Effects of novel dual COX-2 inhibitors/TP receptor antagonists on human endothelial cell permeability and angiogenesis

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a hallmark of cancer that is crucial in its development and progression. Indeed, angiogenesis is essential for supplying oxygen and nutrients to rapidly growing tumors and for facilitating metastasis. A pivotal role for cyclooxygenase-2 (COX-2) overexpression has been observed in promoting angiogenesis in cancer. This overexpression leads to increased production of prostanoids, including prostaglandin E2 (PGE2) and thromboxane A2 (TXA2), which are involved in cancer progression. In this project we focused on the role of TXA2 in the angiogenic process. TXA2 promotes endothelial permeability that may be considered the first step of angiogenic process. Some in vitro studies reported that micromolar concentrations of TXA2 inhibit Vascular Endothelial Growth Factor (VEGF)-induced endothelial cell (EC) migration and formation of capillary tube-like structures, whereas others showed that TXA2 cooperates with VEGF to promote angiogenesis. Overall, the relative contribution of TXA2 to the angiogenic process has been evaluated using micromolar concentrations of thromboxane receptor (TP) agonists and/or against growth factors, such as VEGF. In a collaborative project with the Universities of Milan and Turin, we developed new bifunctional compounds combining COX-2 inhibition with TP receptor antagonism. These compounds are expected to maintain the beneficial effects of COX-2 inhibition while reducing the cardiovascular hazard via TP receptor antagonism, thus improving the well-established beneficial effects of COX-2 inhibition with NSAIDs and coxibs in cancer treatment. Unpublished results from our lab showed that test compounds inhibit arachidonic acid-induced breast cancer cell invasion and MMP-9 activity via COX-2-mediated PGE2 inhibition. The aim of this thesis was to evaluate the effects of SWE96, the bifunctional COX-2 inhibitor/TP antagonist showing the highest potency as a TP antagonist, on different steps of the angiogenic process directly triggered by nanomolar concentrations of the TP agonist U46619. To this end, we used several in vitro assays that recapitulate the most important steps of the angiogenic process including EC permeability, proliferation, migration and formation of capillary-like structures. Our results show that TP receptor activation promoted EC contraction and permeability, whereas SWE96 (100 nM, IC80) counteracted both effects. Using pharmacological inhibition and protein silencing we also found that activation of Rho/ROCK pathway is involved in TP-mediated EC contraction evaluated by confocal microscopy. Moreover, TP receptor agonists drove EC migration through activation of PI3K/Akt signalling. Conversely, the Rho/ROCK pathway did not affect HUVEC migration based on experiments with the ROCK inhibitor Y27632 as well as RhoA silencing. Finally, our data show that TXA2-induced cell migration was inhibited by SWE96. TP activation with U46619 did not affect HUVEC proliferation and capillary-like structures formation. These in vitro data suggest that blocking TP receptor activation in ECs could impair tumor-induced angiogenesis by counteracting EC permeability and migration. In conclusion, these findings in ECs combined with previous data in cancer cells lay the ground to further studies to evaluate the efficacy of bifunctional COX-2 inhibitors/TP antagonists in impairing breast cancer progression.