Metformin Sensitizes Glucose Starved Microvascular Endothelial Cells to Death by Inhibition of the mTOR Pathway and Autophagy
Authors: Samuel Samson Mathews , Ghosh Suparna , Triggle Chris R. 
Published: November 2023

Background & objectiveGlucose deprivation/starvation (GS) is a characteristic feature of the tumor microenvironment caused by reduced blood supply and relatively high rate of consumption of glucose. Cancer cells are metabolically reprogrammed to derive energy by metabolizing glucose through enhanced glycolysis even in the presence of an ample amount of oxygen (Warburg effect), rendering them resistant to chemo‐ and radiation therapy. Therefore targeting cancer cell metabolism by using drugs, which in combination with other therapeutic strategies that cause energy stress in cancer cells should have a synergistic effect. Metformin, the most frequently prescribed, anti‐diabetic drug has been shown to exhibit anti‐cancer activity in different types of cancer cells.Materials & methodsIn the present study mouse microvascular endothelial cells (MMECs) were subjected to GS for 24h and 48h in the presence & absence metformin (50μM & 2mM) & western blot analysis was performed to assess the status of endoplasmic reticulum (ER) stress, autophagy, cell survival and apoptotic markers. Immunofluorescence staining followed by confocal microscopy was also done to assess LC3B punctae staining in cells, indicating autophagy.ResultsGS significantly increased the levels of ER stress markers ‐ GRP78, ATF4 and CHOP when compared to cells exposed to 11mM glucose. ER stress activation was followed by activation of autophagy in glucose‐starved cells as evidenced by the significant increase in the levels of LC3A‐II and LC3B‐II compared to normal glucose exposed MMECs. Immunofluorescence staining for LC3B followed by confocal microscopy revealed distinct LC3B stained punctae in glucose starved MMECs compared to normal glucose exposed cells, thereby confirming the activation of autophagy. Treatment with 2mM metformin, but not 50μM, markedly reversed the effect of glucose starvation on ER stress and autophagic markers and markedly reduced the formation of LC3B stained punctae, when compared to non‐treated glucose starved cells. AMPK knockdown studies revealed that this effect of metformin on GS induced ER stress and autophagy is independent of AMPK. Treatment with 2mM metformin, for 24h in GS conditions, however, did not show a marked change in cell viability. Extending the study to 48h, we found that persistent ER stress and metformin associated inhibition of autophagy significantly decreased cell viability, caused G2/M cell cycle arrest and increased the number of cells in sub‐G0/G1 phase of cell cycle. Furthermore, treatment with metformin for 48h in MMECs subjected to GS significantly reduced levels of Sirt1 and pAkt (S473), which in turn will increase the levels of acetylated‐p53 and decrease pmTOR (S2448). Inhibition of mTOR pathway was also confirmed by significant decreases in the levels of p4E‐BP1 (T36/47), pS6 (S235/236) and pS6 (240/244) in metformin (2mM) treated glucose–starved MMECs when compared to non‐treated MMECs subjected to GS.ConclusionOur findings show that using metformin in combination with agents that modify cancer cell metabolism should sensitize cancer cells to chemo/radiation therapy and promote cell death.Support or Funding InformationSupported by Qatar National Research Fund (QNRF) grants: NPRP‐4‐910‐3‐244 (awarded to Dr. Chris R. Triggle) and JSREP‐3‐016‐3‐009 (awarded to Dr. Samson Mathews Samuel)