Measuring NQO1 Bioactivation Using [2H7]Glucose

Abstract

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.

Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency

“…The cell pellets were washed twice in ice-cold PBS, and final pellets were flash frozen in liquid nitrogen and stored frozen at −80 °C until processing. Cell pellets and media samples were processed and analyzed by GC-MS exactly as in published methods ( 34 ). In brief, cell pellets were spiked with DL-norleucine internal standard, extracted with acetonitrile:isopropanol:water (3:3:2 by volume), dried with a speedvac, reconstituted in acetonitrile:water (1:1 by volume), and dried in GC-MS reaction v-vials.…”

Synergistic Effect of β-Lapachone and Aminooxyacetic Acid on Central Metabolism in Breast Cancer

“…One way to significantly offset and further drive this mechanism is to target other metabolic pathways that are highly dependent on NAD + /NADH. Our previous work has shown a significant reduction in flux through metabolic pathways that are vulnerable to NAD + /NADH imbalance including branched-chain amino acid metabolism and the malate-aspartate shuttle [ 23 ]. In this work, we targeted the malate-aspartate shuttle with AOA as a strategy to disconnect cytoplasmic and mitochondrial NADH pools in order to further disturb cellular redox balance and central metabolism.…”

“…Chemotherapeutic treatment of cancers with β-lap bioactivates NQO1, yielding a highly unstable hydroquinone that auto-oxidizes in an aggressive futile cycle and generates large amounts of reactive oxygen species (ROS) that are ultimately transformed to hydrogen peroxide [ 19 , 20 , 21 ]. The accelerated accretion of hydrogen peroxide causes overwhelming DNA damage, which hyperactivates poly-ADP-ribose polymerase-I (PARP1), inducing substantial global depletion of NAD + and ATP, thus hindering glycolysis, cellular redox balance, and downstream metabolism [ 22 , 23 ]. Through this mechanism, cancer cells that overexpress NQO1 consequently die from NAD + -keresis [ 24 ].…”

“…The fractional enrichment of citrate, glutamate, and succinate isotopologues demonstrated a synergistic downregulation of central metabolism as determined through the calculation of the coefficient of drug interactions. We have previously shown a significant β-lap-dependent reduction in isotopic labeling in citrate and other central metabolites utilizing a [ 2 H 7 ]glucose tracer [ 23 ]. Here, we enhance the metabolic effects of β-lap by combining treatment with a sublethal dose of AOA and demonstrate a proof of principle for a plausible and novel combination strategy targeting breast cancer metabolic vulnerability.…”

Enrichment of hepatic glycogen and plasma glucose from H₂18O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice

“…Fifty microliters of MTBSTFA + 1% TBDMS reagent was added to the vial and incubated at 60°C for 1 h. The reaction solution was transferred into a sample vial and GC–MS analysis was performed using the same instrument as for the glucose aldonitrile pentapropionate derivative. The following parameters were used for GC–MS analysis of glycerol 3‐TBDMS derivative: the initial oven temperature of GC was 60°C for 1 min followed by a ramp of 10°C/min up to 325°C and finally a hold time of 5 min 22 . The isotopic 18 O and natural abundance correction was performed as for the glucose aldonitrile pentapropionate fragments.…”