Treatment with a Combination of Metformin and 2-Deoxyglucose Upregulates Thrombospondin-1 in Microvascular Endothelial Cells: Implications in Anti-Angiogenic Cancer Therapy

Abstract

Metformin, the most widely used anti-diabetic drug, also exhibits anti-cancer properties; however, the true potential of metformin as an anticancer drug remains largely unknown. In this study using mouse microvascular endothelial cells (MMECs), we investigated the effects of metformin alone or in combination with the glycolytic inhibitor, 2-deoxyglucose (2DG), on angiogenesis-a process known to be an integral part of tumor growth, cancer cell survival and metastasis. MMECs were exposed to 2DG (1–10 mM) for 48 h in the absence or presence of metformin (2 mM). The status of angiogenic and anti-angiogenic marker proteins, proteins of the mTOR pathway and cell-cycle-related proteins were quantified by Western blot analysis. Assays for cell proliferation, migration and tubulogenesis were also performed. We observed robust up-regulation of anti-angiogenic thrombospondin-1 (TSP1) and increased TSP1-CD36 co-localization with a marked decrease in the levels of phosphorylated vascular endothelial growth factor receptor-2 (pVEGFR2; Y1175) in 2DG (5 mM) exposed cells treated with metformin (2 mM). Additionally, treatment with metformin and 2DG (5 mM) inhibited the Akt/mTOR pathway and down-regulated the cell-cycle-related proteins such as p-cyclin B1 (S147) and cyclins D1 and D2 when compared to cells that were treated with either 2DG or metformin alone. Treatment with a combination of 2DG (5 mM) and metformin (2 mM) also significantly decreased cell proliferation, migration and tubulogenic capacity when compared to cells that were treated with either 2DG or metformin alone. The up-regulation of TSP1, inhibition of cell proliferation, migration and tubulogenesis provides support to the argument that the combination of metformin and 2DG may prove to be an appropriate anti-proliferative and anti-angiogenic therapeutic strategy for the treatment of some cancers.

Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms

“…Interestingly, the presence of metformin shifted the 2-DG response of prostate cancer cell lines from autophagy to apoptosis [114], and also triggered apoptosis in ovarian cancer cells and esophageal squamous cell carcinoma [158,159]. This combination also caused a decrease in angiogenesis in endothelial cells more efficiently than the drugs alone [160], in line with other investigations supporting the use of 2-DG to inhibit angiogenic potential [102,[161][162][163]. However, recent results showed that this combination induced a detachment of breast cancer cells in vitro that remain viable [110].…”

Metformin: The Answer to Cancer in a Flower? Current Knowledge and Future Prospects of Metformin as an Anti-Cancer Agent in Breast Cancer

“…Dosage of metformin administration as a monotherapy or in combination with other drugs or therapeutic modalities is critical to achieve therapeutic efficiency in the treatment with minimal side effects. When used orally in the treatment of diabetes, the anti-hyperglycemic effects of metformin have been reported at plasma concentrations ranging from around from 10-100 µM [68,137,203]. Interestingly, epidemiological, observational, and meta-analysis reports suggest that oral administration of metformin (normal initial oral dose for; 'immediate release' is 500 mg twice/day or 850 mg once/day, with 500 mg increments weekly as tolerated, maximum dose of 2550 mg/day; 'extended release' is 500 to 1000 mg once/day, with 500 mg increments weekly as tolerated, maximum dose of 2000 mg/day) in type 2 diabetic patients significantly reduced the risk of many different cancers when compared to diabetic patients on other anti-hyperglycemic treatment regimens [35,37,204,205].…”

“…Metformin use showed no effect on the incidence of cancers in studies that avoided these biases [201,206]. In the majority of in vitro/cancer cell based experiments, however, metformin treatment-related inhibitory effect on tumor cell proliferation, activation of apoptotic cell death, and inhibition of tumor progression was only observed at >2-5 mM concentrations of metformin [68,137,138,203,207]. Further studies are warranted to provide a clear insight into the possible dose-dependent effect of metformin on cancer prevention/risk reduction in 'diabetic' patients and cancer treatment in 'diabetic-cancer' patients.…”

Metformin: The next angiogenesis panacea?

“…In athymic BALB/c nu/nu mouse xenograft models (induced by injecting BIU-87 cells) treated with metformin combined with cisplatin, it markedly inhibited AKT/mTOR signaling pathway and its angiogenesis. 36 The analysis of Mathews Samuel et al 37 in mouse microvascular endothelial cells (MMECs) supports the possibility that metformin combined with 2-deoxyglucose (2DG) may be an appropriate anti-proliferative and anti-angiogenic treatment strategy. In gastric cancer (GC) cells, metformin was found to efficiently inhibit protein tyrosine phosphatase receptor delta (PTPRD)-loss-induced angiogenesis and reverse the decrease in PTPRD expression.…”