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Metformin prevents NGF-dependent proliferative and pro-angiogenic effects in epithelial ovarian cancer and endothelial cells

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Metformin prevents NGF-dependent proliferative and pro-angiogenic effects in epithelial ovarian cancer and endothelial cells

Maritza Garrido1,2, Margarita Vega1,2, Andrew Quest3, Carmen Romero1,2,3

1Laboratory of Endocrinology an Biology Reproduction, Clinical Hospital, University of Chile, Santiago.2Obstetric and Gynecology Department, Faculty of Medicine, University of Chile, Santiago; 3Advanced Center for Chronic Diseases (ACCDIS)

Introduction

Epithelial Ovarian Cancer (EOC) is detected in advanced stages with poor prognosis and is characterized by high angiogenesis, that promotes tumor growth and metastasis and are regulated by angiogenic and growth factors1. Nerve Growth factor (NGF) and its high affinity receptor TRKA increase during EOC progression2. NGF/TRKA increases survival and proliferation markers involving PI3K/AKT and MAPK/ERK signaling pathways in EOC explants3. Moreover, NGF increases vascular endothelial growth factor (VEGF) levels in EOC cells4. Endothelial cells express TRKA receptor2; then we postulate that NGF is a direct and indirect angiogenic factor. Currently available therapies yield modest results in patients making it necessary to develop new alternative therapies. Metformin is of interest in this context, because it has been attributed anti-carcinogenic effects in many types of cancers5,6, but the underlying mechanisms remain unknown. The aim of this study was to determine the effects of the anti-diabetic drug metformin on NGF-dependent proliferation in epithelial ovarian cancer cells and angiogenesis potential in endothelial cells

Methods

Cellular lines A2780 (primary EOC cells), HOSE (non-carcinogenic Human Ovarian Surface Epithelium cells) and EA.hy926 cells (human endothelial cells) were stimulated with metformin (0.5, 1, 5 and 10 mM) and NGF (50 and 100 ng/mL) for 6, 8 or 48 hours. Cells were count with trypan blue exclusion and LUNA cell counter; cell viability was measure by cell cytotoxicity assay. Additionally, cell cycle was evaluated by flow cytometry using propidium iodine and Ki-67 immunodetection. Also, cell mortality was evaluated by flow cytometry.  Vasculogenesis assays were performed (matrigel differentiation assay) and angiogenic score of EA.hy926 cells was calculated. Parameters as junctions or meshes were obtained by Image J Angiogenesis Analyzer software. Migration was evaluated by transwell assay, where migrated cells were strained with violet crystal and counted with Image J cell counter. Statistical analysis: Kruskal-Wallis test

Results

NGF (50 and 100 ng/mL) increased the number of A2780, HOSE and EA.hy926 cells beyond baseline values after 48 hours of stimulation (p<0.05 and p<0.01; figure 1A), while metformin (10 mM) decreased cell number in A2780 and EA.hy926 (p<0.01), without changes in HOSE cells (figure 1B). Also, NGF increased the immunostaining of the cell cycle marker Ki 67 to 93,6% in A2780 cells (p<0.01), 63.3% in HOSE cells (p<0.05) and 50.0% in EA.hy926 cells (p<0.05) (figure 1C) while metformin decrease Ki 67 presence only in A2780 (p<0.01) and EA.hy926 cells (p<0.05; figure 1D). Moreover, NGF increased the percentage of cells in S and G2/M phases in all cellular lines (figure 1E) and metformin shows the contrary effect, decreasing cells in S phase and increasing cells in G0/G1 phase (figure 1F). Importantly, in the presence of GW441756 (GW), a specific TRKA inhibitor, or a neutralizing antibody against NGF (Ab), NGF-induced effects were reversed (figures 1C,1E). Taken together, these results show that NGF increases A2780, HOSE and EA.hy926 cell proliferation via a TRKA-dependent mechanism and metformin decreases A2780 and EA.hy926 cell  proliferation.

On the other hand, NGF (100 ng/mL) increased the angiogenic score of EA.hy926 cells (p<0.05), the average number of junction structures (multicellular joints) and average number of polygonal structures, referred to as “meshes” (p<0.05; figure 2A). On the other hand, metformin (5 and 10 mM) decreased angiogenic score and meshes structures of EA.hy926 cells. (p<0.05; figure 2B).

The co-treated with NGF (100 ng/ml) and metformin (10 mM) blocks the NGF-increased cell count and cell viability in A2780, HOSE and EA.hy926 cells (p<0.05 and p<0.01; figures 3A-3C). Similar results were obtained with cell cycle evaluation: metformin prevented NGF-enhanced Ki 67 immunostaining in all three cell lines (p<0.001; figure 4) and prevented the NGF-induced changes in the different phases of cellular cycle (p<0.05; figure 3D). Additionally, we also evaluated cell death in all three lines; where NGF and metformin did not induce significant changes regarding the basal condition, although NGF and metformin co-treatment increased the number of A2780 cells undergoing cell death (p<0.05; figure 3E).

Furthermore, NGF (100 ng/mL) increased the migration of EA.hy926 cells by 75.5% (p<0.001) and metformin shows opposite effects, decreasing migration rate of EA.hy926 cells (p<0.001; figure 4B). Finally, we evaluated the effect of co-treatment with NGF and metformin on the angiogenic score and migration of EA.hy926 cells, where metformin completely prevented the NGF-induced increase in the angiogenic score (p<0.05) and migration of EA.hy926 cells (p<0.0001; Figure 4)

Conclusions and Perspectives

Metformin precludes pro-angiogenic and proliferative effects of  NGF in EOC cells. Given that NGF plays an important role in EOC progression and our finding here showing that metformin blocked NGF induced effects, we anticipate that metformin holds considerable promise in the future for the treatment of ovarian cancer.

Grants: FONDECYT #1160139, CONICYT-FONDAP #15130011 and CONICYT scholarship # 21150360

Bibliography: 1: Auersperg N et al. Endocr Rev 2001; 2:Tapia V et al. Gynecol Oncol 2011; 3: Urzua U et al. Horm Metab Res 2012; 4: Vera C et al. J Ovarian Res 2014;      5: Evans J et al. BMJ 2005; 6: Dowling R et al. BMC  Medicine 2011.

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