Inhibition of Cancer and Drug-Associated Thrombosis Using Novel Inhibitors of NF-κB and Oxidative Stress Pathways.

Abstract

Background: Venous thromboemblism is associated with a systemic hypercoagulable state that is secondary to tumor or proinflammatory activation of procoagulant mechanisms, as well as down-regulation of anticoagulant mechanisms. In the present investigation, the potential antithrombotic efficacy and mechanism for OT-304 analogs, novel small molecules targeting NFkB and oxidative stress pathways, was examined in various cellular systems.Methods: Human monocytic cells were incubated with OT-304 analogs for 3 hours and then stimulated with LPS (25 ng/ml) for another 3 hours. Real-time PCR for NF-κB (P50/P65 complex) nuclear translocation dynamics, TNF-alpha, EGR1, and tissue factor (TF) was then performed. Additionally, the effect of OT-304 analogs on oxidative stress, inflammation, and procoagulant pathways in human monocytes and endothelial cells were performed using Affymatrix Microarray. Furthermore, the anticoagulant efficacy of OT-304 analogs as compared to classical anticoagulants such as Low Molecular weight Heparins (LMWH) was determined in proinflammatory-mediated hypercoagulable state, cancer, and/or chemotherapy-associated platelet/fibrin clot formation assays using thrombelastography (TEG). TEG was performed using blood from healthy volunteers. Clot dynamics were initiated using different activators of coagulation that included different cancer types, different chemotherapeutic agents, and standard anti-angiogenesis agents such as bevacizumab, ranibizumab and others in human blood. Additionally, effects of OT-304 analogs as compared to unfractionated heparin and the LMWH enoxaparin on activated partial thromboplastin time (aPTT) in human plasma was carried out using a fibrometric method.Results: OT-304 analogs effectively inhibited NF-κB and oxidative stress pathways leading to down-regulation of cytokines and chemokines, including TNF-alpha. OT-304 analogs effectively inhibited EGR1, which was associated with down-regulation of the key procoagulant mediator tissue factor (TF). In human blood, OT-304 effectively inhibited hypercoagulation and prothrombotic states mediated by proinflammatory stimuli including LPS, NF-κB activators such as ceramide, different cancer types such as pancreatic, glioma and lung cancer, different chemotherapeutic agents such as doxorubicin and etopocide, and angiogenesis inhibitors such as bevacizumab (Avastin) or ranibizumab (Lucentis) as evident from the inhibition of platelet-fibrin clot dynamics. The IC50 required for blocking cancer or drug-associated thrombosis ranged from 1–3 uM. OT-304 analogs did not have any effect on aPTT up to concentrations ranging from 1– 100 uM in human plasma. In contrast, heparin or LMWH resulted in a dose-dependent (0.001 – 1 ug/ml) increase in aPTT. These data suggest OT-304 effectively inhibits prothrombotic events, regardless of the stimulus, with potentially no effect on hemostasis.Conclusions: OT304 analogs, by virtue of their ability to target and modulate more than one pathway, may represent a promising therapeutic strategy for inhibiting thrombosis associated with inflammation, cancer, and chemotherapy plus other adjunct therapies without compromising hemostasis.