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theses

Prostate Cancer is one of the leading diagnosed cancers and sixth leading cause of cancer deaths worldwide. Prostate cancer has a variety of risk factors including age, diet susceptibility genes and somatic gene defects, all of which can drive prostate carcinogenesis. Phytochemicals have been shown to act as a chemoprevention agent in carcinogenesis, where the chemicals "delay" or inhibit cancer at any step of initiation, promotion, progression or metastasis. The phytochemical curcumin has been shown to possess chemopreventive anti-cancer and anti-inflammation properties, both of which have been indicated in prostate cancer models. The master regulator of anti-oxidative stress, nuclear-factor erythroid 2 related factor (Nrf2) is responsible for many detoxifying and anti-oxidation enzymes. In prostate carcinogenesis, oxidative stress and inflammation are major factors in the development of prostate cancer and nrf2 is necessary to maintain a stable, non-toxic environment. In the present study, curcumin derivative FN1 was used to investigate its potential in restoring Nrf2 activity through modulation of epigenetic enzymes in mouse prostate cancer TRAMPC1 cells. Compounds were first tested on the HepG2-C8 stably transfected ARE-luciferase cell line to determine its Nrf2/ARE induction potential. Real time PCR and western blot were performed to determine levels of expression of Nrf2 related enzymes at protein and mRNA level. Bisulfite genomic sequencing (BGS) and methylated DNA immunoprecipitation (MeDIP) were used to determine methylation status of Nrf2 CpG islands. Anchorage-independent colony-formation analysis was performed to examine the compounds potential as a tumor inhibitor. FN1 revealed to be a significant inhibitor of colony formation. FN1 was found to be a more potent inducer of the Nrf2/ARE pathway than curcumin. Expression of Nrf2 and its downstream targets were increased by FN1. Epigenetic modifying enzymes were also found to be downregulated. Methylation status of Nrf2 through BGS and MeDIP revealed reduced methylation of CpG sites. These results indicate that in TRAMPC1 cells, FN1 can increase level of Nrf2 and its downstream genes by activation of the Nrf2/ARE pathway. In the next study, curcumin derivatives BDMC and DMC were used to investigate potential epigenetic activation of Nrf2 in human prostate cancer cells. LNCaP cells were treated with varying concentrations of the compound to determine cell viability. Real time PCR and western blot were performed to determine levels of expression of Nrf2 related enzymes as well as various epigenetic modifying enzymes, at protein and mRNA level. Bisulfite genomic sequencing (BGS) was used to determine methylation status of the human Nrf2 promoter region CpG islands. mRNA expression of Nrf2 and its downstream genes were induced by both curcumin derivatives. BDMC and DMC treatment resulted in inhibition of DNMT1 and DNMT3B while expression of DNMT3A was increased. Western blot analysis reflected mRNA results with increases of Nrf2 and its downstream targets at the protein level. HDAC proteins expression was inhibited by the curcumin derivatives. Methylation of Nrf2 CpG sites were not reduced by BDMC and DMC treatment. The derivatives of curcumin have shown to activate Nrf2 and its phase II detoxifying genes, by which mechanism, remains unclear and requires further investigation. In summary, the potential of these curcumin derived chemicals to act as a chemoprevention agents in prostate cancer is high. FN1 displayed the ability to restore expression of Nrf2 and its downstream genes via hypomethylation of Nrf2’s promoter CpG sites. BDMC and DMC displayed a potential to activate Nrf2, however, the two compounds were stronger inducers of Nrf2’s downstream genes, HO-1 and NQO-1.

ETD_7812

M.S.

Includes bibliographical references

by Douglas Pung

doi:10.7282/T3542R2S

ETD graduate

The author owns the copyright to this work.