Anti-tumor effects of everolimus and metformin are complementary and glucose-dependent in breast cancer cells

Abstract

BackgroundClinical efficacy of the mTOR inhibitor everolimus is limited in breast cancer and regularly leads to side-effects including hyperglycemia. The AMPK inhibitor and anti-diabetic drug metformin may counteract everolimus-induced hyperglycemia, as well as enhancing anti-cancer efficacy. We investigated the glucose-dependent growth-inhibitory properties of everolimus, metformin and the combination in breast cancer cell lines.MethodsThe breast cancer cell lines MCF-7, MDA-MB-231 and T47D were cultured in media containing 11 mM or 2.75 mM glucose with 21% or 1% oxygen. Everolimus and metformin treated cells were subjected to cytotoxicity and clonogenic assays, western blotting, FACS and metabolic measurements.ResultsEverolimus was less effective in MCF7 cells under low glucose conditions compared to high glucose conditions (IC50 of >50 nM vs 29.1 ± 1.4 nM) in a short-term survival assay, while sensitivity of MDA-MB-231 and T47D cells to everolimus was lost under low glucose conditions. In contrast, metformin was more effective in low than in high glucose conditions in MCF7 (IC50 of 1.8 ± 1.2 mM vs >5 mM) and MDA-MB231 cells (1.5 ± 1.3 mM vs 2.6 ± 1.2 mM). Metformin sensitivity of T47D cells was independent of glucose concentrations. Everolimus combined with metformin additively inhibited cell survival, clonogenicity, mTOR signaling activity and mitochondrial respiration. These effects were not the result of enhanced autophagy or apoptosis induction. Similar results were observed under hypoxic conditions.ConclusionMetformin-induced effects are additive to the anti-proliferative and colony inhibitory properties of everolimus through inhibition of mitochondrial respiration and mTOR signaling. These results warrant further in vivo investigation of everolimus combined with metformin as a putative anti-cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3230-8) contains supplementary material, which is available to authorized users.

Comparison of the effects of metformin on MDA-MB-231 breast cancer cells in a monolayer culture and in tumour spheroids as a function of nutrient concentrations

“…Limited availability of glucose and/or other nutrients increases the sensitivity of cancer cells to metformin [17,22,23,26,31–39,46,47]. However, excepting glucose, the role of other nutrients and their combinations on the sensitivity of MDA-MB-231 cells to metformin was not examined in detail.…”

“…Metabolic pathways of cancer cells in vitro vary also between monolayer cultures and physiologically more relevant tumour spheroids [9–12]. Nutrient availability and metabolic phenotype can modify the sensitivity of cancer cells to pharmacological compounds that target cancer cell metabolism, such as metformin [17,22,23,26,31–39,46,47]. Recently, we have shown that maintaining constant glucose concentrations by daily medium renewal blocks the effects of metformin on MDA-MB-231 cells [31].…”

“…Recently, we have shown that maintaining constant glucose concentrations by daily medium renewal blocks the effects of metformin on MDA-MB-231 cells [31]. In contrast, MDA-MB-231 cells are sensitive to metformin in glucose-depleted conditions [31,32,36–39,47]. In the present study, we compared the effects of three major nutrients, glucose, glutamine and pyruvate, on the sensitivity of MDA-MB-231 cells to metformin in a monolayer culture and in tumour spheroids.…”

Novel application of metformin combined with targeted drugs on anticancer treatment

“…42,43 In breast cancer cells, cotreatment with metformin and everolimus intensified the inhibition of cell proliferation and colony formation. 44 Metformin and everolimus significantly suppressed obesity-induced tumor growth (0.5-fold and 0.3-fold). 45 It was also reported that the effect of everolimus might be associated with insulin resistance, manifesting in impaired glucose tolerance or hyperglycemia, whereas metformin could restore it.…”

“…In breast cancer cells, cotreatment with metformin and everolimus intensified the inhibition of cell proliferation and colony formation . Metformin and everolimus significantly suppressed obesity‐induced tumor growth (0.5‐fold and 0.3‐fold) .…”