Javier Stelling Férez¹, Santiago López Miranda², José Antonio Gabaldón Hernández², FRANCISCO JOSE NICOLAS VILLAESCUSA³

(1) Unidad de Investigación - Laboratorio de Regeneración, Oncología Molecular y TGFß, 30507, España (Región de Murcia)
(2) Departamento de Nutrición y Tecnología de los Alimentos - UCAM, 30107, España (Región de Murcia)
(3) TRASPLANTE HEMATOPOYÉTICO / TERAPIA CELULAR, IMIB-Arrixaca, España

Epithelial cells migration is essential for wound closure and rely on key regulator proteins. Natural triterpenoids, particularly oleanolic acid (OA), have shown wound healing properties. We studied the molecular pathways that were activated by this triterpenoid and we found that both activation of SAP/JNK and MAP kinases pathways were activated by the stimulation with OA. The co-stimulation with EGF did not produce any additional effect to cell migration, suggesting that OA and EGF were triggering similar events at the molecular level. Indeed, OA stimulation induced a phosphorylation on the receptor for EGF that was consistent with these phenomenon and independent of protein synthesis. Additionally, the use of an inhibitor of the EGF receptor was able to abolish completely the effect of OA on cell migration. Despite all this results, we need to focus deeper in which molecular pathways are activated by OA. What's more, OA has a given difficulties to be delivered to cells due to its water insolubility.

To understand OA effect on wounds, we have studied OA-triggered molecular mechanisms in two epithelial cell lines: Mv1Lu and MDA-MB-231, by using in vitro wound healing scratch assays. Besides these, we have studied pro-migratory proteins expression and its subcellular localization. Finally, we performed OA soluble complexes with modified beta- and gamma-cyclodrextrins, which are well known to improve molecules solubility and bioavailability.

We have shown that OA induces the activation, at cells of the wound edge, of c-Jun; a master regulator of migration. On top of that, cell migration is mediated by dynamic changes in cell architecture: cytoskeleton reorganization and focal adhesion (FA) remodeling. Thus, we have shown that OA activates focal adhesion kinase (FAK), a critical factor for FA remodeling, and also the reorganization of paxillin and actin, indicating that OA promotes a remodeling of the cell architecture. On the other hand, study of different signaling kinases inhibitors had been used to dissect the contribution of different pathways to the activation of migration key elements such as FAK. In addition, we have performed scratch assays in human fibroblasts, which are involved in tissue remodeling during wound healing. Similarly, OA enhances fibroblast migration. . Due to its lipophilic nature, OA delivery to cells is difficult. However, we used cyclodextrins to improve its lipophilic nature. Strikingly, we have shown in scratch wound healing assays that these complexes facilitate OA in vitro use and enhance its cell migration properties.

• The treatment with OA causes the reorganization of migration machinery and a differential expression of proteins required for cell migration, specially at the wound edge. • Cyclodextrins improved solubility of OA, also improving the migration of cells in scratch assays. • Fibroblasts, a relevant cell type for wound healing, are able to migrate in response to the stimulation with OA. Additionally, several migration related pathways in this cell line are stimulated by OA. • All these results encourage us to pursue on OA/cyclodextrins as a way of improving OA application in wounds.