

Protective effect of polysaccharides from Sargassum horneri against oxidative stress in RAW264.7 cells

This study was designed to investigate chemical composition and the protective effects of polysaccharides isolated from Sargassum horneri against hydrogen peroxide (H2O2)-induced oxidative injury in RAW264.7 cells. Results showed that isolated polysaccharides (SHSc) and the major fractions (SHS1, SHS0.5) contained sulfate ester, and SHS1 was high fucose-containing sulfated polysaccharide. After preincubation with three isolated polysaccharides, RAW264.7 cells viability were significantly restored and decreased in cellular LDH release (P<0.05). SHS1 and SHS0.5 decreased intracellular ROS level, intracellular NO and malonic dialdehyde (MDA) level (P<0.05), restoring activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) (P<0.05), decreasing inducible nitric oxide synthase (iNOS) (P<0.05). Moreover, preincubation of SHS1 with RAW264.7 cells resulted in the increase of the gene expression level of endogenous antioxidant enzymes such as MnSOD and GSH-Px (P<0.05). These results clearly showed that SHSc and its fractions could attenuate H2O2-induced stress injury in RAW264.7 cells, and a similar efficiency in protecting RAW264.7 cells against H2O2-induced oxidative injury between SHS1 and Vitamin C. Taken together, our findings suggested that SHS1 can effectively protect RAW264.7 cells against oxidative stress by H2O2, which might be used as a potential natural antioxidant in the functional food and pharmaceutical industries.

Polyopes lancifolia extracts protect INS-1 pancreatic β cells against high glucose level induced apoptosis

Polyopes lancifolia extracts were investigated for protection of INS-1 pancreatic β cells against high glucose level-induced apoptosis using cell viability, lipid peroxidation generation, intracellular ROS, and NO assays. Expressions of the Bax and Bcl-2 proteins in INS-1 cells were evaluated using western blotting. Apoptosis induced by high glucose levels was determined based on analysis of FITC-annexin V/PI stained cells. A high glucose level-treatment induced INS-1 cells death. Cells treated with P. lancifolia extracts showed markedly reduced cytotoxicity in a dose-dependent manner, compared to the high glucose level-treated cells. Treatment with extracts dose-dependently decreased lipid peroxidation and intracellular ROS and NO levels, and increased cell viability in high glucose level-pretreated INS-1 cell, compared to the high glucose level-treated cells. Extract treatment significantly (p<0.05) reduced the Bax protein level and increased the anti-apoptotic Bcl-2 protein level, compared with the high glucose leveltreated cells. P. lancifolia extract treatment reduced the apoptosis that is induced by a high level glucose-treatment, based on annexin V/PI staining.

Regulation of flow‐induced intracellular NO levels by endothelial surface glycocalyx

To test the hypothesis that endothelial surface glycocalyx (ESG) plays a role in mechanosensing and transduction of the microvessel wall, we used fluorescence microscopy to measure the NO levels in the post‐capillary venules of rat mesentery under low and high flow conditions and with/out enzyme treatment for removing heparan sulfate (HS) of the ESG. Rats (SD, 250–300g) were anesthetized with pentobarbital sodium given subcutaneously, the mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. After perfusion for 1 hr with 1% BSA Ringer for the control or with 50 mU/mL heparanase III for the treatment, an individual post‐capillary venule (35–50 μm) was loaded for 45 min with 5 μM 4, 5‐Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO was measured for ~10min under a low flow (&lt; 300 μm/s) as the baseline and for ~60min under a high flow (1500–2000 μm/s mean velocity). In 1 min after switching to the high low, NO increased by 1.14 ± 0.05‐fold (n=3) under control and 1.15 ± 0.04‐fold (n=3) under enzyme treatment. NO continuously increased after 1 min under high flow, reached a plateau of 1.42 ± 0.02‐fold in ~40 min under control and of 1.22 ± 0.03 in ~15min under treatment. The results suggest that ESG participate in EC mechanosensing and transduction through its HS and other components. Supported by NIH R01HL094889–01.

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin

Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

P048 Suppression of proinflammatory cytokines by mulberry derivatives

Introduction The mulberry belongs to the Morus genus of the Moraceae family. There are 24 species of Morus, with at least 100 known varieties. Methods RAW 264.7 cells were pretreated with mulberry or mulberry derivatives for 2 h, and then stimulated with lipopolysaccharide (LPS). Gene expression of proinflammatory cytokines were determined by RT-PCR. The levels of NO were determined by measuring the amount of nitrate using Griess reagents, and absorption was measured at 570 nm. Intracellular ROS levels were evaluated using a DCFH-DA fluorescent probe by flow cytometry. Results We found that mulberry derivatives inhibited the gene expression of proinflammatory cytokines including IL-6, IL-1b, TNF-aand COX-2 in Raw264.7 cells stimulated with lipopolysaccharide (LPS). In addition, LPS-induced intracellular ROS and NO levels were decreased in Raw264.7 cells treated with mulberry derivatives compared with those in mulberry. Conclusion These data suggest that mulberry derivatives showed anti-inflammatory activity by reducing intracellular ROS and NO production via suppression of proinflammatory cytokine expression, and mulberry derivatives may be useful for the prevention and therapy of inflammation-related diseases.

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells

BackgroundNitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC).MethodsProstate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay.ResultsThe NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway.ConclusionsOur results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC.

Protein nitration and nitrosylation by NO-donating aspirin in colon cancer cells: Relevance to its mechanism of action

Nitric oxide-donating aspirin (NO-ASA) is a promising agent for cancer prevention. Although studied extensively, its molecular targets and mechanism of action are still unclear. S-nitrosylation of signaling proteins is emerging as an important regulatory mechanism by NO. Here, we examined whether S-nitrosylation of the NF-κB, p53, and Wnt signaling proteins by NO-ASA might explain, in part, its mechanism of action in colon cancer. NO-ASA releases significant amounts of NO detected intracellularly in HCT116 and HT-29 colon cells. Using a modified biotin switch assay we demonstrated that NO-ASA S-nitrosylates the signaling proteins p53, β-catenin, and NF-κB, in colon cancer cells in a time- and concentration-dependent manner. NO-ASA suppresses NF-κB binding to its cognate DNA oligonucleotide, which occurs without changes in the nuclear levels of the NF-κB subunits p65 and p50 and is reversed by dithiothreitol that reduces ―S―NO to ―SH. In addition to S-nitrosylation, we documented both in vitro and in vivo widespread nitration of tyrosine residues of cellular proteins in response to NO-ASA. Our results suggest that the increased intracellular NO levels following treatment with NO-ASA modulate cell signaling by chemically modifying key protein members of signaling cascades. We speculate that S-nitrosylation and tyrosine nitration are responsible, at least in part, for the inhibitory growth effect of NO-ASA on cancer cell growth and that this may represent a general mechanism of action of NO-releasing agents.

Antioxidant and signal modulation properties of plant polyphenols in controlling vascular inflammation

Oxidized low-density lipoproteins (oxLDL) play a critical role in the initiation of atherosclerosis through activation of inflammatory signaling. In the present work we investigated the role of antioxidant and signal modulation properties of plant polyphenols in controlling vascular inflammation. Significant decrease in intracellular NO level and superoxide overproduction was found in human umbilical vein endothelial cells (HUVEC) treated with oxLDL, but not with LDL. The redox imbalance was prevented by the addition of quercetin or resveratrol. Expression analysis of 14 genes associated with oxidative stress and inflammation revealed oxLDL-mediated up-regulation of genes specifically involved in leukocyte recruitment and adhesion. This up-regulation could be partially avoided by the addition of verbascoside or resveratrol, while treatment with quercetin resulted in a further increase in the expression of these genes. Lipopolysaccharide (LPS)-treated HUVEC were also used for the evaluation of anti-inflammatory potency of plant polyphenols. Significant differences between HUVEC treaded with oxLDL and LPS were found in both the expression pattern of inflammation-related genes and the effects of plant polyphenols on cellular responses. The present data indicate that plant polyphenols may affect vascular inflammation not only as antioxidants but also as modulators of inflammatory redox signaling pathways.

Modification of Intracellular Level of Free Radicals and Apoptosis in Cultured Human Endotheliocytes and Carcinoma Cells

The intracellular levels of superoxide O(2)(-)and nitric oxide NO were directly measured under intravital conditions in cultured human endotheliocytes ECV 304 and carcinoma cells HeLa G-63. Comparative analysis of changes in the intracellular levels of superoxide and NO induced by ascorbic acid revealed a negative correlation between NO and O(2)(-)levels, whose strength depended on concentration of the acid. In pharmacological concentrations, ascorbic acid induced apoptotic death of carcinoma cells, but did not trigger apoptosis of endotheliocytes. The study demonstrated a possible way of cytotoxic action of ascorbic acid in pharmacological concentrations.

Dynamics of Intracellular Superoxide and NO Content in Human Endotheliocytes and Carcinoma Cells after Treatment with NO Synthase Inhibitors

The dynamics of intracellular levels of superoxide and NO after cell treatment with NO synthase inhibitors were studied in human cells expressing various NOS isoforms: endotheliocytes and ECV-304 (eNOS) and carcinoma cells and HeLa-G63 (iNOS). Cytometric analysis of changes in the cell fluorescence intensity was carried out using superoxide and NO fluorescent indicators (dihydroethidene and DAF-2-DA, respectively). Intracellular levels of superoxide decreased in HeLa-G63 and ECV-304 cells after their incubation in medium with aminoguanidine, L-NAME, and D-NAME. Intracellular NO level decreased only in HeLa-G63 cells after incubation in medium with aminoguanidine and L-NAME, but not D-NAME. The level of NO returned to normal after 7-h culturing in inhibitor-free medium, while the level of superoxide increased and remained high throughout 3 generations. Incubation of cells with D-NAME did not increase the intracellular level of superoxide. Presumably, high prolonged generation of superoxide is a delayed result of inhibition of NO synthesis in HeLa-G63 cells.