ABSTRACT
BACKGROUND: Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells. METHODS: Live whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]D-glucose to [2H]-labelled L-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET). RESULTS: Intracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected. CONCLUSIONS: Glycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the 'centrality of the [Na+]i-NKA nexus' in the mechanistic basis of the Warburg effect.
ABSTRACT
Several specific metallic elements must be present in the human body to maintain health and function. Maintaining the correct quantity (from trace to bulk) and location at the cell and tissue level is essential. The study of the biological role of metals has become known as metallomics. While quantities of metals in cells and tissues can be readily measured in biopsy and autopsy samples by destructive analytical techniques, their trafficking and its role in health and disease are poorly understood. Molecular imaging with radionuclides - positron emission tomography (PET) and single photon emission computed tomography (SPECT) - is emerging as a means to non-invasively study the acute trafficking of essential metals between organs, non-invasively and in real time, in health and disease. PET scanners are increasingly widely available in hospitals, and methods for producing radionuclides of some of the key essential metals are developing fast. This review summarises recent developments in radionuclide imaging technology that permit such investigations, describes the radiological and physicochemical properties of key radioisotopes of essential trace metals and useful analogues, and introduces current and potential future applications in preclinical and clinical investigations to study the biology of essential trace metals in health and disease.
