Loveridge, Carolyn Janice

2018-03-29T12:18:20Z

2018-03-29T12:18:20Z

2007

There is substantial evidence that the use of aspirin and related nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with colorectal cancer prevention. Although some data indicates a role for inhibition of cyclooxygenase (COX) enzymes, the underlying mechanisms of the anti-tumorigenic effects of NSAIDs are complex and poorly understood. It has previously been shown by the host laboratory that aspirin activates the nuclear factor-kappa B (NF-kB) pathway via phosphorylation and degradation of the NF-kB inhibitor protein, IkBcl (Stark et al., 2001). More recent work has established that nucleolar translocation of the RelA component of NF-kB in response to aspirin is causally involved with repression of basal NF-kB transcriptional activity and apoptosis (Stark and Dunlop, 2005). The first research strand in this thesis was to determine whether non-aspirin NSAIDs also induce apoptosis of colorectal cancer cells via modulation of the NFkB pathway and to elucidate the upstream mechanisms involved. Experimental evidence presented here shows that sulindac, sulindac sulfone and indomethacin cause NF-kB pathway activation. It was found that non-aspirin NSAIDs induce nucleolar translocation of the NF-kB component, RelA, repression of NF-kB driven transcriptional activity and apoptosis. Furthermore, in a similar manner to aspirin, nucleolar translocation of RelA was demonstrated to be absolutely required for the apoptotic effects of sulindac, sulindac sulfone and indomethacin. Flowever, in contrast to aspirin, activation of the NF-kB pathway by the non-aspirin NSAIDs was independent of IkBcx degradation. By means of phospho-specific antibodies, chemical inhibition and genetic intervention, the tyrosine kinase c-Src was found to 1 be activated in response to NSAIDs and this activation was causally involved in the apoptotic mechanism of sulindac, sulindac sulfone and indomethacin. The host laboratory was the first to demonstrate nucleolar sequestration of RelA and so the second research strand in this thesis was to further understand the mechanisms involved. Beta catenin (P-catenin) has previously been shown to interact with RelA and modulate nuclear NF-kB activity (Deng et al., 2002). Therefore, the role of the ß-catenin pathway in NSAID-effects on the NF-kB pathway and apoptosis was investigated. Aspirin, sulindac, sulindac sulfone and indomethacin all induced activation of the (ß-catenin pathway and nucleolar sequestration of ß-catenin. Blocking aspirin-induced nucleolar translocation of RelA blocked nucleolar localisation of p-catenin, suggesting a possible interaction between the NF-kB and ßcatenin pathways. It is increasingly apparent that post-translational modifications of RelA, particularly acetylation and ubiquitination, are implicated in nuclear regulation of NF-kB. The final strand of research in this thesis was therefore to determine whether these modifications were involved in aspirin effects on NF-kB activity and apoptosis. By means of immunoprecipitation and Western blot analysis, aspirin was found to cause an increase in ubiquitinated RelA. Furthermore, the proteasome inhibitor, MG132, mimicked the effects of aspirin in terms of nucleolar translocation of RelA and P-catenin, repression of NF-kB activity and increased ubiquitinated RelA. Moreover, both aspirin and MG132 mediated a reduction in proteasome levels and caused proteasomes to localise to the nucleolus, providing support for the notion that RelA and/or P-catenin are subject to ubiquitin-mediated proteolysis in the nucleolus. Additionally, the finding that there is a decrease in acetylated RelA after aspirin treatment provides further evidence that acetylation is important in the response to aspirin. Collectively, the data in this thesis establishes a novel mechanism whereby NSAIDs induce apoptosis of colorectal cancer cells. Activation of the NF-kB pathway is critical in this process, although the upstream mechanism of activation differs between NSAIDs. Cross-talk between the NF-kB and P-catenin pathways could represent an important regulatory mechanism in response to NSAIDs and the nucleolus might be a key site for such cross-regulation. Moreover, post-translational modifications, including ubiquitination and acetylation, seem to play a significant role in sequestration to the nucleolus of key NF-kB and P-catenin pathway components. Elucidating the mechanisms whereby NSAIDs induce apoptosis of colorectal cancer cells is of vital importance for the rational design of new therapeutic agents.

http://hdl.handle.net/1842/29229

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 17

Already catalogued

Effects of nonsteroidal anti-inflammatory drugs on nuclear factor-kappaB and beta-catenin signaling in colorectal cancel cells

The effects of nonsteroidal anti-inflammatory drugs on nuclear factor-kappaB and beta-catenin signaling in colorectal cancel cells

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy