Measuring NQO1 Bioactivation Using [²H₇]Glucose

Treatment of cancers with β-lapachone causes NAD(P)H: quinone oxidoreductase 1 (NQO1) to generate an unstable hydroquinone that regenerates itself in a futile cycle while producing reactive oxygen species (ROS) in the form of superoxide and subsequently hydrogen peroxide. Rapid accumulation of ROS damages DNA, hyperactivates poly-ADP-ribose polymerase-I, causes massive depletion of NAD⁺/ATP, and hampers glycolysis. Cells overexpressing NQO1 subsequently die rapidly through an NAD⁺-keresis mechanism. Assessing changes in glycolytic rates caused by NQO1 bioactivation would provide a means of assessing treatment efficacy, potentially lowering the chemotherapeutic dosage, and reducing off-target toxicities. NQO1-mediated changes in glycolytic flux were readily detected in A549 (lung), MiaPaCa2 (pancreatic), and HCT-116 (colon) cancer cell lines by ²H-NMR after administration of [²H₇]glucose. The deuterated metabolic products ²H-lactate and HDO were quantified, and linear relationships with glucose consumption for both products were observed. The higher concentration of HDO compared to ²H-lactate allows for more sensitive measurement of the glycolytic flux in cancer. Gas chromatography-mass spectrometry analysis agreed with the NMR results and confirmed downregulated energy metabolism in NQO1⁺ cells after β-lapachone treatment. The demonstrated method is ideal for measuring glycolytic rates, the effects of chemotherapeutics that target glycolysis, and has the potential for in vivo translation.