Abstract
Triple naegative breast cancer has an increased rate of distant metastasis and consequently poor prognosis. To metastasize, breast cancer cells must detach from the main tumour mass and resist anoikis, a programmed cell death induced by lack of cell-extracellular matrix communication. Although cancer cells must detach to metastasize in vivo, the viability of floating cancer cells in vitro is rarely investigated. Here we show that co-treatment of anoikis-resistant MDA-MB-231 cells with metformin and 2-deoxy-D-glucose (2-DG) increased the percentage of floating cells, of which about 95% were viable. Floating cells resumed their proliferation once they were reseeded in the pharmacological compound-free medium. Similar effects on detachment were observed on anoikis-prone MCF-7 cells. Co-treatment of MDA-MB-231 cells with metformin and 2-DG induced a strong activation of AMP-activated protein kinase (AMPK), which was reduced by AMPK inhibitor compound C that prevented detachment of MDA-MB-231 cells. However, direct AMPK activators A-769662 and AICAR did not have any major effect on the percentage of floating MDA-MB-231 cells, indicating that AMPK activation is necessary but not sufficient for triggering detachment of cancer cells. Our results demonstrate that separate analysis of floating and attached cancer cells might be important for evaluation of anti-cancer agents.
Highlights
Metabolic adaptations enable survival of cancer cells in an anchorage-independent condition[6, 15,16,17,18]
Our results show that combined treatment of MDA-MB-231 cells with metformin and 2-deoxy glucose (2-DG) results in substantial number of viable floating cells, which is in contrast to commonly accepted belief that the floating cells in vitro are dead
To inhibit glycolysis in the medium with glucose (5.6 mM), we used 600 μM 2-deoxy glucose (2-DG), a concentration that can be achieved in human serum after oral administration of 2-DG36
Summary
Metabolic adaptations enable survival of cancer cells in an anchorage-independent condition[6, 15,16,17,18]. Oncogenes restore redox and energy balance and prevent anoikis in breast cancer cells[18] Another way to prevent anoikis in cancer cells is by antioxidant treatment or through the upregulation of NADPH producing pathways[15, 18]. While supraphysiological AMPK activation suppresses growth of fast proliferating cancer cells[20, 21], it enables survival of non-proliferating cancer cells in unfavourable conditions by stimulating fatty acid oxidation through inhibition of acetyl-CoA carboxylases (ACC)[15]. We have found that combined treatment with metformin and low concentrations of 2-DG induces detachment of adherent MDA-MB-231 cells from the bottom of standard cell culture plates in vitro. Our results show that combined treatment of MDA-MB-231 cells with metformin and 2-DG results in substantial number of viable floating cells, which is in contrast to commonly accepted belief that the floating cells in vitro are dead
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