Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling

Abstract

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.

A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression

“…This can be observed in a range of DMOG concentrations commonly used to inhibit PHD (0.1-1 mM). We show that 1 mM DMOG reduces basal respiration and respiratory responses of HCT116 cells to sustained mitochondrial uncoupling with FCCP, and transient cytosolic Na + influx triggered by chelation of extracellular Ca 2+ with EGTA [49,58]. Under moderate hypoxia (e.g., 8% O 2 ) cells treated with DMOG cannot become hypoxic as they normally do due to active O 2 consumption (Fig.…”

“…To explore this novel effect in detail, we compared deoxygenation profiles in DMOG (+) and DMOG (−) cells transferred from higher (20.9%) to lower (8%) atmospheric O 2 . In conditions of decreased O 2 supply, iO 2 changes to another steady state level at a rate which correlates with the OCR [58]. We applied two respiration media: with glucose (regular) and galactose (enhanced OCR) and found that in both conditions DMOG greatly affected iO 2 dynamics (Fig.…”

“…2A). To better visualise the effect, we treated the cells with FCCP or EGTA which continuously (FCCP) or transiently (EGTA) increase cell respiration and deoxygenation [49,58] (Figs. 2A-B).…”

Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

“…no 5030) reconstituted in deionised water and filter-sterilised was used to prepare 12 different working media (WM) by adding 10 mM D-glucose (Glc), 10 mM D-galactose (Gal), 2 mM Gln, 1 mM pyruvate (Pyr) and 100 nM NGF, respectively [49]. Except for ECA rate analysis, all media contained 20 mM HEPES buffer (pH 7.2).…”

Geração de linhagens celulares HEK293 knockdown para as proteínas p53, ATM, mTOR e PGC1α e estudo do papel de p53 na resposta ao estresse oxidativo provocado por azul de metileno

“…Essa é uma estratégia bastante recente e interessante, pois pode contribuir para a melhor compreensão e tratamento de doenças, tais como o câncer. Em um estudo recente, foi demonstrado como a disponibilidade de substratos metabólicos pode modular a resposta respiratória frente ao desacoplamento mitocondrial em células transformadas (Zhdanov et al, 2014).…”