Anti‐angiogenic Effects of Metformin in 2‐Deoxyglucose Treated Microvascular Endothelial Cells: Role of Thrombospondin‐1
Authors: Samuel Samson Mathews , Ghosh Suparna , Majeed Yasser , Ding Hong , Triggle Chris R 
Published: November 2023

Background & objective

The biguanide metformin, which is widely used in the management of type 2 diabetes, has received considerable interest as a potential anti‐cancer agent in many forms of cancer. However, the effect of metformin in tumor endothelial cells (TECs) has not been studied. TECs play a key role in tumor angiogenesis thereby supporting tumor growth, cancer cell survival and metastasis and hence targeting TECs in order to inhibit angiogenesis could prove to be a potential anti‐angiogenic cancer therapy in a wide range of cancers. Reports show that metformin increases the levels of anti‐angiogenic thrombospondin‐1 (TSP1) in the serum of women with polycystic ovarian syndrome (1). Data from our preliminary studies in Mouse Microvascular Endothelial Cells (MMECs) that overexpress angiogenic VEGF (CRL‐2460; ATCC; sarcoma cell line; cell type: endothelial) has revealed that metformin (2mM) significantly increased TSP1 in glucose‐starved cells when compared to metformin treated glucose exposed cells. We therefore investigated the effects of metformin on angiogenesis, in MMECs in combination with the glycolytic inhibitor, 2‐deoxyglucose (2DG).

Materials & methods

MMECs were treated with 2DG (5mM) for 48h in the presence or absence of metformin (2mM) & western blot analysis was performed to assess the status of angiogenic and anti‐angiogenic marker proteins. Alternatively, the putative AMPK activator, A769662 (150mM), was also used instead of metformin. Cell proliferation assay, cell migration assays and wound healing assays were also performed.

Results

We observed a significant up‐regulation of TSP1, while the levels of pVEGFR2 (Y1175) were 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. A769662, did not show any effect on TSP‐1 levels in normal glucose or 2DG‐exposed cells. Furthermore, treatment with metformin (2mM) in 2DG exposed cells significantly increased the levels of pRap (S792), which in turn should have caused the inhibition of the mTOR pathway as evidenced by the significant decrease in the levels of pmTOR (S2448), p4E‐BP1 (T36/47), pS6 (S235/236) and pS6 (240/244) when compared to metformin treated normal glucose exposed cells. Levels of cell cycle related proteins such as pCycB1 (S147), CycD1 and CycD2 significantly decreased in cells treated with a combination of 2DG and metformin when compared to cells that were treated with either 2DG or metformin alone. The rate of cell proliferation and endothelial cell migration also significantly decreased in cells treated with a combination of 2DG and metformin when compared to cells that were treated with either 2DG or metformin alone.

Conclusion

Our findings show that using metformin in combination with 2DG has an anti‐angiogenic activity associated with a significant up‐regulation of thrombospondin‐1 and could prove to be therapeutic strategy in a wide range of cancers.

Support or Funding Information

This publication was made possible by JSREP grant [3‐016‐3‐009] awarded to Dr. Samson Mathews Samuel and NPRP grant [4‐910‐3‐244] awarded to Prof. Chris R. Triggle from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the authors.