Abstract B25: Meformin and LY294002 treatment regulate angiogenesis process through PI3K-MAPK/AKT/mTOR signaling pathways in mammary tumors
Authors: Moschetta Marina Gobbe , Leonel Camila , Maschio-Signorini Larissa Bazela , Borin Thaiz Ferraz , Gelaleti Gabriela Bottaro , Ferreira Lívia Carvalho , Jardim-Perassi Bruna Victorasso , Sonehara Nathália Martins , Zuccari Debora Aparecida Pires de Campos 
Published: May 2019

Abstract
Introduction: The angiogenesis process is regulated by numerous factors, especially the hypoxia-inducible factor 1alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). The HIF-1 is a central regulator of pathophysiologic response of cells to hypoxia conditions, able to activate transcription of the gene that promotes the induction of VEGF, which in turn promotes angiogenesis through its ability to stimulate growth, migration, and invasion of endothelial cells, leading to the formation of new blood vessels and subsequent tumor growth. Metformin has demonstrated its ability to reduce the incidence of cancer in diabetic patients with positive results, especially in breast cancer, inhibiting cell growth by AMPK/Akt/mTOR signaling pathway. Likewise, LY294002, a PI3K signaling pathway inhibitor, has antiangiogenic features. In this context, the aim of this study was to evaluate the effectiveness of metformin and LY294002 inhibitor treatment in angiogenesis as a therapeutic strategy in mammary tumors in an in vitro and in vivo study.
Materials and Methods: In the in vitro study, canine mammary tumor cell line (CF41) was cultured in DMEM high-glucose culture medium, at 37°C in 5% CO2. Cell viability was measured by MTT assay after treatment with different concentrations of metformin and LY294002 inhibitor. Once established the concentration of 5 mM for metformin and 5 µM of LY294002, the protein and gene expression of HIF-1alpha and VEGF were detected by immunocytochemistry and real-time PCR, respectively. For an in vivo study, CF41 were injected in nude athymic female mice and treated with metformin (200 mg/kg i.p.) for 4 weeks and LY294002 (7.5 mg/kg intratumorally) every 3 days for three times. At the end, mice were euthanized and the tumors were collected to determine the protein and gene expression of HIF-1alpha and VEGF and microvessel density by immunohistochemistry for CD31. 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 Ct method.
Results: There was a significant decrease of cell viability after treatment with different concentrations of metformin and LY294002 in 24 hours. Both HIF-1alpha and VEGFA protein and gene expression significantly decreased after treatment with metformin and LY294002 or in combination. Furthermore, in animals, there was a decrease in tumor size, protein and gene expression of HIF-1alpha and VEGFA, in addition to a decrease of CD31 expression after all treatments.
Conclusion: Our results suggest the potential effectiveness of metformin and LY294002 acting in angiogenesis process of mammary tumors.
Financial support: FAPESP.
Citation Format: Marina Gobbe Moschetta, Camila Leonel, Larissa Bazela Maschio-Signorini, Thaiz Ferraz Borin, Gabriela Bottaro Gelaleti, Lívia Carvalho Ferreira, Bruna Victorasso Jardim-Perassi, Nathália Martins Sonehara, Debora Aparecida Pires de Campos Zuccari. Meformin and LY294002 treatment regulate angiogenesis process through PI3K-MAPK/AKT/mTOR signaling pathways in mammary tumors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B25.