Abstract A10: Simultaneous block of angiogenesis through PI3K-AMPK/AKT/mTOR signaling pathways after treatment with metformin and LY294002 in canine mammary tumor cell line

Introduction: The PI3K-AMPK / Akt / mTOR signaling pathways are the most important signaling pathways involved in the angiogenesis process, which is regulated by numerous factors, being the most important the hypoxia-inducible factor - 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Metformin, the prescribed drug for patients with metabolic syndrome, has been suggested as a new therapeutic agent with potential anti-neoplastic action capable to inhibit cell growth by AMPK signaling pathway. In the same way, the LY294002, an important inhibitor of PI3K / Akt / mTOR signaling pathway, has anti- angiogenic properties able to decrease the release of growth factors, such as VEGF. In this context, the aim of this study was to evaluate the effectiveness of treatment with metformin and LY294002 in angiogenesis process. Methods: Canine mammary tumor cell line ER-positive (CMT-U229) was cultured in HAM-F12 culture medium, at 37°C in 5% CO2. Cell viability was measured by MTT assay after treatment with varying concentrations of metformin and LY294002. Once stablished the concentration of metformin (1mM) and LY294002 (5µM) treatment, the protein and gene expression of HIF-1α and VEGF were detected by immunocytochemistry and real time PCR, respectively. The immunostaining was quantified by optical densitometry technique, slides were analyzed and photographed in Nikon Eclipse E200 microscope at 40X objective, and proteins were quantified by the ImageJ software analysis. For real time PCR, the relative expression of the genes of interest was determined by DataAssist v3.0 software, using ΔΔCt method. Results: There was a significantly decrease of cell viability after treatment with all concentrations (1mM, 2mM, 3mM and 5mM) of metformin, 5µM and 10µM of LY294002 (p