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