Graduation Date

Fall 12-18-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Rebecca Oberley-Deegan

Abstract

Prostate cancer patients are often treated with radiotherapy. MnTE-2-PyP, is a superoxide dismutase (SOD) mimic and a known radioprotector of normal tissues. Our recent work demonstrates that MnTE-2-PyP also inhibits prostate cancer progression with radiotherapy; however, the mechanisms remain unclear. In this thesis, we identified that MnTE-2-PyP-induced intracellular H2O2 levels are critical in inhibiting growth of prostate cancer cells. We found that MnTE-2-PyP induced protein oxidations in PC3 cells and one major group of oxidized protein targets were involved in energy metabolism. The oxidative phosphorylation rates were significantly enhanced in both PC3 and LNCaP cells with MnTE-2-PyP treatment, but mitochondrial membrane potential was unaffected. In addition, MnTE-2-PyP significantly increased NAD(P)+/NAD(P)H ratios in PC3 and LNCaP cells in a dose-dependent manner, which was mainly due to a reduction of cellular NAD(P)H pool. Correspondingly, we observed a significant decrease of activity in glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), which are major cellular NADPH producing enzymes in pentose phosphate pathway. A decrease of GSH/GSSG ratios were confirmed in MnTE-2-PyP-treated prostate cancer cells, which may result from the decreased glutathione reductase (GR) activity due to NADPH depletion. We also identified the oxidation of Ser/Thr protein phosphatase 1 beta catalytic subunit (PP1CB), and a decrease of PP1CB activity in MnTE-2-PyP-treated prostate cancer cells. One key protein, pRB, regulates cell cycle progression that is downstream target of PP1CB was hypophosphorylated in MnTE-2-PyP-treated prostate cancer cells. Significant increase of Ki67-negative populations were observed in both PC3 and LNCaP cells but overall cell cycle progression was not altered, which indicates v interfering cell cycle progression is not the major mechanism of MnTE-2-PyP-induced cell growth inhibition. Besides protein oxidation, MnTE-2-PyP also caused nuclear abnormalities in prostate cancer cells. High H2O2 levels by MnTE-2-PyP treatment induced nuclear fragmentation in PC3 cells, which could be synergistically enhanced with radiotherapy both in vitro and in vivo. In LNCaP cells, disturbing H2O2 balance may contribute to the bi-nucleation phenomenon. The increased H2O2 levels, protein oxidative modifications, mitotic catastrophe, cellular energy metabolism alterations, and NAD(P)H depletion caused by MnTE-2-PyP are all likely factors contributing to prostate cancer cell growth inhibition.

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