Combination Therapy with DETA/NO and Clopidogrel Inhibits Metastasis in Murine Mammary Gland Cancer Models via Improved Vasoprotection

Vascular endothelial dysfunction and platelet activation play a key role in tumor metastasis, and therefore both of these processes are considered important therapeutic targets in cancer. The aim of our studies was to analyze anti-metastatic activity of combination therapy using nitric oxide donor DETA/NO and antiplatelet drug clopidogrel. Nitric oxide acts as a vasoprotective mediator, while clopidogrel inhibits ADP-mediated platelet aggregation. 4T1-luc2-tdTomato cell line transplanted intravenously (i.v.) and 4T1 cell line transplanted orthotopically were used as metastatic mammary gland cancer models. Moreover, anti-aggregation action of compounds was tested ex vivo on the blood samples taken from breast cancer patients. We have shown that in selected dosage regimes DETA/NO combined with clopidogrel significantly reduced lung metastatic foci formation in i.v. model and such inhibition was transiently observed also in orthotopic model. Anti-metastatic effect correlated with significant increase of prostacyclin (PGI2) metabolite and reduction of endothelin-1, sE-selectin, sI-CAM and TGF-β plasma level and with decreased V-CAM expression on the endothelium. Combination therapy decreased fibrinogen binding to the resting platelets at the early stage of tumor progression (day 14). However, at the later stages (day 21 and 28) the markers of platelets activation were detected (increased JON/A antibody bound, P-selectin level, binding of fibrinogen and vWf). Decreased aggregation as well as lower release of TGF-β were detected in platelets from metastatic breast cancer patients incubated ex vivo with compounds tested. Although combination therapy increases E-cadherin, the increase of N-cadherin and α-SMA in tumor tissue was also observed. The results showed that at the early stages of tumor progression combined therapy with DETA/NO and clopidogrel improves vasoprotective and antiplatelet activity. However, in advanced tumors some adverse effects towards platelets activation can be observed.