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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

BMC Cancer 1/2017

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

BMC Cancer > Ausgabe 1/2017
Gerke Ariaans, Mathilde Jalving, Emma Geertruida Elisabeth de Vries, Steven de Jong
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12885-017-3230-8) contains supplementary material, which is available to authorized users.



Clinical 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.


The 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.


Everolimus 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.


Metformin-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.
Additional file 1: Figure S3. Uncoupling of the mitochondrial respiration with FCCP after treatment with metformin and everolimus for 48 h. Using the seahorse XF analyzer, the basal OCR and maximal OCR after uncoupling of mitochondrial respiration with FCCP of MCF-7 (A) and MDA-MB-231 cells (B) in response to 48 h of metformin or everolimus treatment was determined. Basal OCR in untreated cells was set at 100% as a reference to which all other mitochondrial respiration values of the same glucose concentration group were correlated. Data are presented as mean ± SD of three different experiments. (PDF 69 kb)
Additional file 2: Figure S1. Glucose concentration of cell culture medium during 4 days of metformin treatment. MCF-7 and MDA-MB-231 cells were plated at a concentration of 30.000 (MCF-7) or 80.000 (MDA-MB-231) cells in medium containing 11 mM or 2.75 mM glucose. Additionally, cells were plated in medium containing 2.75 mM glucose and replenished with 2.75 mM glucose every 24 h (2.75 mM glucose replenished). Cells were treated with 5 mM metformin for 4 days. 20 μl medium samples were taken for glucose concentration measurements every day. In the glucose-supplemented condition, this was done after addition of glucose. Data are presented as mean ± SD of three different experiments. (PDF 35 kb)
Additional file 3: Figure S2. Metformin and everolimus inhibit colony formation of breast cancer cell lines independently of glucose concentration. MCF-7 cells were plated in medium containing 11 mM or 2.75 mM glucose at a concentration of 500 cells/well. A subset of cells plated in 2.75 mM glucose was also replenished with 2.75 mM glucose every 48 h (2.75 mM glucose replenished). Cells were treated with indicated concentrations of everolimus and metformin for 8 days and colonies were counted. Data are presented as mean ± SD of three different experiments. (PDF 33 kb)
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