Tracking the Biochemical Activities in Cultured Cancer Cells Using Nuclear Magnetic Resonance

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August 2022

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Abstract

The human body, on average, is made up of approximately 30 to 40 trillion cells which are divided into subgroups as organs and tissues with specific roles and functions, along with a regular and controlled growth mechanism. Cancer forms when some of these cells mutate and become rebellious, manifesting in uncontrollable growth and abnormally rapid proliferation. As cancer cells multiply rapidly, there is an immediate need for new raw materials and nutrients to sustain its hyperactive metabolic machinery. Thus, most of the biochemical pathways in cancer are abnormally hyperactive to satisfy its voracious appetite to multiply into new cells. This Ph.D. dissertation entails a discussion of using nuclear magnetic resonance (NMR) spectroscopy to track the aberrant biochemical activities of cancer cells at the molecular level. Chapter 1 of this dissertation includes an introductory discussion on the 3 cancer cell types that I have investigated: pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and glioblastoma multiforme (GBM) cell lines, and the experimental techniques that I used to study these cells: NMR spectroscopy, electron spin resonance (ESR), Western blot, and the NMR enhancing technique Overhauser effect dynamic nuclear polarization (DNP). In this chapter, I also discussed the fundamental principles of NMR and the basic details of the other experimental techniques. Chapter 2 of this thesis is about the effect of the chemotherapeutic drug beta-lapachone on the metabolism of the biochemical tracer [1,3-13C2] ethyl acetoacetate (EAA) in PDAC and CRC cells. The main finding of this study was that the metabolism of EAA is reasonably rapid in these cells, with acetate and beta-hydroxybutyrate as some of the metabolic byproducts. The enzyme NQO1 converts β-lapachone into a cancer-killing reactive oxygen species; details of its effects on EAA metabolism in PDAC and CRC cells will be discussed. Chapter 3 discusses the use of the biochemical tracer [1-13C1]α-ketoisocaproate (KIC) to study the hyperactivity of the branched-chain amino acid transferase (BCAT) enzyme in glioblastoma cells. 13C NMR results revealed that 13C-labeled KIC was converted abundantly into the amino acid leucine due to overexpressed and hyperactive BCAT enzymatic activity in SFxL glioblastoma cells. Further study was done using a BCATc inhibitor in which 13C NMR unveiled specific details as to the disruption of this metabolic pathway by this inhibitor ting details from microscopy and Western blot results. Chapter 4 details the fundamentals, instrumental setup, and preliminary results of the Overhauser effect DNP. Herein, the mechanism of electron spin polarization transfer to the nuclear spins is provided as well as the detailed instrumental assembly and setup of the homebuilt Overhauser DNP machine. This DNP setup is a combination of NMR and microwave technologies in pursuit of enhancing the NMR signals at room temperature. This project is still ongoing, but preliminary proton NMR results are presented. The rest of the dissertation entails technical details on the operation and data acquisition of various instruments and techniques used in this thesis. Overall, this PhD thesis provides a compilation of research works on tracking the abnormal biochemical activities in cancer cells using 13C NMR spectroscopy to turn these metabolic aberrations into diagnostic advantages for early detection and metabolic assessment of this disease.

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Physics, Nuclear, Physics, Radiation

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