| Summary: | Nrf2 (Nuclear factor erythroid 2-related factor 2), a redox-sensitive transcription factor, plays a critical role in the regulation of cellular defence and contributes to a number of cellular processes. Nrf2 is regulated through an interplay of complex transcriptional and post-translational mechanisms that modulates its activity during cellular perturbations or other biological processes thereby ensuring cellular homeostasis is maintained through the orchestration of adaptive responses. However, there is mounting evidence that constitutive upregulation of the Nrf2 pathway drives the enhanced proliferation and chemoresistance of various cancers. Therefore, an ability to modulate the activity of the Nrf2 pathway holds promise as a therapeutic strategy in certain disease settings. The work presented in this thesis showed that CDDO-Me provoked the induction of the Nrf2 pathway in C57BL6J WT and Nrf2 KO mice and CD1 WT mice. Analysis of CDDO-Me induced gene expression changes in both WT and Nrf2 KO mice showed a significant increase in the relative mRNA levels of ARE-dependent genes in the livers of CDDO-Me treated WT animals. Notably, CDDO-Me also provoked the accumulation of Nrf2 and NQO1 in human PBMCs and PHHs demonstrating its translational relevance. The mechanism of action of CDDO-Me as an inducer of Nrf2 is poorly understood. It was shown here that CDDO-Me post-transcriptionally evoked concentration and time-dependent, accumulation of Nrf2 protein in Hepa1c1c7 cells. Furthermore, CDDO-Me was shown to stabilize Nrf2 protein independently of the modulation of protein kinases and other signalling pathways that are purported to regulate Nrf2 activity. The work here also provides in vitro insights into the molecular mechanism of Nrf2 inhibition by the quassinoid brusatol. Brusatol post-transcriptionally evoked concentration- and time-dependent, yet transient, depletion of basal and inducible protein levels of Nrf2 in Hepa-1c1c7 cells. Furthermore, the ability of brusatol to inhibit Nrf2 was not affected by siRNA depletion of Keap1. In keeping with the latter observation, brusatol induced the depletion of Nrf2 independently of the proteasome and autophagic degradation machineries. Thus, these findings indicate that brusatol exploits a previously unknown mechanism of Nrf2 degradation. By examining the molecular mechanisms underlying the activation of Nrf2 by CDDO-Me and its inhibition by brusatol, this work reveals novel aspects of regulation within this important cellular pathway, and informs the design of new pharmacological inducers and inhibitors, which hold promise as therapeutic agents in a number of diseases.
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