JULIA PEÑAS MARTINEZ¹, Ginés Luengo Gil¹, SALVADOR ESPÍN GARCÍA¹, Carmen Ortega Sabater², MARÍA DEL CARMEN RODENAS BLEDA¹, David Zaragoza Huesca³, NATALIYA BOHDAN ¹, Vicente Vicente García¹, ALBERTO CARMONA BAYONAS¹, IRENE MARTINEZ MARTINEZ¹

(1) HEMATOLOGÍA Y ONCOLOGÍA MÉDICA CLÍNICO-EXPERIMENTAL, IMIB-Arrixaca, España
(2) NUTRICIÓN, IMIB-Arrixaca, España
(3) Centro Regional de Hemodonación. IMIB-Arrixaca. Servicio de Hematología y Oncología Médica. Universidad de Murcia., 30730, España (Región de Murcia)

Glioblastoma multiforme remains a poor prognosis tumour, where research into new treatments is urgently required.It has been shown that antithrombin, the main physiological inhibitor of the coagulation cascade,has an anti-tumor effect on glioblastoma multiforme cells. Interestingly, antithrombin adopts different conformations with different features: native, activated by heparin, prelatent, latent and cleaved. Prelatent form has lower heparin affinity than native but greater than latent, it maintains its inhibitory capacity unlike latent conformation, and it has the greatest anti-angiogenic ability. Given this special properties,we decided to investigate the effect of this prelatent conformation on tumorigenic processes of glioblastoma multiforme cells.

Native and prelatent antithrombins were purified by chromatography. After prelatent AT characterization as previously described, enteropeptidase and hepsin inhibition by antithrombin was evaluated by chromogenic/fluorogenic assays and western blot. Wound healing and matrigel invasion assays were carried out to evaluate migration and invasion processes, respectively. Expression of E-cadherin, Vimentin, VEGF-A, AKT, STAT3 and ERK1/2 wereevaluated by western blot and/or qRT-PCR. Statistical analysis was performedusing non-parametric tests.

Prelatent antithrombin was able to inhibit enteropeptidase and hepsin, two serine proteases related with migration and invasion, but with less efficiency than native conformation. Interestingly, migration and invasion of glioblastoma multiforme cells both were significantly reduced after treatment with prelatent antithrombin and, in the case of migration, more efficiently thanafter treatment with native antithrombin. Additionally, prelatent antithrombin treatment downregulated the expression of VEGF-A (Fig. 1), STAT3 (Fig. 2A), and ERK1/2 (Fig. 2B) in glioblastoma multiforme cells, which may have an impact on cell survival and angiogenesis.

Prelatent antithrombin shows anti-tumor properties on glioblastoma multiforme cells by reducing migration and invasion, and the expression of proteins that promote tumor progression, resistance to anti-tumor therapies and angiogenesis, key in the glioblastoma multiforme due to its high vascularity.