Antiangiogenic Properties of a New VEGF Inhibitor Family : The Segetalins.

Angiogenesis is a physiologic process involved in neovessel formation fundamental to the process of tumor growth, invasion, and metastatic dissemination. Various data, including the presence of estrogen receptors in endothelium, suggest that estrogens can mediate endothelial proliferation and differentiation. Therefore, it is likely that anti-estrogenic drugs can also exert their effects in endothelial cells. Some analogs or metabolites derived from estrogens have been demonstrated with angiogenic inhibitor properties. Segetalins are a group of natural cyclohexapeptides isolated from the seeds of Vaccaria segetalis that have been previously suspected to possess phytoestrogenic activities. The aim of this study was: i) to synthesize segetalins and analogs with antiangiogenic properties by using molecular modeling as a predictive tool, ii) to study biological properties of these new molecules on angiogenesis including cell migration, tube formation and proliferation, iii) to test the most active molecules on VEGF expression and AKT/ERK pathways activation, in different conditions.

We found that changes in the conformational rigidity of the natural molecules were potentially able to modify the estrogen-like activity of the segetalins. Natural segetalins (SA, SB, and SG) and their respective analogs (S1, S2 and S3) were synthesized using a classic peptide synthesis method (ABI Fast-Moc protocol). A natural segetalin and its analog differ one from the other by only one aminoacid substitution. For example, SA and S1 differ by an Ala > Trp substitution in position 4 of the cyclohexapeptide skeleton. All the cyclohexapeptides were obtained with an average yield of 55% and without racemisation. The presence of stable conformers on the NMR time scale was displayed by the occurrence of well-resolved sharp signals.

1) Segetalins and analogs did not show any effect on HUVECs, MCF7 and MDA MB-321 cells proliferation and direct survival. 2) Using HUVECs, only S1 was shown to reduce cell migration (haptotactic migration assay) and capillary tube formation (matrigel assay) by 55 % (from10^-5 to 10^-6 M). 3) Conditioned media from SA or S1 treated MCF7 or MDA MB-231 cells inhibited endothelial cell proliferation by 25% vs vehicule (p<0.0l). 4) We investigated the effect of SA and S1 on VEGF secretion in the presence of variable estrogen concentrations (from 10^-12 to 10^-5 M) and we found that both SA and S1 act as an estrogen antagonist on VEGF secretion. 5) SA and S1 decreased VEGF secretion in both MCF7 and MDA MB-231 cell supernatant by about 50% vs vehicule (p<0.01). The half maximal inhibitory concentration (IC) was found to be 10^-8 M for all cell lines. 6) Quantitative real time PCR analysis and western blotting demonstrated that SA and S1 specifically reduced VEGF 165 (p< 0.004) and VEGF 121 (p<0.008) isoform expression in MCF7 and MDA MB-231 cell lines. The inhibition effect was greater for S1 (60% decrease in VEGF 121 expression and 45% decrease in VEGF 165 expression) compared to SA (35 % decrease expression for both VEGF isoforms). 7) AKT and ERK pathways are also the downstream effectors of VEGF-Receptor2 which is highly expressed on the surface of HUVECs. We found that the phosphorylation of AKT and ERK proteins was decreased by about 40% in the early time phase (5 mns) in HUVECs treated with conditioned media from SA and S1 pretreated MCF7 and MDA MB-231 cells.

This is the first documented report on the biological properties of the Segetalin family. We show that S1, a structural analog of the natural segetalin A, displays prominent antiangiogenic properties on HUVECs. Furthermore experimental evidence show that S1 significantly reduces VEGF 121 and 165 isoform expression in cancer cells and suggest that both the AKT and ERK pathway might have an important function in Segetalin - inhibition VEGF expression.

No relevant conflicts of interest to declare.