Background: Cancer is one of the leading causes of mortality. Among women the most common cancer is breast cancer, which represents 21 % of all cancers. Metabolic disorders, such as obesity and type 2 diabetes mellitus, are important risk factors for development of breast cancer. On the other side, metabolic changes are one of the hallmarks of cancer cells, so the substances that affect metabolism, could be used in treatment of breast cancer. One of those substances is metformin, which is used for the treatment of diabetes. Aim: It was recently discovered that metformin, which inhibits complex I of respiratory chain in combination with 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, causes detachment of cancer cells in cell culture. In contrary with general accepted belief, that detached cells are dead, high percent of those cells are viable and able of continuing proliferation. Aim of our work was, to find out if other inhibitors of respiratory chain work on breast cancer cells, like metformin. We used MDA-MB-231 cell, the cell line of triple negative breast cancer as our experimental model. Hypotheses: (1) For survival of floating and attached MDA-MB-231 cells, glycolysis is more important than oxidative phosphorylation. (2) Combined treatment with metformin and glycolysis inhibitor 2-DG has a similar effect on MDA-MB-231 cells as combination of other inhibitors of the respiratory chain and 2-DG. (3) Hypoxia induces expression of HIF-1α in floating and attached MDA-MB-231 cells. (4) Glycolysis inhibition with 2-DG impair survival of floating and attached MDA-MB-231 cells in real hypoxia, and not in chemically-induced hypoxia. Methods: We evaluated the cell number and survival of MDA-MB-231 cells with propidium iodide staining and the method of flow cytometry. For detection of reactive oxygen species, we used CM-H2DCFDA staining and the method of flow cytometry. Expression of proteins and signal pathways were evaluated with western blot. Results: The analysis of the floating MDA-MB-231 cells has shown that 20 to 95 % of cells remain viable when different metabolism inhibitors are used. Survival of cells was lower with inhibition of glycolysis with 2-DG, than with inhibition of respiratory chain with metformin or others respiratory chain inhibitors. Inhibition of respiratory chain has significantly lowered the survival of MDA-MB-231 cells only when the cells were grown in the absence of glucose, indicating that MDA-MB-231 cells have a functional respiratory chain, but their survival is more dependent on glycolysis than oxidative phosphorylation. We thus confirmed our first hypothesis. Combined treatment with 2-DG and metformin or 2-DG and other mitochondrial respiratory chain inhibitors, like antimycin A, sodium azide and oligomycin, had a similar synergic effect on detached living MDA-MB-231 cells. This result support hypothesis two. In attached MDA-MB-231 cells hypoxia (1 % O2) induced expression of transcription factor HIF-1α, while in floating MDA-MB-231 cells HIF-1α expression was reduced in both normoxia and hypoxia. This result partially confirmed our third hypothesis. We also found that the inhibition of glycolysis with 2-DG reduced survival of attached MDA-MB-231 cell in real and chemically-induced hypoxia, induced with CoCl2, which does not support our fourth hypothesis. Conclusions: Inhibition of the respiratory chain and/or glycolysis cause the living cancer cell to detach. Functional characteristic in floating cancer cells are different than in attached cancer cells. While studying metabolism modulators and their effect on cancer cells, it is important that we analyze both, floating and attached cells.
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