Nitrite-Mediated Modulation of HL-60 Cell Cycle and Proliferation: Involvement of Cyclin-Dependent Kinase 2 Activation

ABSTRACT Recent research suggests the vivid possibility of using nitrite therapy against various pathological conditions. Moreover, chronic nitrite therapy offers protection against ischemia and augments endothelial cell proliferation through unknown mechanisms. Nitrite-mediated augmentation in the number of circulating neutrophils has also been reported; however, the exact mechanism is not known. In the present study, we have investigated the effect of nitrite (0.5-10 mM) on the proliferation of the neutrophilic cell line HL-60 and also explored the underlying mechanism. Treatment of HL-60 cells with sodium nitrite (0.5-5 mM) led to an increase in cell proliferation, which was confirmed by cell cycle analysis and 5-bromo-2-deoxyuridine and thymidine incorporation, whereas cells accumulated in the G 0 /G 1 phase after treatment with 10 mM nitrite. Experiments on the synchronized cells exhibited similar effect, which seems to be nitric oxide (NO)-dependent, because carboxyl-1H-imidazol-1-yloxy,2-(4-carboxyphenyl)-4,5-dihydro 4,4,5,5-tetramethyl-3-oxide abolished nitrite-mediated proliferative effect. Moreover, the NO donor sodium nitroprusside at micromolar concentrations also exhibited similar effects. Nitrite induced augmentation in S phase, and intracellular reactive oxygen species (ROS) generation was prevented by ROS scavenger/inhibitors. Moreover, mitochondrial blockers, rotenone and antimycin A, also reduced nitrite-mediated cell proliferation. Assessment of the cell cycle regulators cyclin-dependent kinase 2 (Cdk2), Cdk4, cyclin A, cyclin D, cyclin E, and p21 suggested augmentation in the expression and interaction of Cdk2/cyclin E and Cdk2 activity, whereas p21 was down-regulated. Indeed proliferative effect of nitrite was blocked by roscovitine, a Cdk2 inhibitor. The results obtained demonstrate that the proliferative effect of nitrite on HL-60 cells seems to be NO-mediated, redox-sensitive, and Cdk2 activation-dependent, warranting detailed studies before initiating its clinical use

Anti-thrombotic efficacy of S007-867: Pre-clinical evaluation in experimental models of thrombosis in vivo and in vitro.

Pharmacological inhibition of platelet collagen interaction is a promising therapeutic strategy to treat intra-vascular thrombosis. S007-867 is a novel synthetic inhibitor of collagen-induced platelet aggregation. It has shown better antithrombotic protection than aspirin and clopidogrel with minimal bleeding tendency in mice. The present study is aimed to systematically investigate the antithrombotic efficacy of S007-867 in comparison to aspirin and clopidogrel in vivo and to delineate its mechanism of action in vitro. Aspirin, clopidogrel, and S007-867 significantly reduced thrombus weight in arterio-venous (AV) shunt model in rats. In mice, following ferric chloride induced thrombosis in either carotid or mesenteric artery; S007-867 significantly prolonged the vessel occlusion time (1.2-fold) and maintained a sustained blood flow velocity for >30 min. Comparatively, clopidogrel showed significant prolongation in TTO (1.3-fold) while aspirin remained ineffective. Both S007-867 and aspirin did not alter bleeding time in either kidney or spleen injury models, and thus maintained hemostasis, while clopidogrel showed significant increase in spleen bleeding time (1.7-fold). The coagulation parameters namely thrombin time, prothrombin time or activated partial thromboplastin time remained unaffected even at high concentration of S007-867 (300 µM), thus implying its antithrombotic effect to be primarily platelet mediated. S007-867 significantly inhibited collagen-mediated platelet adhesion and aggregation in mice ex-vivo. Moreover, when blood was perfused over a highly thrombogenic combination of collagen mimicking peptides like CRP-GFOGER-VWF-III, S007-867 significantly reduced total thrombus volume or ZV50 (53.4 ± 5.7%). Mechanistically, S007-867 (10-300 μM) inhibited collagen-induced ATP release, thromboxane A2 (TxA2) generation, intra-platelet [Ca+2] flux and global tyrosine phosphorylation including PLCγ2. Collectively the present study highlights that S007-867 is a novel synthetic inhibitor of collagen induced platelet activation, that effectively maintains blood flow velocity and delays vascular occlusion. It inhibits thrombogenesis without compromising hemostasis. Therefore, S007-867 may be further developed for the treatment of thrombotic disorders in clinical settings

Cdk2 nitrosylation and loss of mitochondrial potential mediate NO-dependent biphasic effect on HL-60 cell cycle

Nitric oxide (NO), a multifaceted signaling molecule, regulates a wide array of cell functions, including proliferation, differentiation, cytostasis, and apoptosis, which depend on the cell type and redox status. This study systematically explores the effects of NO donors on promyelocytic HL-60 cell proliferation and apoptosis. The NO donor DETA-NO modulated the HL-60 cell cycle in a biphasic manner. DETA-NO in lower concentrations (1-100 μM) had a proliferative effect as investigated by [3H]thymidine incorporation, BrdU labeling, and cell cycle analysis, whereas cells treated with higher concentrations (250 μM-1 mM) showed cytostasis, apoptosis, mitochondrial membrane potential loss, caspase-3 activity, and dUTP nick-end labeling. The proliferative effect of DETA-NO was NO dependent and redox sensitive, as the effect was abolished by cPTIO and DTT pretreatment, respectively. Expression of various cell cycle regulators such as Cdk2, cyclin B, and cyclin E was significantly augmented in cells treated with 10-50 μM DETA-NO. The proliferative effect of NO was blocked by roscovitine, a Cdk2 inhibitor. S-nitrosylation of Cdk2 and an increase in the Cdk2-associated kinase activity was observed for the first time in DETA-NO-treated cells. This study demonstrates that the DETA-NO-mediated biphasic effect was dependent on Cdk2 nitrosylation/activation and the loss of mitochondrial potential at low and high concentrations, respectively

Biphasic regulation of cell cycle by nitric oxide donors in promyelocytic HL-60 cell line

Nitric oxide (NO) regulates a wide array of cell functions, proliferation, cytostasis and apoptosis. Role of NO in cell proliferation is however is less explored, which has been suggested to be dependent on the NO concentration, cell type and intracellular redox status. Present study explores the effect of NO donors [DETA-NONOate (DETA-NO) and SNAP] on the promyelocytic HL-60 cells. NO induced proliferation of HL-60 cell at lower concentration (1-100 µM), which was evaluated by Thymidine incorporation, BrdU labelling and cell cycle analysis regardless of any apoptosis, while higher concentration (250µM-1mM) promoted apoptosis as evident by change in mitochondrial potential, Caspase activation, nick and PI labelling. To investigate further the mechanism involved, expression profile of various CDK/ cyclins including CDK2, CDK6, Cyclin A, Cyclin B, Cyclin D1, p15, p27 was monitored, Expression of Cyclin B, CDK2 was increased at 10-50 µM by DETA-NO, while most of these cell cycle regulators were reduced at 1 mM DETA-NO. This proliferative effect seems to be redox sensitive as effect was abolished by pretreatment of cells with DTT (2mM). NO (10-50µM) also augmented nitrosylation of various proteins suggesting nitrosylation of important targets. The results obtained thus suggest that NO in a concentration dependent manner modulated cell cycle in the HL-60 cells

Response of Populus deltoides Bartr. ex Marsh genes under elevated CO2 through Next-Generation Sequencing (NGS)

The impact of climate change has attracted considerable attention globally. Atmospheric Carbon Dioxide (CO2) is expected to increase to 900 μmol mol-1 from present level of 400 μmol mol-1 by the end of 21st century. CO2 is a greenhouse gas that leads climate change have significantly affected structure and function of the terrestrial ecosystem, global carbon, water balance, and also crop productivity. These responses of the plant appear by altering gene expression pattern of different genes involved in anabolic and catabolic processes. We have conducted a study to see the response of genes to elevated CO2 inside open top chambers on Populus deltoides. Onemonth- old ramets were exposed for 180 days to treatment (CO2 800 μmol mol-1) and control (CO2 ~400 μmol mol-1). After completion of treatment, leaf tissues were outsourced to Sci-genome for transcriptome sequencing. This study demonstrated, higher (1754) number of transcript expression in treatment (119,306) compared to control (121,060). Differential gene expression analysis shown 1951 transcripts were down regulated while 2603 transcripts up regulated and 159,982 transcripts have no significance in treatment. Our results show that plants growing in an environment where atmospheric CO2 is higher may alter plant adaptation, productivity, vegetation and ecosystem health by changing; the first, number of genes and second, altering gene expression patterns. Such behavior may be a good indicator of developing adaptation strategies of the plant

Nitric oxide synthesis in neutrophil precursor cells and effect of nitric oxide modulators on their maturation

Neutrophil maturation in bone marrow and their release in circulation have been shown to be modulated by nitric oxide (NO). Present study was undertaken to further assess the role of NO in neutrophil maturation in vitro. Neutrophil precursor cells were isolated from Sprague Dawley rat (200-250 g) bone marrow by Percoll density gradient in myeloblasts/promyelocytes rich band 3, myelocytes/metamyelocytes rich band 2 and band cells/segmented Neutrophils rich band 1, purity was confirmed by Giemsa staining/CD markers, NADPH oxidase and myeloperoxidase activity. Intracellular NO level (assessed by DAF-2DA), total nitrite (measured by Griess reagent) content, NOS activity (using 3H-L-arginine) and nNOS protein (Western blotting) were highest in band 1 and lowest in band 3. We also investigated effect of NO donor (SNP 1µM-1mM) and NOS inhibitor (L-NAME 1mM) on cell cycle by using Propidium iodide and BrdU staining during their culture for 24 hrs. SNP treated cells exhibited an increase in apoptosis, while viable cells showed a marginal increase in the S phase in band 2 and band 3. NOS inhibitor (L-NAME) however increased the survival of cells without any modulation of cell cycle. Results obtained indicate that NO content augment with neutrophil maturation and seems to play an important role in the maturation of neutrophils

NO synthesis and its regulation in the arachidonic-acid-stimulated rat polymorphonuclear leukocytes

Nitric oxide (NO) synthesis and free radical generation from polymorphonuclear leukocytes (PMNs) play an important role in several pathological conditions. In the present study, regulation of NO synthesis has been investigated in the unstimulated and arachidonic-acid (AA)-stimulated rat PMNs. L-Citrulline formation or nitrite content was used as a marker of NO synthesis, while AA-induced free radical generation was assessed by flow cytometry using a dye, 2',7'-dichlorofluoreseindiacetate. L-Citrulline formation in the unstimulated PMNs increased in a time-dependent manner for up to 120 min. The increase was significantly less (25-55%) in AA-stimulated PMNs at all the time points. AA-induced free radical generation was maximum during the first 15 min followed by a time-dependent decrease. Interestingly, similar experiments under hyperoxic conditions did not exhibit any decrease in L-citrulline and nitrite formation after AA stimulation even though the free radical generation further increased. Scavenging or inhibition of free radicals by several types of interventions increased NO generation from AA-stimulated PMNs. The results of the present study suggest that the availability of oxygen, a common substrate for both NADPH oxidase and NOS, can inversely affect the synthesis of NO and PMNs seem to prefer oxygen utilization over NO synthesis for free radical generation

New insight into the NO-mediated signaling to modulate neutrophil free radical generation

Neutrophils (PMNs) and nitric oxide (NO) play an important role in pathogen killing as well as in various pathological conditions associated with inflammation, hypoxia/ischemia and reoxygenation injury by generating reactive oxygen (ROS) and reactive nitrogen (RNS) species. Previous work from this lab has shown that NO modulates neutrophil NADPH-oxidase (NOX) activity and thus modulates ROS production. The present study has been undertaken to assess signalling mechanisms involved in NO mediated effect on PMNs by flowcytometry (DCF-DA, DHE, propidium iodide, CBA Flex array), biochemical (H2O2, hypochlorous acid [HOCl], nitryl chloride [NO2Cl], dityrosine) and molecular (Western blotting) measurements. NO donors, SNP and SNAP (10 µM to 1 mM) inhibited the NADPH-oxidase activity but enhanced DCF-DA fluorescence in a concentration and time dependent manner, with no adverse effect on the cell viability. The effect was significantly blocked by NOS inhibitors and was partially blocked by ERK and MPO inhibitors. NO at the concentrations used seems to activate futile cycle generating peroxynitrite (ONOO)/NO3−, which subsequently uncouples NOS and generate superoxide radicals and H2O2. H2O2 thus formed leads to the formation of HOCl by MPO catalysis, which further reacts with nitrite (NO2−) to generate NO2Cl, a more potent oxidant and nitrating agent. The results obtained thus suggest that NO mediated augmentation in neutrophil free radical production involves ERK signalling