The role of heat shock proteins in colorectal diseases

Introduction: This thesis examines the roles of Hsp10, Hsp60, and Hsp7O in colorectal diseases, in particular Inflammatory Bowel Disease, Colonic Polyps, and Colorectal Cancer. HSPs are reported to be elevated in a number of human diseases, including autoimmune diseases and cancers. On a daily basis the colon is exposed to a number of harsh stresses, including fermentation of foodstuffs as well as a high bacterial load. These stresses are thought to possibly induce the expression of HSPs in colonic mucosal cells, where it is thought that they may influence cellular function. HSPs are known to regulate apoptosis and coordinate certain immune functions that may provide either beneficial or detrimental effects depending upon the situation. Deranged apoptosis and inappropriately coordinated immune responses, possibly as a consequence of HSP activity, are thought to be implicated in the pathogenesis of some colorectal diseases. This project aims to find correlations in the concentration of a number of important HSPs in colorectal mucosa with colorectal diseases to enhance our knowledge of the roles that HSPs might play in pathogenesis of inflammatory bowel disease, colorectal polyps and colorectal cancer. Methods: 81 subjects were recruited prior to colonoscopy or sigmoidoscopy and consented to the removal of blood and colorectal mucosal biopsies for the purpose of this study. Colonic mucosal biopsies were analysed for protein levels of Hsp70 by ELISA, in addition the mRNA expression of HsplO, Hsp60 and Hsp70 was quantified by RT-PCR in colonic mucosal specimens. A further 20 patients were venesected so that whole lysed blood could be analysed by flow cytometry to determine the cellular localisation of Hsp70 concentration in neutrophils, monocytes and lymphocytes, to understand the involvement of Hsp70 in the coordination of immune responses appropriate to colorectal diseases. Kruskal-Wallis, Mann-Whittney, and Linear regressional analysis were performed to determine statistical significance. Results: Endoscopic appearance and pathological diagnosis separated the 81 patients into the following groups: normals (n = 42), inflammatory lesions (n = 23), polyps (n = 10), and colorectal cancer (n = 6). Hsp70 protein expression appears to be most elevated in normal mucosa, while lower levels are measured in inflammatory lesions and polyps, and minimal levels seen in colorectal cancer specimens (P-value = 0.447). A similar pattern is seen in the levels of Hsp70 mRNA expression, again with lowest levels measured in the colorectal cancer specimens (P-value = 0.528). The gene expression of both HsplO (P-value = 0.977) and Hsp60 (P-value = 0.245) is raised in inflammatory and polypoid lesions, but as with Hsp70, is lower in colorectal cancer specimens than in healthy colonic mucosa. Too few patients with colorectal disease were evident among the 20 patients for whom blood was sampled for flow cytometry therefore statistical significance was not achieved. The percentage of neutrophils and monocytes expressing Hsp7O was maximal in the colorectal cancer patient and lowest among healthy patients, however the average concentration of Hsp70 on these cells was lower in the colorectal cancer patient than healthy patients. Discussion: Pathological group sizes were a representation of the epidemiology of colorectal diseases and hence some sample sizes, particularly the colorectal cancer group, were too small to obtain significant conclusions. Further work with larger groups is therefore required to build upon the results of this pilot study. Contrary to what was predicted, high levels of Hsp70 in healthy bowel mucosa may be a cytoprotective mechanism ensuring the survival of the cell. In contrast, cells with lower levels of Hsp70 may be more likely to undergo spontaneous mutations this could explain lower Hsp70 levels measured in the pathological groups. Alternatively, low levels of Hsp70 in the pathological groups could be due to active secretion as part of a 'danger signal' by these cells to mount an effective immune response