Metformin Mediated Inhibition of the mTOR Pathway Promotes Death in Glucose Starved Micro-Vascular Endothelial Cells
Authors: Samuel Samson Mathews , Ghosh Suparna , Majeed Yasser , Triggle Chris R. 
Published: July 2019

Background & objectiveIn order to survive the effects of the stress conditions, such as hypoxia and glucose starvation (GS) that exist in a tumor microenvironment, the regulatory mechanisms that control metabolism in cancer cells undergo change so that sufficient energy sources are available for proliferation, migration and invasion - thus facilitating metastasis. Via what is referred to as The Warburg Effect cancer cells primarily derive energy by metabolizing glucose through enhanced glycolysis even in the presence of an ample amount of oxygen. Thus, in principle, the non-specific cytotoxicity of traditional cancer chemotherapeutic agents will be reduced via a therapeutic strategy that targets the altered metabolism that is unique to cancer cells. Therefore, we predict that by using drugs, targeting cancer cell metabolism in combination with therapeutic strategies, which cause energy stress in cancer cells will result in a synergistic effect and enhance the likelihood of selectively killing cancer cells. Metformin, the most frequently prescribed, anti-diabetic drug has been shown to exhibit anti-cancer activity in different types of cancer cells thus supporting epidemiologic data that is suggestive that type 2 diabetic patients treated with metformin seem to be less likely to develop various types of cancers. Since angiogenesis is a key function of endothelial cells and aberrant angiogenesis is key to survival and growth of a tumor, we studied the effect of metformin on glucose-starved (GS) cancer microvascular endothelial cells with special reference to the Akt/mTOR pathway that is known to be dysregulated in many forms of cancer.Materials & methodsIn the present study mouse mile sven 1 vascular endothelial growth factor endothelial cells (MS1-VEGF; CRL-2460,from ATCC, USA, of micro-vascular endothelial origin) cells were subjected to GS for 48 h in the presence & absenceof metformin (2 mM). Metformin treated and non-treated normal glucose (11 mM) exposed cells were used as suitable controls. MS1-VEGF cells were produced by overexpressing the primate VEGF-121 in the MS1 endothelial cell line that was derived from mice pancreatic microvasculature and immortalized with temperature sensitive SV40 large T antigen (1). These cells generate well-differentiated angiosarcomas in nude mice model (1). Following the experimental protocol cell lysates were prepared, total protein estimation was carried out and thereafter western blot analysis was performed to assess the levels of Sirt1, pAkt (S473), acetylated-p53 (K379), pmTOR (S2448), pRaptor (S792), p4E-BP1 (T36/47), pS6 (S235/236), pS6 (S240/244) and cleaved caspase-3. The band densities of the western blot images obtained were then quantified using the basic Quantity One software (Biorad, Inc. CA, USA). Trypan blue exclusion assay was carried out for cell viability analysis while MTS assay was carried out to analyze the cell proliferation status. Propidium iodide staining followed by FACS analysis on a BD LSRFortessa system (BD Biosciences, CA, USA) was performed for cell cycle analysis. All the data was analyzed using the statistical software GraphPad Prism 5.0 (GraphPad Software, Inc. CA, USA). Data is presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) and post-hoc comparisons between groups were performed by Tukey's multiple comparison tests. ‘p’ values less than 0.05 were considered to be statistically significant.ResultsGlucose starvation for 48 h in MS1-VEGF cells reduced cell proliferation and showed a significant increase in the levels of pAkt (S473) and a significant decrease in the levels of acetylated-p53 when compared to normal glucose exposed cells. mTOR is known to be phosphorylated at S2448 by Akt. The GS induced increase in the levels of pAkt (S473) can be related to the observed significant increase in the levels of pmTOR (S2448). The increase in the levels of pmTOR (S2448) also caused increase in the levels of downstream pS6 (S235/236) and pS6 (S240/244) in the glucose starved cells when compared to normal glucose exposed cells. Treatment with metformin (2 mM) for 48 h in MS1-VEGF cells subjected to GS significantly reduced cell viability. This can be related to the significant decrease in the levels of Sirt1 and pAkt (S473), which in turn would contribute to the increase in the levels of acetylated-p53 and decrease in pmTOR (S2448) levels in the metformin treated glucose starved cells when compared to non-treated glucose starved cells. Inhibition of mTOR pathway was confirmed by the significant decrease in the levels of downstream p4E-BP1 (T36/47), pS6 (S235/236) and pS6 (240/244) in metformin (2 mM) treated glucose–starved MS1-VEGF cells when compared to non-treated cells subjected to GS. In addition, the levels of pRap (S792), a negative regulator of mTOR activation, significantly increased in metformin treated glucose starved cells when compared to non-treated glucose starved cells. Inhibition of the Akt/mTOR pathway provides an explanation for the decrease in cell viability as evident by the accumulation of cells in the G2/M phase of the cell cycle. In addition to G2/M arrest, a significant decrease in the G0/G1 phase was observed while the cells in the sub-G0/G1 phase increased indicating cell death in the metformin treated glucose starved cells when compared to the non-treated glucose starved cells. The significant increases in the levels of acetylated-p53 and cleaved caspase-3 indicate that the cells have entered the apoptosis pathway. Treatment with 2 mM metformin also reversed the glucose starvation induced pro-survival autophagic response as evidenced by a decrease in the levels of LC3A-II and LC3B-II and marked reduction in the formation of LC3B stained punctae, when compared to non-treated glucose starved cells. Knockdown of AMPK revealed that this effect of metformin on GS induced autophagy is independent of AMPK.ConclusionOur findings indicate that using metformin in combination with agents that modify cancer cell metabolism (such as glycolytic inhibitors) is a therapeutic strategy that will selectively promote cancer cell death.This work was supported by the Qatar National Research Funds (QNRF): National Priorities Research Program (NPRP: 4-910-3-244, awarded to Dr. Chris R. Triggle), a Junior Scientist Research Experience Program (JSREP 03-016-3-009, awarded to Dr. Samson Mathews Samuel). We thank Ms. Aleksandra M. Liberska (Flow cytometry supervisor) and the Flow Cytometry Facility within the Microscopy Core at Weill Cornell Medicine-Qatar for contributing to these studies. The Core is supported by the “Biomedical Research Program at Weill Cornell Medicine-Qatar”, a program funded by Qatar Foundation.Reference[1] Arbiser, J. 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