Metformin Induces Thrombospondin‐1 in Metabolically Challenged Endothelial Cells: Implications in Anti‐angiogenic Cancer Therapy
Authors: Samuel Samson Mathews , Ghosh Suparna , Majeed Yasser , Triggle Chris R 
Published: November 2023

Background & objectiveMetformin has been shown to exhibit anti‐cancer properties indifferent types of cancer. Recent studies from our laboratory with mousemicrovascular endothelial, MS1‐VEGF cells (capable of generating well differentiated angiosarcomas in mice), indicate that metformin (2mM) has adifferential effect depending on whether these cells are maintained undernormal glucose or glucose‐starved conditions. Metformin (2mM), significantly reversed glucose starvation‐induced endoplasmic reticulum (ER) stress andautophagy, while 50μM metformin (concentration of metformin that is therapeutically achievable in the treatment of type 2 diabetes) produced no effect. Additionally, metformin(2mM) caused G2/M cell cycle arrest, decreased cell viability, decreased the levels of Sirt1 and pAkt (S473) while increasing the levels of acetylated p53 and activated caspase 3, leading to cell death (increased number of cells insub‐G0/G1 phase) in glucose‐starved cells. However, metformin's potential, as an anti‐angiogenic cancer therapy remains largely unknown. Angiogenesis is an integral part of tumor growth, cancer cell survival and metastasis; Thus, we investigated the effects of metformin on angiogenesis in MS1‐VEGF cells under glucose‐starved conditions and in combination with the glycolytic inhibitor,2‐deoxyglucose (2DG).Materials &methodsMS1‐VEGF cells were subjected to glucose starvation or treated with 2DG (1mM, 2mM, 5mM, 7.5mM and 10mM) for 48h in the presence or absence of metformin (50μM & 2mM). Western blot analysis was performed to assess the status of angiogenic and anti‐angiogenic marker proteins. Cell proliferation assay was also performed.ResultsCell proliferation rate markedly decreased in cells treated with a combination of 2DG and metformin (2mM) when compared to cells that were treated with either 2DG or metformin alone. Additionally, we observed robust up‐regulation of anti‐angiogenic thrombospondin‐1, while the levels of pVEGFR2 (Y1175) markedly decreased, in 2DG exposed cells treated with metformin (2mM) when compared to cells maintained in normal glucose exposed cells that were treated with metformin. In addition, treatment with metformin(2mM), reversed 2DG induced ER stress and autophagy, decreased the levels of Sirt1 and pAkt (S473) and increased the levels of acetylated‐p53 in 2DG treated cells. Furthermore, treatment with metformin (2mM) decreased the levels of pmTOR (S2448). Inhibition of mTOR pathway was also confirmed by a significant increase in the mTOR inhibitory Raptor phosphorylation (pRap; S792)and a significant decrease in the levels of p4E‐BP1 (T36/47), pS6 (S235/236)and pS6 (240/244) in 2DG exposed cells treated with metformin (2mM) when compared to normal glucose exposed cells that were treated with metformin. All the effects of metformin that we observed in 2DG treated MS1‐VEGF cells were comparable to the effect of metformin in glucose‐starved cells.ConclusionOur findings show that using metformin in combination with 2‐DG could prove to be a potential anti‐angiogenic cancer therapy owing to the up‐regulation of thrombospondin‐1.Support or Funding InformationThis publication was made possible by NPRP grant [4‐910‐3‐244] awarded to Prof. Chris R. Triggle and JSREP grant [3‐016‐3‐009] awarded to Dr. Samson Mathews Samuel, from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the authors.