Date on Master's Thesis/Doctoral Dissertation

8-2013

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Committee Chair

Fan, Teresa

Author's Keywords

Tumor metabolism; Lung cancer; Metabolomics; O-GlcNAc modification; Selenite chemoprevention; Pathway modeling

Subject

Lungs--Cancer--Treatment

Abstract

The following dissertation was aimed at characterizing the metabolic disruptions that occur with selenite treatment in human lung cancer. Specifically, the pathways of glycolysis, TCA cycle, nucleotide metabolism, pentose phosphate pathway, hexosamine biosynthetic pathway, and glutaminolysis were examined thoroughly. A stable isotope-resolved metabolomic (SIRM) approach utilizing the tracers 13C6-glucose and 13C5,15N2-Gln was employed in order to trace the flow of atoms through the metabolic network. By utilizing this approach and a combination of NMR and MS analyses, the metabolic pathways perturbed by selenite were reconstructed based on measured isotopologues and isotopomers and known biochemical reaction mechanisms. These methods were employed in both cell-based and individualized ex vivo lung cancer tissue models developed from an ongoing NSCLC patient study. In the A549 cell model, glycolysis was upregulated by selenite treatment with a corresponding decrease in flux through the TCA cycle and increase in flux through lactate dehydrogenase. These effects were propagated through nucleotide synthesis by inhibiting the rate of carbon and nitrogen incorporation into nucleobases. Furthermore, UDP-GlcNAc synthesis was diminished by selenite treatment, particularly in terms of its uracil subunit and acetyl group incorporation. These findings were specific to cancer cells and not observed in non-transformed NHBE cells and were recapitulated in the patient data. The KGA splice variant, GAC, was greatly reduced by selenite treatment, which was consistent with the metabolic data and reduction in glutaminase activity. Again, this effect was not observed in NHBE cells. Proliferation of A549 cells was at least partially restored upon supplementation of selenite-treated cells with Glu, the product of the glutaminase reaction, validating the importance of glutaminase in selenite-mediated cytotoxicity, including the synthesis of glutathione for detoxifying excess ROS. Finally, findings from the tissue slice data suggested that selenite induced a decrease in the synthesis of glycogen. The brain form of glycogen phosphorylase (PYGB) was suppressed using shRNA in order to characterize the metabolic consequences. Glycogen was identified as an important source of fuel for A549 cells, since suppression of PYGB resulted in reduced growth and colony formation accompanied by decreased nucleobase synthesis, TCA cycle activity, and amino acid synthesis.

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