| Summary: | The first research strand in this thesis was to determine whether non-aspirin NSAIDs induce apoptosis of colorectal cancer cells via modulation of the NF-κB pathway and to elucidate the upstream mechanisms involved. Experimental evidence presented here shows that sulindac, sulindac sulfone and indomethacin cause NF-κB pathway activation. It was found that non-aspirin NSAIDs induce nucleolar translocation of the NF-κB component, RelA, repression of NF-κB driven transcriptional activity and apoptosis. Furthermore, in a similar manner to aspirin, nucleolar translocation of Re1A was demonstrated to be absolutely required for the apoptotic effects of sulindac, sulindac sulfone and indomethacin. However, in contrast to aspirin, activation of the NF-κB pathway by the non-aspirin NSAIDs was independent of IκBα degradation. The tyrosine kinase c-Src was found to be activated in response to NSAIDs and this activation was causally involved in the apoptotic mechanism of sulindac, sulindac sulfone and indomethacin. The role of the β-catenin pathway in NSAID-effects on the NF-κB pathway and apoptosis was investigated. Aspirin, sulindac, sulindac sulfone and indomethacin and induced activation of the β-catenin pathway and nucleolar sequestration of β-catenin. Blocking aspirin-induced nucleolar translocation of Re1A in response to aspirin treatment blocked nucleolar targeting of β-catenin, suggesting an interaction between the NF-κB and β-catenin pathways. It is increasingly apparent that post-translational modifications of Re1A, particularly acetylation and ubiquitination, are implicated in nuclear regulation of NF-κB. The final strand of research in this thesis was therefore to determine whether these modifications were involved in aspirin effects on NF-κB activity and apoptosis. By means of immunoprecipitation and western blot analysis, aspirin was found to cause an increase in ubiquitinated Re1A. Furthermore, the proteasome inhibitor, MG132, mimicked the effects of aspirin in terms of nucleolar translocation of Re1A and β-catenin, repression of NF-κB activity and increased ubiquitinated Re1A. Furthermore, both aspirin and MG132 mediate a reduction in proteasome levels and caused proteasome to localise to the nucleolus, strongly suggesting that Re1A and/or β-catenin are subject to ubiquitin-mediated proteolysis in the nucleolus. The finding that there is a decrease in acetylated Re1A after aspirin treatment and also implicates a role for acetylation in response to aspirin. Elucidating the mechanisms whereby NSAIDs induce apoptosis of colorectal cancer cells is of vital importance for the rational design of new therapeutic agents.
|
|---|
