Abstract
To fully understand the glycolytic behavior of cancer cells, it is important to recognize how it is linked to pH dynamics. Here, we evaluated the acute effects of mild acidification and alkalization on cancer cell glucose uptake and glycolytic flux and investigated the role of hexokinase (HK). Cancer cells exposed to buffers with graded pH were measured for 18F-fluorodeoxyglucose (FDG) uptake, lactate production and HK activity. Subcellular localization of HK protein was assessed by western blots and confocal microscopy. The interior of T47D breast cancer cells was mildly alkalized to pH 7.5 by a buffer pH of 7.8, and this was accompanied by rapid increases of FDG uptake and lactate extrusion. This shift toward glycolytic flux led to the prompt recovery of a reversed pH gradient. In contrast, mild acidification rapidly reduced cellular FDG uptake and lactate production. Mild acidification decreased and mild alkalization increased mitochondrial HK translocation and enzyme activity. Cells transfected with specific siRNA against HK-1, HK-2 and voltage-dependent anion channel (VDAC)1 displayed significant attenuation of pH-induced changes in FDG uptake. Confocal microscopy showed increased co-localization of HK-1 and HK-2 with VDAC1 by alkaline treatment. In isolated mitochondria, acidic pH increased and alkaline pH decreased release of free HK-1 and HK-2 from the mitochondrial pellet into the supernatant. Furthermore, experiments using purified proteins showed that alkaline pH promoted co-immunoprecipitation of HK with VDAC protein. These findings demonstrate that mild alkalization is sufficient to acutely trigger cancer cell glycolytic flux through enhanced activity of HK by promoting its mitochondrial translocation and VDAC binding. This process might serve as a mechanism through which cancer cells trigger the Warburg effect to maintain a dysregulated pH.
Highlights
The Warburg effect refers to the inclination of cancer cells to produce energy predominantly through a heightened rate of glycolysis and lactate production [1]
Intracellular pH could be accurately measured using emitted fluorescence ratios from BCECF-AM indicators that closely correlated to pH standards applied to cell interior (Fig 1A)
When this technique was applied to T47D cells exposed for 30 min to graded pH, intracellular pH was shifted toward the applied buffer pH in a close linear manner
Summary
The Warburg effect refers to the inclination of cancer cells to produce energy predominantly through a heightened rate of glycolysis and lactate production [1]. This likely represents a response to an increased demand for energy and biomass substrates to promote their survival and proliferation [2,3]. Cancer cells have a reversed pH gradient with a slightly elevated intracellular pH despite an acidic microenvironment [6], and this property has a central role in tumor biology. Contrariwise, the Warburg effect serves a functional role in maintaining pH dysregulation by augmenting acid generation through a shift of metabolism toward glycolysis. It has been observed that cellular acidification and alkalization stimulates shifts of metabolic patterns in a rapid manner [18,19,20], which cannot be explained by the delayed effects of transcriptional control
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