Modulation of Cancer Cell-Associated Procoagulant Activity (PCA) by a Novel, Small Molecule, Orally Active Inhibitor of Oxidative Stress Pathways

Background: The association between malignancy and venous thromboembolism (VTE) is well-recognized. VTE is associated with a systemic hypercoagulable state that is secondary to cancer, cancer-associated therapy and the host inflammatory response. We have tested the ability of OT-304, which is a dual inhibitor of reactive oxygen species (ROS)-mediated activation of both the EGR-1 and NF-κB pathways, to inhibit the PCA of tumor cells.

Methods: Molecular measurements of NF-kB (p50/p65), IkBa, TNF-a, EGR1 and tissue factor (TF) were performed on the human monocytic cell line THP-1, and human umbilical vein endothelial cells (HUVECs) using Affymetrix micro-array, RTPCR, Western blot, ELISA and gel shift assays. The cell-based, anticoagulant efficacy of OT-304 at 1–30 mM was determined in in vitro models of both pro-inflammatory and cancer cell-associated hypercoagulable states, using thrombelastography (TEG) in human blood and in a rabbit model of cell-based coagulation activation.

Results: OT-304 effectively inhibited LPS-mediated TNF-α production in human blood and in THP-1 cells, with an IC₅₀ of 6.7 mM. Similarly, in mice, in a dose-dependent manner, OT-304 at 0.1–50mg/kg IP or 1.0–150 mg/kg oral, inhibited LPS-induced NF-κB activation-associated up-regulation of various chemokines and cytokines. At the molecular level, OT-304 demonstrated effective inhibition of LPS-induced NF-κB activation, nuclear translocation of p50/p65, degradation of IKBa, without any effect on nuclear p50/p65 DNA binding. OT-304 appeared to induce modest down-regulation of the key procoagulant mediator TF, presumably via the inhibition of EGR1. In human whole blood subjected to TEG, OT-304 inhibited the procoagulant effect induced by LPS, lipid hydroperoxide, and ceramide, without directly affecting factor Xa, factor IIa or other coagulation factors. Additionally, OT-304 at 0.1–10 mM inhibited the PCA of glioma, lung, and pancreatic cancer cells. In comparison to the low-molecular-weight heparin, enoxaparin, OT-304 (1–30 ug/ml) was without any significant effect on soluble blood coagulation, as measured by the activated partial thromboplastin time. Repeated administration of OT-304 to mice (10 mg/kg, IP daily for up to 14 days), was without any hemorrhagic toxicity as compared to enoxaparin administered at the same dose. In a mouse xenograft model of human glioma, OT-304 (30 mg/kg, IP, daily for 15 days) resulted in significant inhibition of tumor growth, tumor angiogenesis, and glioma-associated elevation of plasma D-dimer. In rabbits, OT-304 (10 mg/kg IP) resulted in significant inhibition of LPS-induced PCA.

Conclusions: OT-304, by virtue of its ability to target and modulate more than one pathway, may represent a promising anti-cancer therapy, capable of inhibiting tumor growth, inflammation and cancer-associated thrombosis without compromising hemostasis.