High Levels Of Nitric Oxide Research Articles

Ozone therapy in the treatment of the elderly

Aging is a universal process of constant reducing of the functioning of a complex system over time, affecting all levels of its organization that impede self-renewal processes. In the structure of the geroprotective therapeutic effect, the main role is given to combat oxidative stress. The most important natural remedy without drugs with this property is the ozoneoxygen mixture, whose effect on the human body is verified by studies conducted both nationally and internationally. 119 patients who underwent outpatient treatment at the Ozone Center in Nizhny Novgorod, Russia, were examined, the basic diagnosis was coronary heart disease, stable angina CF II-III. Parenteral administration of ozonated saline normalizes the pro- and antioxidant serum balance of elderly patients with coronary heart disease. After a course of ozone therapy, there is a decrease in the level of lipid metabolism - total cholesterol, LDL-C, triglycerides. Intravenous administration of low ozone levels improves endothelial function by lowering high levels of nitric oxide and endothelin. Ozone therapy reduces the ability of platelets to aggregate and improves the rheological properties of the blood of elderly patients with coronary heart disease.

Novel nitric oxide-releasing derivatives of pyranocarbazole as antitumor agents: Design, synthesis, biological evaluation, and nitric oxide release studies

In this study, a series of novel furoxan-based nitric oxide (NO) releasing derivatives of pyranocarbazole alkaloids were designed, synthesized, and biologically evaluated against human cancer cell lines. The derivatives showed considerable antiproliferative activities (IC50 = 0.05–7.55 μM) and most compounds showed higher activity in MDA-MB-231 than H460 and HeLa. Especially, the most active derivative 7a (IC50 = 0.05 μM) against MDA-MB-231 was about 60 times stronger than lead compound, as well as equivalent to positive control taxol, and produced high levels of NO in MDA-MB-231. Furthermore, 7a could significantly inhibit the growth of MDA-MB-231 tumors in vivo with low toxicity and the PI3K/Akt signaling pathway. These results indicated that compound 7a could be a promising lead for further studies.

Effects of dulaglutide on endothelial progenitor cells and arterial elasticity in patients with type 2 diabetes mellitus

BackgroundRandomised controlled trial showed that dulaglutide can reduce the risk of atherosclerotic cardiovascular disease (ASCVD) in patients with type 2 diabetes mellitus (T2DM), but the underlying mechanisms remain unclear. This study aimed to investigate the effect of dulaglutide on the number and function of endothelial progenitor cells (EPCs) in the peripheral blood of patients with T2DM and its role in improving arterial elasticity, so as to determine potential mechanisms of preventive effect of dulaglutide on ASCVD.MethodsSixty patients with T2DM were treated with 1000 mg/day of metformin and randomly divided into two groups for 12 weeks: metformin monotherapy group (MET group, n = 30), and metformin combined with dulaglutide group (MET-DUL group, n = 30). Before and after treatment, the number of CD34+CD133+KDR+ EPCs and the brachial–ankle pulse wave velocity (baPWV) of the participants were measured, and EPC proliferation, adhesion, migration, and tubule formation were assessed in vitro.ResultsThere were no significant differences in the number and function of EPCs and baPWV changes in MET group (P > 0.05). In MET-DUL group, nitric oxide (NO) levels and the number of EPCs increased after treatment (P < 0.05), while the levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), advanced glycation end products (AGEs), and baPWV decreased (P < 0.05). EPC proliferation, adhesion, migration, and tubule formation abilities were significantly enhanced (P < 0.05). Correlation analysis showed that in MET-DUL group, the changes in CRP, IL-6, TNF-α, and AGEs were negatively correlated with the number of EPCs and their proliferation and migration abilities (P < 0.05). Body weight, NO, CRP, and IL-6 levels were independent factors affecting the number of EPCs (P < 0.05). The changes in number of EPCs, proliferation and migration abilities of EPCs, and NO and IL-6 levels were independent influencing factors of baPWV changes (P < 0.05).ConclusionDulaglutide can increase the number and function of EPCs in peripheral blood and improve arterial elasticity in patients with T2DM; it is accompanied by weight loss, inflammation reduction, and high NO levels. Dulaglutide regulation of EPCs may be a mechanism of cardiovascular protection.

Effect of zinc oxide nanoparticles on cellular stress in Ehrlich ascites carcinoma

Background Cancer cells are characterized by high cellular stress that is considered crucial for cancer cell survival and metastasis. Aim In this study, we assessed cellular stress in Ehrlich ascites carcinoma (EAC) cells before and after treatment with intraperitoneal zinc oxide nanoparticles. Materials and methods This experiment was performed on six groups of mice: 90 female albino mice were included in this study and were divided into six groups (n=15 per group): C [saline treated mice (20 ml/Kg intraperitoneal (i.p.)], CZn20/2d [ZnO NP-treated mice (20 mg/kg i.p. every other day], EC [untreated EAC-bearing mice], EZn 10/d [EAC-bearing mice administered with ZnO NPs 10 mg/kg everyday], EZn 10/2d [EAC-bearing mice administered with ZnO NPs 10 mg/kg every other day], EZn20/2d [EACs-bearing mice administered with ZnO NPs 20 mg/kg every other day]. After 2 weeks, the ascitic fluid and mammary gland were collected to measure the level of nitric oxide (NO) and peroxynitrite. Results Based on this results, there was a significantly high level of NO and peroxynitrite in the EAC groups compared with the control group. Zinc oxide nanoparticles significantly decreased the level of NO and peroxynitrite in the EAC groups compared with the control group. Conclusion This study implied that cancer cells have higher cellular stress, and it was efficiently decreased by treatment with zinc oxide nanoparticles indicating its efficacy against cancer cells.

Differential immune response to two Staphylococcus aureus strains with distinct adaptation genotypes after experimental intramammary infection of dairy cows

The aim of this study was to evaluate and compare the ability of two S. aureus strains with different adaptation genotypes (low and high) to the bovine mammary gland (MG) to establish an intramammary infection (IMI) and induce an immune response after an experimental challenge in lactating cows. Two isolates (designated 806 and 5011) from bovine IMI with different genotypic profiles, harboring genes involved in adherence and biofilm production, belonging to different capsular polysaccharide (CP) type, accessory gene regulator (agr) group, pulsotype (PT) and sequence type/clonal complex (ST/CC) were selected. Strains 806 and 5011 were associated with low (nonpersistent-NP) and high (persistent-P) adaptation to the MG, respectively. Strain 806 (NP) was characterized as agr group II, cap5 positive and ST350; strain 5011 (P) agr group I, cap8 positive and CC188. Three groups of clinically healthy cows, 4 cows/treatment group, were inoculated by the intramammary route with strain 806 (NP), strain 5011 (P) and pyrogen-free saline solution. All mammary quarters challenged with strain 806 (NP) developed mild clinical mastitis between 1 and 7 d post inoculation (pi). Quarters challenged with strain 5011 (P) developed a persistent IMI; bacteria were recovered from milk from d 7 pi and up to d 56 pi. In quarters inoculated with strain 806 (NP) the inflammatory response induced was greater and earlier than the one induced by strain 5011 (P), since a somatic cell count (SCC) peak was observed at d 2 pi, while in quarters inoculated with strain 5011 (P) no variations in SCC were observed until d 4 pi reaching the maximum values at d 14 pi; indicating a lower and delayed initial inflammatory response. The highest levels of nitric oxide (NO) and lactoferrin (Lf) detected in milk from quarters inoculated with both S. aureus strains coincided with the highest SCC at the same time periods, indicating an association with the magnitude of inflammation. The high levels of IL-1β induced by strain 806 (NP) were associated with the highest SCC detected (d 2 pi); while quarters inoculated with strain 5011 (P) showed similar IL-1β levels to those found in control quarters. In quarters inoculated with strain 806 (NP) two peaks of IL-6 levels on d 2 and 14 pi were observed; while in quarters inoculated with strain 5011 (P) IL-6 levels were similar to those found in control quarters. The strain 806 (NP) induced a higher total IgG and IgG1 response; while strain 5011 (P) generated a higher IgG2 response (even against the heterologous strain). The present study demonstrated that S. aureus strains with different genotype and adaptability to bovine MG influence the local host immune response and the course and severity of the infectious process.

Chemical Composition, Antitumor Potential, and Impact on Redox Homeostasis of the Essential Oils of Orlaya grandiflora from Two Climate Localities

It is well known that abiotic components can affect biosynthetic pathways in the production of certain volatile compounds. The aim of this study was to compare the chemical composition of essential oils obtained from Orlaya grandiflora (L.) Hoffm. collected from two localities in Serbia (continental climate, OG1) and Montenegro (Mediterranean climate, OG2) and to assess their antitumor potential on the human colon cancer HCT-116 and breast cancer MDA-MB-231 cell lines. EOs obtained by hydrodistillation were analyzed using GC-MS and GC-FID methods. The results indicate considerable differences in the chemical compositions of the two samples. Although in both samples the main class of volatiles observed was sesquiterpenes (47.5% for OG1 and 70.1% for OG2), the OG1 sample was characterized by a high amount of monoterpene hydrocarbons (29.3%), and sesquiterpene germacrene D (29.5%) as the most abundant compound. On the other hand, the OG2 sample contained a high quantity of oxygenated sesquiterpenes (20.6%), and β-elemene (22.7%) was the major constituent. The possible antitumor mechanisms of these EOs in the HCT-116 and MDA-MB-231 cell lines were examined by means of cell viability, apoptosis, redox potential, and migratory capacity. The antiviability potential appeared to be dose dependent, since the results showed that both EOs decreased the viability of the tested cells. Stronger antitumor effects were shown in MDA-MB-231 cells after short-term treatment, especially at the highest applied concentration, where the percentage of viability was reduced by over 40%. All tested concentrations of EOs exhibited proapoptotic activity and elevated activity of caspase-3 in a dose- and time-dependent manner. The results also showed decreased concentrations of superoxide anion radical in the treated cells, which indicates their significant antioxidative role. Long-term treatments showed mild recovery effects on cell viability in both cell lines, probably caused by the balancing of redox homeostasis. Elevated levels of nitrites indicate high levels of nitric oxide (NO) production and suggest its higher bioavailability due to the antioxidative environment. The tested EOs also induced a drop in migratory capacity, especially after short-time treatments. Taken together, these results suggest considerable antitumor activity of both EOs, which could have potential therapeutic applications.

Experimental and Numerical Study on the Effect of Nitric Oxide on Autoignition and Knock in a Direct-Injection Spark-Ignition Engine

&lt;div class="section abstract"&gt;&lt;div class="htmlview paragraph"&gt;Nitric Oxide (NO) can significantly influence the autoignition reactivity and this can affect knock limits in conventional stoichiometric SI engines. Previous studies also revealed that the role of NO changes with fuel type. Fuels with high RON (Research Octane Number) and high Octane Sensitivity (S = RON - MON (Motor Octane Number)) exhibited monotonically retarding knock-limited combustion phasing (KL-CA50) with increasing NO. In contrast, for a high-RON, low-S fuel, the addition of NO initially resulted in a strongly retarded KL-CA50 but beyond the certain amount of NO, KL-CA50 advanced again. The current study focuses on same high-RON, low-S Alkylate fuel to better understand the mechanisms responsible for the reversal in the effect of NO on KL-CA50 beyond a certain amount of NO. Experiments were conducted to measure the responses of KL-CA50 and trace-autoignition CA50, the latter being indicative of CA50 at which end-gas autoignition starts to become measurable from the apparent heat-release rate. Chemical-kinetics simulations were conducted to reveal the role of NO for end-gas autoignition, with a specific focus on sequential autoignition in a thermally stratified end-gas.&lt;/div&gt;&lt;div class="htmlview paragraph"&gt;The simulation results reveal that the magnitude of low-temperature heat release (LTHR) generally increases with NO. However, the relative importance of NO for enhancing LTHR diminishes when the LTHR inherent to a fuel’s chemistry is strong, such as at lower temperatures in a thermal boundary layer. This rendered more uniform LTHR within a hypothetical thermal boundary and led to a more sequential (&lt;i&gt;i.e.&lt;/i&gt; slower) autoignition event. It was also revealed that a change in compression ratio influences the importance of intermediate-temperature heat release (ITHR) due to changes of the temperature-pressure history of the end-gas. Together with the condition where end-gas autoignition occurs more sequentially, the shorter time spent in LTHR and ITHR regime can counter the increase in autoignition reactivity at high NO levels and allow KL-CA50 to advance.&lt;/div&gt;&lt;/div&gt;

A flexible and highly sensitive organic electrochemical transistor-based biosensor for continuous and wireless nitric oxide detection

As nitric oxide (NO) plays significant roles in a variety of physiological processes, the capability for real-time and accurate detection of NO in live organisms is in great demand. Traditional assessments of NO rely on indirect colorimetric techniques or electrochemical sensors that often comprise rigid constituent materials and can hardly satisfy sensitivity and spatial resolution simultaneously. Here, we report a flexible and highly sensitive biosensor based on organic electrochemical transistors (OECTs) capable of continuous and wireless detection of NO in biological systems. By modifying the geometry of the active channel and the gate electrodes of OECTs, devices achieve optimum signal amplification of NO. The sensor exhibits a low response limit, a wide linear range, high sensitivity, and excellent selectivity, with a miniaturized active sensing region compared with a conventional electrochemical sensor. The device demonstrates continuous detection of the nanomolar range of NO in cultured cells for hours without significant signal drift. Real-time and wireless measurement of NO is accomplished for 8 d in the articular cavity of New Zealand White rabbits with anterior cruciate ligament (ACL) rupture injuries. The observed high level of NO is associated with the onset of osteoarthritis (OA) at the later stage. The proposed device platform could provide critical information for the early diagnosis of chronic diseases and timely medical intervention to optimize therapeutic efficacy.

Changes in the Oral Cavity Mucosal Surface under the Influence of Wearing Protective Face Masks—Nitric Oxide Concentration Analysis—Preliminary Report

Orders to cover the mouth and nose were introduced as a prophylaxis for COVID-19. The use of face masks, apart from their benefits, has some side effects. It can affect, among other things, the oral cavity mucosa, manifested by its dryness, and can increase the amount of inflammatory markers, for example, nitric oxide (NO). The aim of this research was to determine changes in the oral cavity mucosal surface under the influence of the use of protective face masks based on an innovative measurement of NO levels in the exhaled air of healthcare workers. The people taking part in this study were dental assistants and recorders who used masks during work. The first measurement of NO was carried out before starting work and putting on a mask, and the second measurement was carried out after work. Based on the research, a statistically significant difference was shown in the NO values before putting on a mask and immediately after removing it. Despite the advantages of wearing protective masks, studies have shown that their long-term use has an impact on the oral cavity mucosa, which is reflected in the higher level of NO in exhaled air.

Synthesis and immunogenicity of the linear conjugates of polyacrylic acid and antigenic peptide of human papillomavirus

Polyelectrolytes (polymers carrying positively or negatively charged groups) can be used as adjuvants in subunit vaccine formulations to increase the immunogenicity of antigenic molecules. A vaccine prototype can be obtained by covalently bonding antigenic molecules to polyelectrolytes or using their physical mixtures with polyelectrolytes. Covalent conjugates of polyelectrolytes with antigens are known to exhibit stronger immunogenicity compared to physical mixtures of antigens and polyelectrolytes. In this study, linear conjugates of polyacrylic acid (PAA) with an antigenic peptide sequence (STHVDIRTLEDLLMGW) of the HPV-16 E7 oncoprotein were produced. PAAs of different molecular weights with narrow dispersity were synthesized by RAFT polymerization. The linear conjugates of PAA and the peptide were obtained by covalent bonding via the chain-end thiol group of PAA and N-terminal maleimide group of the peptide using the maleimide-thiol reaction. For this purpose, the trithiocarbonate end group of the polymer was reduced with excess ethylenediamine to the thiol group. The produced PAA-peptide conjugates were characterized by GPC, fluorescence, and NMR spectroscopies. Conjugation efficiency was above 80% for all PAAs. Antigenic peptide conjugates of PAAs of different molecular weights were investigated in MTT and nitric oxide (NO) assays in which the linear PAA-peptide conjugates exhibit high biocompatibility and stimulated the macrophages to release high levels of NO.

Nitric oxide, aging and aerobic exercise: Sedentary individuals to Master's athletes

Aging is associated with a decline in physiological function and exercise performance. These effects are mediated, at least in part, by an age-related decrease in the bioavailability of nitric oxide (NO), a ubiquitous gasotransmitter and regulator of myriad physiological processes. The decrease in NO bioavailability with aging is especially apparent in sedentary individuals, whereas older, physically active individuals maintain higher levels of NO with advancing age. Strategies which enhance NO bioavailability (including nutritional supplementation) have been proposed as a potential means of reducing the age-related decrease in physiological function and enhancing exercise performance and may be of interest to a range of older individuals including those taking part in competitive sport. In this brief review we discuss the effects of aging on physiological function and endurance exercise performance, and the potential role of changes in NO bioavailability in these processes. We also provide a summary of current evidence for dietary supplementation with substrates for NO production — including inorganic nitrate and nitrite, l-arginine and l-citrulline — for improving exercise capacity/performance in older adults. Additionally, we discuss the (limited) evidence on the effects of (poly)phenols and other dietary antioxidants on NO bioavailability in older individuals. Finally, we provide suggestions for future research.

Assessing the interrelation between oestrogen receptor status, heart rate variability and serum nitric oxide in breast cancer patients: Understanding their prognostic relevance.

e12564 Background: Heart Rate Variability (HRV) is a non-invasive procedure to assess cardiac functions and provides an index of autonomic function that intricately controls vascular tone. Studying the alteration in HRV with agents producing oxidative stress will provide useful information about disease progression. Lower HRV indicates low level of Parasympathetic Nervous System activation and dysregulation of cardiac functions. Elements of tumour microenvironment like prostaglandin E2, oestrogen and hypoxia stimulate nitric oxide (NO) production which reacts with free radicals released by tumour tissue forming oxidising agents. NO enhances oxidative stress causing vascular dysfunction. Very few studies have reported an interrelationship between NO and HRV parameters in breast cancer. The aim of the study is to verify the relation between HRV and serum NO levels in ER+ and ER- breast cancer groups. Methods: Study design: Analytical, Cross sectional. Sample Size: 135 women above 20 years of age with and without breast cancer. 69 women with histologically proven breast cancer in different stages and 66 women who were healthy age matched controls were included. Oestrogen Receptor (ER) status was obtained from clinical records. Out of 69 women with breast cancer, 31 were ER- with 15 in stage 1 and 16 in stage 2. 38 women were ER+ with 5 in stage 2 and 33 in stage 3. Serum NO was measured using colorimetry. Heart Rate Variability was measured from lead II ECG using Power Lab 15T and lab chart software. Serum NO levels and HRV parameters were compared between ER groups. Results: ER+ women had higher HRV with parasympathetic predominance compared to ER- with p&lt;0.001. ER+ women also had higher serum NO levels compared to ER- group with p&lt;0.001. Though no direct correlation was noticed between NO and HRV, multiple analysis of variance showed significant (p&lt;0.001) difference of all values across the groups. irrespective of menopausal status. Conclusions: A significant difference was observed in HRV of ER+ and ER- women with breast cancer. Most ER+ women were in stage 3 with significantly higher NO levels. Serum NO is expected to be the factor most responsible for HRV changes particularly in stage 3/ER+. Parasympathetic predominance seen in ER+ women possibly is due to high NO levels.[Table: see text]

ASSOCIATIONS BETWEEN PLASMA METABOLOMICS AND BLOOD PRESSURE ACROSS ETHNICITIES: THE HELIUS STUDY

Objective: Blood pressure (BP) is regulated by plasma metabolites from different neurohumoral and cardiometabolic systems. Since there are well-established differences in hypertension pathogenesis and treatment response between ethnicities, we hypothesized that plasma metabolites may be differently associated with BP across ethnic groups. Design and method: From the HELIUS study, 369 subjects (mean age 52 ± 11 years, 51%F) of African and non-African descent were included. Office systolic and diastolic BP levels were recorded. Plasma metabolites were measured with untargeted LC-MS from fasting plasma samples. Associations between metabolite profiles and BP were assessed with machine learning prediction models. Associations between the resulting best predictors and BP were assessed with linear regression models while adjusting for age, sex, renal function and diabetes, and interactions with ethnicity were tested. Results: Plasma metabolite profiles explained 14.1% of systolic BP variance and 10.6% of diastolic BP variance. Top predictors for both systolic and diastolic BP included N-formylmethionine, several acylcarnitines and polyunsaturated fatty acids such as hexadecadienoate. These metabolites were significantly associated with higher systolic BP with estimates ranging from 3.0–4.5 mmHg per 1 SD increase in the adjusted models. Associations with hexadecadienoate, dihomolinoleate and catecholamine metabolites, including vanillactate had significant interactions with ethnicity and were only significant in subjects of non-African descent. The top predictor N-formylmethionine was the most consistent predictor across ethnicities and sex. This metabolite was associated with higher levels of nitric oxide (NO)-related metabolites, including dimethylarginine (r = 0.42, p &lt; 0.001) and citrulline (r = 0.24, p &lt; 0.0001). Conclusions: Plasma metabolome composition explained a large proportion of BP variance, but best predicting metabolites were differently associated with BP across ethnicities. N-formylmethionine was the most consistent predictor for BP across ethnicities and sex. This metabolite was associated with metabolites in the NO-pathway. We hypothesize that formylmethionine affects BP through the inhibition of endothelial NO formation.

High Nitric Oxide Concentration Inhibits Photosynthetic Pigment Biosynthesis by Promoting the Degradation of Transcription Factor HY5 in Tomato.

Photosynthetic pigments in higher plants, including chlorophyll and carotenoid, are crucial for photosynthesis and photoprotection. Previous studies have shown that nitric oxide (NO) plays a dual role in plant photosynthesis. However, how pigment biosynthesis is suppressed by NO remains unclear. In this study, we generated NO-accumulated gsnor mutants, applied exogenous NO donors, and used a series of methods, including reverse transcription quantitative PCR, immunoblotting, chromatin immunoprecipitation, electrophoretic mobility shift, dual-luciferase, and NO content assays, to explore the regulation of photosynthetic pigment biosynthesis by NO in tomato. We established that both endogenous and exogenous NO inhibited pigment accumulation and photosynthetic capacities. High levels of NO stimulated the degradation of LONG HYPOCOTYL 5 (HY5) protein and further inactivated the transcription of genes encoding protochlorophyllide oxidoreductase C (PORC) and phytoene synthase 2 (PSY2)—two enzymes that catalyze the rate-limiting steps in chlorophyll and carotenoid biosynthesis. Our findings provide a new insight into the mechanism of NO signaling in modulating HY5-mediated photosynthetic pigment biosynthesis at the transcriptional level in tomato plants.

Implication of serum Nitric oxide and IgE in urinary schistosomiasis: The case of Bamendjing, Cameroon

BackgroundUrinary schistosomiasis (US), also known as bilharziasis is a waterborne parasitic infection most common in rural areas of developing countries. The infection is associated with haematuria but little or nothing is known about its association with other urinary parameters, induction of Nitric Oxide (NO) and IgE production around the Bamendjing Dam an area highly suspected of Schistosoma haematobium infections. The study sought to address this problem for possible future interventions and control. Materials and methodsUrine samples were collected from 301 randomly selected participants and analysed for the presence of S. haematobium using the syringe filtration concentration method. Sera from patients infected with S. haematobium and also from some uninfected individuals were analysed for IgE and NO using ELISA and colorimetric methods respectively. FindingsThe results showed a prevalence of 16.9% (51/301). Sixty percent of the 49 patients with nitrite in their urine, were infected with urinary schistosomiasis (US) (30/49; p = 0.00). Meanwhile only 40% of the 15 patients with bilirubinuria were infected with US (6/15; p = 0.0241). The risk of patients with US having leucocytes and nitrites was high (OR of 1.3 and 1.7 respectively). Total IgE serum levels were significantly higher in patients with US (648.872 ± 223.142) compared to uninfected individuals (275.682 ± 181.674) (p = 0.00). Infected persons had heightened mean NO levels (2,583,617.647 ± 1,100,678.786) than non-infected participants (1,689,766.667 ± 1,163,084.729). Urinary Schistosomiasis in association with urinary parameters had a significant impact on mean IgE levels (F = 4.248, p = 0.022). Patients infected with Urinary Schistosomiasis alone had significantly higher mean total IgE levels than non-infected participants (p = 0.004). ConclusionApart from haematuria, this study has demonstrated that Urinary Schistosomiasis is prevalent among inhabitants around the Bamendjing Dam and results in an increase of other urine parameters such as leucocytes and nitrates and high levels of serum NO and total serum IgE in patients. These parameters are important in the screening of patients for treatment and control of urinary schistosomiasis.

INOS is essential to maintain a protective Th1/Th2 response and the production of cytokines/chemokines against Schistosoma japonicum infection in rats.

Humans and a wide range of mammals are generally susceptible to Schistosoma infection, while some rodents such as Rattus rats and Microtus spp are not. We previously demonstrated that inherent high expression levels of nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), plays an important role in blocking the growth and development of Schistosoma japonicum in wild-type rats. However, the potential regulatory effects of NO on the immune system and immune response to S. japonicum infection in rats are still unknown. In this study, we used iNOS-knockout (KO) rats to determine the role of iNOS-derived NO in the immune system and immunopathological responses to S. japonicum infection in rats. Our data showed that iNOS deficiency led to weakened immune activity against S. japonicum infection. This was characterized by the impaired T cell responses and a significant decrease in S. japonicum-elicited Th2/Th1 responses and cytokine and chemokine-producing capability in the infected iNOS-KO rats. Unlike iNOS-KO mice, Th1-associated cytokines were also decreased in the absence of iNOS in rats. In addition, a profile of pro-inflammatory and pro-fibrogenic cytokines was detected in serum associated with iNOS deficiency. The alterations in immune responses and cytokine patterns were correlated with a slower clearance of parasites, exacerbated granuloma formation, and fibrosis following S. japonicum infection in iNOS-KO rats. Furthermore, we have provided direct evidence that high levels of NO in rats can promote the development of pulmonary fibrosis induced by egg antigens of S. japonicum, but not inflammation, which was negatively correlated with the expression of TGF-β3. These studies are the first description of the immunological and pathological profiles in iNOS-KO rats infected with S. japonicum and demonstrate key differences between the responses found in mice. Our results significantly enhance our understanding of the immunoregulatory effects of NO on defensive and immunopathological responses in rats and the broader nature of resistance to pathogens such as S. japonicum.

5-Azacytidine Inhibits the Activation of Senescence Program and Promotes Cytotoxic Autophagy during Trdmt1-Mediated Oxidative Stress Response in Insulinoma β-TC-6 Cells.

5-Azacytidine (5-azaC), a methyltransferase inhibitor and anticancer drug, can promote several cellular stress responses such as apoptosis, autophagy, and senescence. The action of 5-azaC is complex and can be modulated by dose, time of treatment, and co-administration with oxidants. Insulinoma is a rare pancreatic neuroendocrine tumor with limited chemotherapeutic options. In the present study, two cellular models of insulinoma were considered, namely NIT-1 and β-TC-6 mouse cells, to evaluate the effects of 5-azaC post-treatment during hydrogen peroxide-induced oxidative stress. 5-azaC attenuated the development of oxidant-induced senescent phenotype in both cell lines. No pro-apoptotic action of 5-azaC was observed in cells treated with the oxidant. On the contrary, 5-azaC stimulated an autophagic response, as demonstrated by the increase in phosphorylated eIF2α and elevated pools of autophagic marker LC3B in oxidant-treated β-TC-6 cells. Notably, autophagy resulted in increased necrotic cell death in β-TC-6 cells with higher levels of nitric oxide compared to less affected NIT-1 cells. In addition, 5-azaC increased levels of RNA methyltransferase Trdmt1, but lowered 5-mC and m6A levels, suggesting Trdmt1 inhibition. We postulate that the 5-azaC anticancer action may be potentiated during oxidative stress conditions that can be used to sensitize cancer cells, at least insulinoma cells, with limited drug responsiveness.

Synthesis of harmine-nitric oxide donor derivatives as potential antitumor agents

To further improve the anti-tumor activity of Harmine (HM), we took the hybridization approach and synthesized harmine derivatives-furoxan hybrids containing nitric oxide (NO) releasing parts by connecting NO donors with anti-tumor active fragments to harmine. Then, the synthesized compounds were evaluated for their in vitro cytotoxicity against five human cancer cell lines. Among them, compound 10 was found to have the strongest antiproliferative activity against HepG2 (IC50 = 1.79 µM). In addition, compound 10 produced high levels of NO in vitro, verifying that the release of NO was closely correlated to the antiproliferative activity. In addition, Compound 10 also showed good plasma stability. Finally, we also preliminarily investigated the acute toxicity of compound 10 in mice and assessed the absorption of compound 10 by Caco-2 cell permeability assay. In brief, the remarkable biological characteristics of the new harmine derivatives-furoxan hybrids may make them promising candidates for human cancer intervention.

Treatment with Commelina communis Extract Exerts Anti-inflammatory Effects in Murine Macrophages via Modulation of the Nuclear Factor-κB Pathway.

The incidence of severe inflammatory diseases caused by chronic inflammation has increased owing to unprecedented changes brought about by industrialization. In this study, we aimed to assess the effect of treatment of lipopolysaccharide- (LPS-) induced murine macrophages with Commelina communis Linne extract (CCE) on synthesis of nitric oxide (NO), hypersecretion of proinflammatory cytokines, intranuclear transition of the p65 subunit of nuclear factor- (NF-) κB, and degradation of the NF-κB inhibitor IκBα. Notably, CCE treatment did not affect cell viability even at a final concentration of 1.5 mg/mL. At a high concentration of CCE, the LPS-induced high levels of NO, tumor necrosis factor-α, interleukin- (IL-) 1β, and IL-6 were decreased via downregulation of inducible NO synthase and proinflammatory cytokine mRNA expression. Furthermore, phosphorylation of IκBα was significantly decreased upon CCE treatment, and the intranuclear transition of NF-κB p65 triggered by LPS was inhibited at a high concentration of CCE. Polyphenols and flavonoids, secondary metabolites in CCE that regulate the NF-κB pathway, may be responsible for its anti-inflammatory activity. We suggest that CCE has anti-inflammatory effects related to suppression of the NF-κB pathway and can be used to treat chronic inflammation.

Abstract P5-10-08: Inflammation drives NOS2-Akt2 signaling in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is aggressive and represents nearly 20% of all breast cancer cases. It is associated with poor prognosis due to its high metastatic capabilities and lack of targeted therapeutic treatments. To improve TNBC treatment, our goal is to better understand the molecular mechanisms that drive TNBC. Recently, inducible nitric oxide synthase (NOS2), a nitric oxide (NO) producing enzyme, has emerged as a key player in TNBC progression. High NOS2 expression in TNBC patient correlates with poor prognosis marked with increased metastasis and disease recurrence. NOS2 catalyzes L-arginine and dioxygen (O2) to produce citrulline and high, sustained levels of NO which has been shown to alter the activity of numerous signaling pathways. Depending on NO concentration and temporal and spatial levels, it has been reported to have both tumor-promoting and tumor-suppressing functions. However, the mechanism by which NOS2 drives TNBC is unclear. Akt is major oncogenic signaling pathway that regulates various aspects of tumor progression, and it has been shown that NO can activate Akt activity. The Akt family has three members, Akt1, Akt2, and Akt3, that seem to have similar and non-redundant functions in cancer. We have discovered for the first time that NOS2 specifically activates Akt2 in TNBC cells. Thus, we hypothesize that Akt2 plays a major role in NOS2 driven TNBC. Inflammatory cytokines (IFN-γ, IL-1β, and TNF-α) highly induce NOS2 in mouse 4T1 TNBC cells. We developed a real-time NO detection assay to measure NO levels in 4T1 cells and confirmed that these cells produce high levels of NO. By pharmacologically and genetically inhibiting NOS2 in the 4T1 cells, we found that NOS2 suppresses Akt2 phosphorylation but not the total levels. Furthermore, NOS2 had no effect on Akt1. The decrease in Akt2 phosphorylation was accompanied by inhibition of its downstream targets, GSK3β and FOXO3a, suggesting that NOS2 is regulating Akt2 activity. Conversely, treatment of 4T1 and human MDA-MD-231 TNBC cells with DETA/NO, a slow releasing NO donor, resulted in increased Akt2 phosphorylation with no effect on Akt1. Akt2 has been shown to promote migration, invasion, and metastasis. Using 4T1 cells, we are presently studying the function of NOS2-Akt2 signaling in TNBC. We have orthotopically injected 4T1 cells into immunocompetent mice and have found that 4T1 tumors express high levels of NOS2 as well as phosphorylated Akt2. We have knocked out NOS2 in the 4T1 cells to examine the effects of NOS2 inhibition in the mouse tumor model. Understanding the link between NOS2 and Akt2 will give further understanding on how NOS2 drives TNBC. Citation Format: Su Chung, William Montfort. Inflammation drives NOS2-Akt2 signaling in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-08.