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

Abstract

The compound β-lapachone, a naturally derived naphthoquinone, has been utilized as a potent medicinal nutrient to improve health. Over the last twelve years, numerous reports have demonstrated distinct associations of β-lapachone and NAD(P)H: quinone oxidoreductase 1 (NQO1) protein in the amelioration of various diseases. Comprehensive research of NQO1 bioactivity has clearly confirmed the tumoricidal effects of β-lapachone action through NAD+-keresis, in which severe DNA damage from reactive oxygen species (ROS) production triggers a poly-ADP-ribose polymerase-I (PARP1) hyperactivation cascade, culminating in NAD+/ATP depletion. Here, we report a novel combination strategy with aminooxyacetic acid (AOA), an aspartate aminotransferase inhibitor that blocks the malate-aspartate shuttle (MAS) and synergistically enhances the efficacy of β-lapachone metabolic perturbation in NQO1+ breast cancer. We evaluated metabolic turnover in MDA-MB-231 NQO1+, MDA-MB-231 NQO1−, MDA-MB-468, and T47D cancer cells by measuring the isotopic labeling of metabolites from a [U-13C]glucose tracer. We show that β-lapachone treatment significantly hampers lactate secretion by ~85% in NQO1+ cells. Our data demonstrate that combinatorial treatment decreases citrate, glutamate, and succinate enrichment by ~14%, ~50%, and ~65%, respectively. Differences in citrate, glutamate, and succinate fractional enrichments indicate synergistic effects on central metabolism based on the coefficient of drug interaction. Metabolic modeling suggests that increased glutamine anaplerosis is protective in the case of MAS inhibition.

Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines

“…The cells were washed with cold 0.9% saline, then flash frozen in liquid nitrogen, and stored at −80°C as well. Cell pellets and media samples were processed and analyzed by gas chromatography‐mass spectrometry (GC‐MS), as reported previously 24,31,32 . GC‐MS data acquisition was accomplished with a Thermo Scientific Single Quadrupole Mass Spectrometer (ISQ) and Gas Chromatograph (Trace 1310).…”

“…Cell pellets and media samples were processed and analyzed by gas chromatography-mass spectrometry (GC-MS), as reported previously. 24,31,32 GC-MS data acquisition was accomplished with a Thermo Scientific Single Quadrupole Mass Spectrometer (ISQ) and Gas Chromatograph (Trace 1310). Amino acid peak areas were processed with XCalibur Quan Browser software and normalized to the peak area of a DL-norleucine internal standard.…”

Exploring the Significance, Extraction, and Characterization of Plant-Derived Secondary Metabolites in Complex Mixtures

“…GC-MS is one of the most widely utilized chromatographic techniques for the highthroughput screening of complex mixtures [61][62][63]. GC-MS can detect and quantify lipophilic metabolites including fatty acids, alkanes, sterols, phytohormones, and tocopherols [62,63].…”

Metabolomic Profiling of Asparagine Deprivation in Asparagine Synthetase Deficiency Patient-Derived Cells

“…Cell pellets were thoroughly washed with 0.9% sterile saline, flash frozen, and stored at −80 • C. Patient-derived fibroblasts were maintained in DMEM supplemented with 10% FBS, ABAM (streptomycin, penicillin G, and amphotericin B), 1X NEAA, and 2 mM of additional Gln. For experiments, 1 million fibroblasts were plated in 100 mm cell culture dishes and incubated for 24 h to allow adherence, then DMEM ± Asn was added, and cells were incubated for an additional 24 h. At the end of the incubation period, media was collected and stored at −80 • C. Cells were harvested by trypsinization, washed with 0.9% saline, flash frozen, and stored at −80 • C. Cell and media samples were prepared and subjected to GC-MS analysis on a Thermo Scientific Single Quadrupole Mass Spectrometer (ISQ) and Gas Chromatograph (Trace 1310), exactly as reported previously [22,[29][30][31].…”