Date Available

1-29-2017

Year of Publication

2017

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Toxicology and Cancer Biology

Advisor

Dr. Richard M. Higashi

Abstract

Studies were carried out to understand how human lung cancer cells and human ex vivo lung cancer tissues that are metabolically reprogramed compared with analogous non-cancer cells or non-cancer tissues. A Stable isotope resolved metabolomics (SIRM) approach was used to fulfill this aim by employing 13C, 2H or 15N labeled metabolic precursors like 13C6-Glc, 13C2-Gly, 2H2-Gly, 2H3-Ser, 13C5, 15N2-Gln to trace the flow of the labeled atoms into the down stream metabolic network. NMR and mass spectrometry are two major analytical tools utilized in these types of metabolic studies. Regarding mass spectrometry, to be able to resolve neutron mass difference between C, H, N in complex metabolite pools and obtain reliable isotopologue data for each metabolite in trace sample size from human, an ultra-high resolution Fourier transform mass spectrometry (UHR-FTMS) with highly sensitive electro-spray is required. However, direct infusion UHR-FTMS analysis of small polar metabolites has been difficult to achieve.

In this dissertation, analytical derivatization methods were developed for small amino and carboxyl metabolites to make them quantitatively analyzable by UHR-FTMS. These methods were employed on both human lung cell lines and tissues resected from surgery. Metabolic pathways like glycolysis, the Krebs Cycle, one-carbon metabolism, nucleotide synthesis and protein turnover were examined and profiled from the analysis of amino and carboxyl metabolites such as amino acids.

Relevant to this analysis, Ala can be de novo synthesized from pyruvate, which is the product of glycolysis. Asp and Glu can be synthesized from the Krebs Cycle intermediates oxaloacetic acid and α-ketoglutarate. Gly and Serine exchange is part of the one-carbon metabolism and they serve as the precursors of the nucleotide synthesis. As the amino acids are the building blocks of protein, the amino acids turnover in protein can also reveal the protein turnover.

The activities of glycolysis and the Krebs Cycle differ significantly between different lung cell lines. However, by comparing the lung cancer tissue and non-cancer tissue generated from the same patient, it is clear that the glycolysis activity is increased in cancer tissue since the de novo synthesized alanine is increased. The different labeling patterns of aspartate indicate enhanced activity of pyruvate carboxylase in cancer tissues. In addition, the Gly and Ser exchange activity seems more active in cancer tissues. Furthermore, we traced the labeling flow to nucleotide synthesis via the labeled atoms from glycine or serine, and found preferred precursor source for nucleotide synthesis. The tissues were also treated with different drugs to investigate the drug effect on cancer or non-cancer tissues. The maintained 3D structure and micro-environment of tissues present a more convincing result than the cell experiments.

Finally, immortalized human bronchial epithelial BEAS-2B cells are believed to be transforming with chronic arsenic exposure, which were examined with time course experiments to study how the arsenic alters the metabolism chronically in BEAS-2B cells. The effects of arsenic exposure and various culture conditions on the metabolism of BEAS-2B cells were further studied.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.020

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