Aspirin-acetylated COX-2–derived DHA metabolites inhibit angiogenesis
M. Vara-Messler1, A. Trenti1, C. Buccellati2, A. Sala2,3, L. Trevisi1, C. Bolego1.
1.       Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy.
2.       Department of Pharmacological and Biomolecular Sciences, University of Milan, Italy
3.       IBIM, C.N.R., Palermo, Italy.
BACKGROUND: Angiogenesis is a tightly regulated process involving endothelial cell (EC) proliferation, migration and tube formation, and is a key feature of many pathological conditions such as cancer (1). Several COX-2- and/or COX-1-derived arachidonic acid (AA, 20:4 ω-6) metabolites have been demonstrated to promote this process (2,3). It is widely accepted that ASA has antineoplastic effects (4) at least in part mediated by metabolites derived from acetylated COX-2 (5). However, data on the role of metabolites from aspirin (ASA)-acetylated COX-2 in angiogenesis are lacking. Several studies suggest that ω-3 fatty acids exert protective effects against cancer progression, including cancer-related angiogenesis. In particular, docosahexaenoic acid (DHA, 22:6 ω-3) epoxymetabolites have been recently shown to decrease tumor-associated angiogenesis (6). We hypothesized that metabolites derived from COX-2 acetylated by ASA in the presence of either AA or DHA could affect the angiogenic capacity of ECs.
METHOD: HUVECs were used at 2nd to 6th passage. Cells were treated for 24-72h with DHA (1-50μM) or AA (1-50μM) in the presence or absence of ASA (50µM). Selected experiments were performed with 17-R HDoHE (100nM-3µM), a DHA metabolite derived from COX-2 acetylated by ASA. Cell viability was analyzed with MTT assay. EC migration was evaluated using a) the scratch/wound healing assay, where the percent of migrated cells was calculated analyzing an average wound space from 3 fields and b) a micro chemotaxis chamber (Neuro Probe), where 5 unit fields/well were counted with Nikon Eclipse (200X magnification).
RESULTS: HUVECs were maximally viable when treated with DHA or AA 1-30 µM in the presence or absence of ASA for 24, 48 or 72h. Cell viability decreased after 72h treatment with DHA 50 µM (42.68% ±13.83, p<0.05). Challenging the cells for 48h with 17-R HDoHE (100nM-3µM), did not affect cell viability.
EC migration evaluated by wound migration assay was significantly decreased (75% ± 6.8, p<0.01) in presence of 30µM DHA for 24h compared to control cells considered as 100%. After 48h, challenge with 10 or 30µM DHA significantly reduced cell migration (80%±1,54 and 56%±8.9, respectively, p<0.001). Finally, 72h pre-treatment with increasing (1-30 µM) DHA concentrations significantly reduced cell migration with respect to controls. By contrast, AA did not affect HUVEC migration at any concentration tested (1-30μM). Interestingly, 10µM DHA in the presence of 50 µM ASA significantly inhibited EC migration (64%±5.4,p<0.001) already after 24h, while 10 µM AA 100 µM ASA did not show any effect after 24 or 48h treatment. In order to determine the possible role of specific metabolites derived from COX-acetylation, we tested 17-R HDoHE. After 24h, 1 and 3µM 17-R HDoHE decreased EC migration (88%±4.1 and 72%±3.9, p<0.01, respectively). This effect was higher after 48h (78%±3.7 and 63%±7.4 p<0.001, for 1 and 3μM, respectively).
Additionally, EC migration in response to an attractant gradient was evaluated at shorter time points using a micro chemotaxis chamber. After 6h, 17-R HDoHE (1-3µM) reduced FBS-induced EC migration, while DHA did not show any effect.
CONCLUSION: We identified a role for DHA metabolites derived from aspirin-acetylated COX-2 in inhibiting angiogenesis. This data further supports the beneficial effects of both aspirin and ω-3 fatty acids in the context of cancer. Ongoing experiments by mass spectrometry will identify the lipidomic profile of metabolites from acetylated COX-2 in ECs.
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