Cell-type specific anti-cancerous effects of nitro-oleic acid and its combination with gamma irradiation.

Nitro-fatty acids (NFAs) are endogenous lipid mediators capable of post-translational modifications of selected regulatory proteins. Here, we investigated the anti-cancerous effects of nitro-oleic acid (NO2OA) and its combination with gamma irradiation on different cancer cell lines. The effects of NO2OA on cell death, cell cycle distribution, or expression of p21 and cyclin D1 proteins were analyzed in cancer (A-549, HT-29 and FaDu) or normal cell lines (HGF, HFF-1). Dose enhancement ratio at 50 % survival fraction (DERIC50) was calculated for samples pre-treated with NO2OA followed by gamma irradiation. NO2OA suppressed viability and induced apoptotic cell death. These effects were cell line specific but not in general selective for cancer cells. HT-29 cell line exerted higher sensitivity toward NO2OA treatment among cancer cell lines tested: induction of cell cycle arrest in the G2/M phase was associated with an increase in p21 and a decrease in cyclin D1 expression. Pre-treatment of HT-29 cells with NO2OA prior irradiation showed a significantly increased DERIC50, demonstrating radiosensitizing effects. In conclusion, NO2OA exhibited potential for combined chemoradiotherapy. Our results encourage the development of new NFAs with improved features for cancer chemoradiation.

Administration of nitro-oleic acid mitigates radiation-induced hematopoietic injury in mice.

AIMS: Limited number of agents that provide protection against hematopoietic acute radiation syndrome led us to the evaluation of nitro-oleic acid (NO2OA) as a potential protector/mitigator against radiation-induced hematopoietic injury in C57/BL6 mice. MATERIALS AND METHODS: NO2OA was administered before and after sub-lethal total body irradiation (TBI) and hematological parameters were evaluated 3 or 7 days after TBI. KEY FINDINGS: Our results show that NO2OA significantly increase bone marrow cellularity including the granulocyte-macrophage colony-forming cells and erythroid progenitors on the 3rd day after TBI. In addition, NO2OA enhanced recovery of white blood cells (lymphocytes, neutrophils, and monocytes) in peripheral blood 7 days after irradiation. These effects may be in part attributed to NO2OA-induced granulocyte colony-stimulating factor production after TBI. On the other hand, radiation-induced impairment of peripheral red blood cells, hemoglobin, and platelets were not affected with NO2OA treatment up to 7 days post TBI. SIGNIFICANCE: In conclusion, our data show that NO2OA significantly protects hematopoiesis after irradiation, and thus showed a high potential to act as an agent for medical radiation countermeasure.

Analysis of hematological parameters in rheumatoid arthritis patients receiving biological therapy: contribution to prevention of avoidable hematological complications.

Administration of biological therapy (BT) in rheumatoid arthritis (RA) patients is often associated with hematological complications, which result in switching among therapies. Thus, there is an instant need for suitable screening parameters that will help to individualize the therapy and minimize the onset of adverse effects. We analyzed the hematological profile of 99 RA patients receiving TNFα (Adalimumab - ADA, Golimumab - GOL, Etanercept - ETA) or IL-6 receptor (Tocilizumab - TCZ) inhibitors in order to find possible indicators to improve personalization of RA therapy. BTs significantly affect the levels of observed hematological parameters. In contrast to TNF-α inhibitors, TCZ normalized almost all monitored hematological parameters to values of healthy donors. Only GOL from the TNF-α inhibitors studied, was able to normalize neutrophil counts, as well as platelet indicators. Importantly, effects on the blood parameters (e.g. lymphocytes or platelet count) differ even within the same therapeutic group (anti-TNFα). Variable effects of individual biological agents in RA treatment point to importance to evaluate the patient's hematological profile to improve the selection of suitable BT. It will help to personalize the administration of BT and prevent unnecessary switching from an effective therapy just because of provocation of avoidable hematological complications.

Nitro-Oleic Acid Inhibits Stemness Maintenance and Enhances Neural Differentiation of Mouse Embryonic Stem Cells via STAT3 Signaling.

Nitro-oleic acid (NO2-OA), pluripotent cell-signaling mediator, was recently described as a modulator of the signal transducer and activator of transcription 3 (STAT3) activity. In our study, we discovered new aspects of NO2-OA involvement in the regulation of stem cell pluripotency and differentiation. Murine embryonic stem cells (mESC) or mESC-derived embryoid bodies (EBs) were exposed to NO2-OA or oleic acid (OA) for selected time periods. Our results showed that NO2-OA but not OA caused the loss of pluripotency of mESC cultivated in leukemia inhibitory factor (LIF) rich medium via the decrease of pluripotency markers (NANOG, sex-determining region Y-box 1 transcription factor (SOX2), and octamer-binding transcription factor 4 (OCT4)). The effects of NO2-OA on mESC correlated with reduced phosphorylation of STAT3. Subsequent differentiation led to an increase of the ectodermal marker orthodenticle homolog 2 (Otx2). Similarly, treatment of mESC-derived EBs by NO2-OA resulted in the up-regulation of both neural markers Nestin and ß-Tubulin class III (Tubb3). Interestingly, the expression of cardiac-specific genes and beating of EBs were significantly decreased. In conclusion, NO2-OA is able to modulate pluripotency of mESC via the regulation of STAT3 phosphorylation. Further, it attenuates cardiac differentiation on the one hand, and on the other hand, it directs mESC into neural fate.

5-Nitro-2,4-Dichloropyrimidine as an Universal Model for Low-Energy Electron Processes Relevant for Radiosensitization.

We report experimental results of low-energy electron interactions with.

How to measure the immunosuppressive activity of MDSC: assays, problems and potential solutions.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of mononuclear and polymorphonuclear myeloid cells, which are present at very low numbers in healthy subjects, but can expand substantially under disease conditions. Depending on disease type and stage, MDSC comprise varying amounts of immature and mature differentiation stages of myeloid cells. Validated unique phenotypic markers for MDSC are still lacking. Therefore, the functional analysis of these cells is of central importance for their identification and characterization. Various disease-promoting and immunosuppressive functions of MDSC are reported in the literature. Among those, the capacity to modulate the activity of T cells is by far the most often used and best-established read-out system. In this review, we critically evaluate the assays available for the functional analysis of human and murine MDSC under in vitro and in vivo conditions. We also discuss critical issues and controls associated with those assays. We aim at providing suggestions and recommendations useful for the contemporary biological characterization of MDSC.

A Concise Synthesis of Forskolin.

A 24-step synthesis of (±)-forskolin is presented, which delivered hundred milligram quantities of this complex diterpene in one pass. Transformations key to our approach include: a) a strategic allylic transposition, b) stepwise assembly of a sterically encumbered isoxazole ring, and c) citric acid-modified Upjohn dihydroxylation of a resilient tetrasubstituted olefin. The developed route has exciting potential for the preparation of new forskolin analogues inaccessible by semisynthesis.

Nitro-oleic acid regulates growth factor-induced differentiation of bone marrow-derived macrophages.

Many diseases accompanied by chronic inflammation are connected with dysregulated activation of macrophage subpopulations. Recently, we reported that nitro-fatty acids (NO2-FAs), products of metabolic and inflammatory reactions of nitric oxide and nitrite, modulate macrophage and other immune cell functions. Bone marrow cell suspensions were isolated from mice and supplemented with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with NO2-OA for different times. RAW 264.7 macrophages were used for short-term (1-5min) experiments. We discovered that NO2-OA reduces cell numbers, cell colony formation, and proliferation of macrophages differentiated with colony-stimulating factors (CSFs), all in the absence of toxicity. In a case of GM-CSF-induced bone marrow-derived macrophages (BMMs), NO2-OA acts via downregulation of signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) activation. In the case of M-CSF-induced BMMs, NO2-OA decreases activation of M-CSFR and activation of related PI3K and ERK. Additionally, NO2-OA also attenuates activation of BMMs. In aggregate, we demonstrate that NO2-OA regulates the process of macrophage differentiation and that NO2-FAs represent a promising therapeutic tool in the treatment of inflammatory pathologies linked with increased accumulation of macrophages in inflamed tissues.

Pharmacological intervention with oxidative burst in human neutrophils.

In this study we investigated the effect of five therapeutically used drugs and four natural polyphenolic compounds on the mechanism of oxidative burst of human neutrophils concerning their participation in the generation of reactive oxygen species (ROS). The compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin > quercetin > curcumin > arbutin > dithiaden > carvedilol. The generation of intracellular reactive oxygen species in isolated neutrophils decreased in the same rank order, while carvedilol was ineffective. Scavenging of extracellular oxygen radicals followed the rank order of potency: N-feruloylserotonin > curcumin > quercetin > dithiaden. Arbutin and carvedilol had no effect. All compounds tested increased the activity of caspase-3 in cell-free system indicating a positive effect on apoptosis of neutrophils. Activation of protein kinase C was significantly decreased by dithiaden, curcumin, quercetin and N-feruloylserotonin. Carvedilol, dithiaden, quercetin and arbutin reduced activated neutrophil myeloperoxidase release more significantly compared with their less pronounced effect on superoxide generation The presented results are indicative of pharmacological intervention with neutrophils in pathological processes. Of particular interest was the effect of natural compounds. Intracellular inhibition of oxidative burst in isolated neutrophils by the drugs tested and natural antioxidants has to be further analysed since ROS play an important role in immunological responses of neutrophils.

The effects of berberine on reactive oxygen species production in human neutrophils and in cell-free assays.

The health benefits of berberine have been recognized for years. Even so, its effects on human neutrophils, the first line of immune defense, have not been reported. The purpose of this study was to investigate the effects of berberine on the human neutrophil oxidative burst. Reactive oxygen species production was analyzed by luminol-enhanced chemiluminescence. The analysis was performed in spontaneous and stimulated (phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP)) whole blood and isolated neutrophils in the presence or absence of berberine. The effects of berberine on oxidant production in cell-free assays were evaluated using luminescence (H2O2-peroxidase-luminol) and fluorescence (Oxygen Radical Absorbance Capacity - ORAC) techniques. Berberine decreased the production of reactive oxygen species in human whole blood and isolated neutrophils stimulated with either PMA or OZP with a different efficiency (EC50 was 69 µM and 197 µM for PMA and OZP, respectively). The effect was more pronounced in isolated neutrophils. Cell-free assays showed the antioxidant activity of berberine against peroxyl radicals and hydrogen peroxide. Based on our results, we suggest that the effects of berberine on reactive oxygen species production in human neutrophils are due to its antioxidant activity.

HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells.

Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α)-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC) in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7) and with a decreasing percentage of myosin heavy chain α and ß, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and ß) and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha). These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro.

Lung Neutrophilia in Myeloperoxidase Deficient Mice during the Course of Acute Pulmonary Inflammation.

Systemic inflammation accompanying diseases such as sepsis affects primarily lungs and induces their failure. This remains the most common cause of sepsis induced mortality. While neutrophils play a key role in pulmonary failure, the mechanisms remain incompletely characterized. We report that myeloperoxidase (MPO), abundant enzyme in neutrophil granules, modulates the course of acute pulmonary inflammatory responses induced by intranasal application of lipopolysaccharide. MPO deficient mice had significantly increased numbers of airway infiltrated neutrophils compared to wild-type mice during the whole course of lung inflammation. This was accompanied by higher levels of RANTES in bronchoalveolar lavage fluid from the MPO deficient mice. Other markers of lung injury and inflammation, which contribute to recruitment of neutrophils into the inflamed lungs, including total protein and other selected proinflammatory cytokines did not significantly differ in bronchoalveolar lavage fluid from the wild-type and the MPO deficient mice. Interestingly, MPO deficient neutrophils revealed a decreased rate of cell death characterized by phosphatidylserine surface expression. Collectively, the importance of MPO in regulation of pulmonary inflammation, independent of its putative microbicidal functions, can be potentially linked to MPO ability to modulate the life span of neutrophils and to affect accumulation of chemotactic factors at the inflammatory site.

Selective inhibition of extracellular oxidants liberated from human neutrophils--A new mechanism potentially involved in the anti-inflammatory activity of hydroxychloroquine.

Hydroxychloroquine is used in the therapy of rheumatoid arthritis or lupus erythematosus. Although these diseases are often accompanied by activation of neutrophils, there are still few data relating to the impact of hydroxychloroquine on these cells. We investigated the effect of orally administered hydroxychloroquine on neutrophil oxidative burst in rats with adjuvant arthritis. In human neutrophils, extra- and intracellular formation of oxidants, mobilisation of intracellular calcium and the phosphorylation of proteins regulating NADPH oxidase assembly were analysed. Administration of hydroxychloroquine decreased the concentration of oxidants in blood of arthritic rats. The inhibition was comparable with the reference drug methotrexate, yet it was not accompanied by a reduction in neutrophil count. When both drugs were co-applied, the effect became more pronounced. In isolated human neutrophils, treatment with hydroxychloroquine resulted in reduced mobilisation of intracellular calcium, diminished concentration of external oxidants and in decreased phosphorylation of Ca(2+)-dependent protein kinase C isoforms PKCα and PKCßII, which regulate activation of NADPH oxidase on plasma membrane. On the other hand, no reduction was observed in intracellular oxidants or in the phosphorylation of p40(phox) and PKCδ, two proteins directing the oxidase assembly to intracellular membranes. Hydroxychloroquine reduced neutrophil-derived oxidants potentially involved in tissue damage and protected those capable to suppress inflammation. The observed effects may represent a new mechanism involved in the anti-inflammatory activity of this drug.

Molecular pharmacology of antihistamines in inhibition of oxidative burst of professional phagocytes.

Antihistamines of the H1and H3/H4groups interfere with oxidative burst of human professional phagocytes in vitro. In the concentration of 10 µM, H1antihistamines of the 1st and 2nd generation inhibited oxidative burst of human neutrophils in the rank order of potency: dithiaden > loratadine > brompheniramine > chlorpheniramine > pheniramine. Of the H1antihistamines, the most effective was dithiaden in suppressing oxidative burst of whole human blood and dose-dependently the chemiluminescence of isolated neutrophils at extra- and intracellular level. Inhibition of free oxygen radical generation in isolated neutrophils by dithiaden resulted from the inhibition of protein kinase C activation. The potentiation of recombinant caspase-3 by dithiaden is supportive of the antiinflammatory effect of dithiaden and suggestive of increasing the apoptosis of professional phagocytes. Of the H3/H4antihistamines, the most effective was JNJ7777120 in decreasing chemiluminescence in whole blood and also at extra- and intracellular sites of isolated neutrophils. JNJ 10191584 and thioperamide were less effective and the latter significantly potentiated free oxygen radical generation intracellularly. The results demonstrated that, compared with the H3/H4antihistamines investigated, H1antihistamines were much more potent in inhibiting free oxygen radical generation in human professional phagocytes. This finding should be taken into account therapeutically.

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils.

The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 µM), but not histamine (0.1-1 µM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 µM) or JNJ 28610244 (0.1-10 µM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 µM histamine and 10 µM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

On the molecular pharmacology of resveratrol on oxidative burst inhibition in professional phagocytes.

Resveratrol-3,5,4'-trihydroxystilbene-possesses antioxidant activities in vitro. It dose-dependently inhibited the generation of peroxyl, hydroxyl, peroxides, and lipid peroxidation products in cell free systems. Oxidative burst of whole human blood stimulated with PMA, fMLP, OpZ, and A23187 was inhibited in a concentration-dependent way, indicating suppression of both receptor and nonreceptor activated chemiluminescence by resveratrol. Results from isolated human neutrophils revealed that resveratrol was active extracellularly as well as intracellularly in inhibiting the generation of reactive oxygen species. Liberation of ATP and analysis of apoptosis showed that in the concentration of 100 µM, resveratrol did not change the viability and integrity of isolated neutrophils. Western blot analysis documented that resveratrol in concentrations of 10 and 100 µM significantly decreased PMA-induced phosphorylation of PKC α/ß II. Dose-dependent inhibition of nitrite production and iNOS protein expression in RAW 264.7 cells indicated possible interference of resveratrol with reactive nitrogen radical generation in professional phagocytes. The results suggest that resveratrol represents an effective naturally occurring substance with potent pharmacological effect on oxidative burst of human neutrophils and nitric oxide production by macrophages. It should be further investigated for its pharmacological activity against oxidative stress in ischaemia reperfusion, inflammation, and other pathological conditions, particularly neoplasia.

Colloidal polyaniline dispersions: antibacterial activity, cytotoxicity and neutrophil oxidative burst.

Polyaniline colloids rank among promising application forms of this conducting polymer. Cytotoxicity, antibacterial activity, and neutrophil oxidative burst tests were performed on cells treated with colloidal polyaniline dispersions. The antibacterial effect of colloidal polyaniline against gram-positive and gram-negative bacteria was most pronounced for Bacillus cereus and Escherichia coli, with a minimum inhibitory concentration of 3,500 µg mL(-1). The data recorded on human keratinocyte (HaCaT) and a mouse embryonic fibroblast (NIH/3T3) cell lines using an MTT assay and flow cytometry indicated a concentration-dependent cytotoxicity of colloid, with the absence of cytotoxic effect at around 150 µg mL(-1). The neutrophil oxidative burst test then showed that colloidal polyaniline, in concentrations <150 µg mL(-1), was not able to stimulate the production of reactive oxygen species in neutrophils and whole human blood. However, it worked efficiently as a scavenger of those already formed.

Study of possible mechanisms involved in the inhibitory effects of coumarin derivatives on neutrophil activity.

To specify the site of action of the synthetic coumarin derivatives 7-hydroxy-3-(4'-hydroxyphenyl) coumarin (HHC) and 7-hydroxy-3-(4'-hydroxyphenyl) dihydrocoumarin (HHDC), we evaluated their effects on extra- and intracellular reactive oxygen species (ROS) formation in phorbol-myristate-13-acetate (PMA) stimulated human neutrophils. We studied also the effects of HHC and HHDC on possible molecular mechanisms which participate in the activation of NADPH oxidase, that is, on PKC activity, on phosphorylation of some PKC isoforms (α, ßII, and δ), and on phosphorylation of the NADPH oxidase subunit p40(phox). Without affecting cytotoxicity, both coumarines tested were effective inhibitors/scavengers of ROS produced by neutrophils on extracellular level. HHC markedly diminished oxidant production and also, intracellularly, decreased PKC activity and partly phosphorylation of PKCα, ßII. On the other hand, we did not observe any effect of coumarin derivatives on phosphorylation of PKC δ and on phosphorylation of the NADPH oxidase subunit p40(phox), which were suggested to be involved in the PMA-dependent intracellular activation process. In agreement with our previous findings, we assume that the different molecular structures of HHC and HHDC with their different physicochemical and free radical scavenging characteristics are responsible for their diverse effects on the parameters tested.

The effects of pterostilbene on neutrophil activity in experimental model of arthritis.

It has been demonstrated that pterostilbene inhibits reactive oxygen species production in neutrophils in vitro. However, little is known about its effects on neutrophils during inflammation in vivo. In this study, the effect of pterostilbene on neutrophil activity was investigated in experimental arthritis model. Lewis rats were injected by a single intradermal injection of heat-killed Mycobacterium butyricum in Freund's adjuvant to develop arthritis. Another group of arthritic animals received pterostilbene 30 mg/kg, daily, p.o. The number and activity of neutrophils in blood were measured on a weekly basis during the whole experiment. Moreover, the total radical trapping potential in plasma was measured at the end of the experiment. In the pterostilbene treated arthritic group, the treatment significantly lowered the number of neutrophils in blood on days 14 and 21 without significant downregulation of neutrophil oxidative burst. Pterostilbene nonsignificantly increased total radical trapping potential in arthritic animals. These results indicate that the promising effects of pterostilbene on reactive oxygen species operate by different mechanisms in vitro and in the animal model of inflammation. In conclusion, the positive effects of pterostilbene in the model of arthritis may be attributed to regulation of neutrophil number.

The natural stilbenoid piceatannol decreases activity and accelerates apoptosis of human neutrophils: involvement of protein kinase C.

Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role) were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40(phox)-a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular) membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC)-the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α , ß II, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.