M.Tech.

Master of Science - Science / Normal colonic epithelium bombarded by a range of molecular changes, affecting cell proliferation and apoptosis, result in the initiation of an adenoma and consequently an invasive carcinoma, which is usually lethal. One of the main characteristics of tumour progression is the loss of regulation between the cell cycle and apoptosis. Under normal circumstances, these processes are strictly controlled by a number of regulators and inhibitors. Previous studies have implicated the novel Domain With No Name gene in apoptosis. This study aimed to characterize the expression patterns and levels of the gene in colon cancer and to determine its role in apoptosis. In situ hybridisation, immunocytochemistry and quantitative PCR localised the gene and its products in cancerous and normal colon tissue. Combined with apoptosis detection studies, proliferation assays and Bcl-2 assays, the results suggest that the gene is involved in promoting apoptosis in cancerous cells i.e. the targeting of undesirable cells. Helicobacter pylorus was implicated in the progression of noninvasive colon cancer to the invasive state. From this study DWNN is proposed to be a pro-apoptotic participant in programmed cell death and classification studies such as these allow for potential manipulation of the apoptotic system to serve as a therapeutic corridor.

As céluals tumorais estão constantemente expostas a flutuações nas concentrações de nutrientes e oxigênio no microambiente tumoral. Através da ativação de diferentes vias de sinalização, as células tumorais sofrem uma reprogramação metabólica a fim de suportar as condições hostis impostas pelo microambiente tumoral. Uma das vias de sinalização ativada nessas condições é a autofagia, a qual tem sido considerada um dos principais mecanismos de sobrevivência celular em condições de estresse. Além disso, muitos medicamentos anti-câncer, como os agentes alquilantes, tem sido implicados na indução de autofagia. Apesar da autofagia contribuir com a sobrevivência da célula, uma superativação da via autofágica por períodos prolongados pode contribuir com a morte celular, o que faz com que o papel da autofagia no câncer seja ainda bastante debatido. Neste trabalho, buscou-se avaliar o papel da autofagia induzida em células de câncer colorretal da linhagem HCT116 continuamente cultivadas em baixa concentração de glicose e submetidas ao tratamento com o agente alquilante oxaliplatina. Os resultados demonstraram que a autofagia induzida em células HCT116 nessas condições exerce um papel citoprotetor, contribuindo para a resistência ao tratamento com oxaliplatina. A ativação da autofagia pelo tratamento com oxaliplatina em baixa concentração de glicose foi capaz de manter os níveis intracelualres de ATP e de reduzir a morte celular por apoptose. A utilização de inibidores ou de um ativador farmacológico da via autofágica, em combinação com a oxaliplatina, foi capaz de sensibilizar células HCT116 tratadas em baixa concentração de glicose, aumentando a morte celular por apoptose. Além disso, a indução de autofagia pela oxaliplatina foi mediada pela ativação de AMPK e inibição de mTOR. Estes dados demonstram que a ativação da autofagia em células de câncer colorretal HCT116 expostas a baixa concentração de glicose contribui para resistência ao tratamento com oxaliplatina. Estes dados também sugerem que a manipulação da via autofágica, pela sua inibição ou superativação, pode fornecer um maneira eficiente de limitar a progressão do tumor e aumentar a eficiência dos regimes quimioterápicos. / Tumor cells are constantly exposed to nutrients and oxygen concentration fluctuations at tumor microenvironment. Through activation of different signaling pathways, tumor cells undergo metabolic reprogramming to tolerate hostile conditions imposed by tumor microenvironment. One of the activated signaling pathways in such conditions is autophagy, which has been considered one of the central mechanisms of cell survival in stress conditions. Furthermore, many anticancer drugs, like alkylanting agents, have been implicated in autophagy induction. Despite autophagy contribution to cell survival, the autophagic pathway activation for prolonged periods may contribute to cell death, which creates extensively debates about the role of autophagy in cancer. In the present work we intended to evaluate the role of autophagy induced in colorectal cancer cells HCT116 grown continuously in low glucose concentration and treated with the alkylating agent oxaliplatin. Our results showed that autophagy induced in HCT116 cells in these conditions plays a cytoprotective role, contributing to oxaliplatin resistance. The activation of autophagy by oxaliplatin in low glucose concentration was able to maintain the intracellular levels of ATP and to reduce apoptotic cell death. The combined use of pharmacologic inhibitors or an activator of autohophagy with oxaliplatin was capable to sensitize HCT116 cells treated in low glucose concentration, increasing apoptotic cell death. Moreover, autophagy induction by oxaliplatin was mediated by the activation of AMPK and inhibition of mTOR. Our data demonstrate that autophagy activation in colorectal cancer cells HCT116 exposed to low glucose concentration contributes to the tolerance of oxaliplatin. These data also suggest that the manipulation of autophagic pathway, through its inhibition or hyperstimulation, may provide an effective manner of limiting tumor progression and increase chemotherapy effectiveness.

Introduction: Short chain fatty acids (SCFA) are produced by large bowel fermentation of dietary carbohydrates including resistant starch (RS) and non-starch polysaccharides (NSP). SCFA (particularly butyrate) play a major role in maintaining large bowel function and may reduce the incidence of colonic disease. Butyrate is the preferred metabolic substrate of colonocytes and is believed to play a key role in modulating epithelial cell cycle, mucosal immune response and gut motility. Increasing large bowel butyrate supply requires intakes of NSP or (RS) much higher than those currently consumed in western diets. Recent studies have shown large bowel butyrate is increased by the ingestion of butyrylated starch but the characteristics and physiological effects of its ingestion in animal models of colonic disease have not yet been investigated. Aims and Methods: The experiments in in vitro and in rats described in this thesis examined: the effects of production techniques and cooking on the capacity of butyrylated starch to deliver butyrate to the large bowel. They investigated the effects of increased butyrate levels on large bowel function in: (i) normal rats, (ii) the dextran sulphate sodium (DSS) rat model of ulcerative colitis (UC) and (iii) the high dietary protein rat model of colonocyte genetic damage. Results: Starch type, pre-treatment and the degree of butyrylation influenced the in vitro digestion and fermentation characteristics of butyrylated starch before and after cooking. Butyrylated starch was less susceptible to small intestinal digestion RS as than high amylose maize starch (HAMS) in vitro. Feeding diets containing 10% cooked butyrylated starch delivered significantly greater amounts of butyrate to the large bowel of rats than 10% raw or cooked HAMS. Butyrate did not influence colonocyte proliferation throughout the large bowel of the rat but increased distal colonic IL-18 concentrations and decreased longitudinal smooth muscle contractility. Feeding HAMS or butyrylated HAMS (HAMSB) to rats during DSS induced UC and during 7 days of recovery resulted in increased mucosal damage compared to low amylose maize starch (LAMS) fed rats. When rats were fed HAMS or HAMSB during the 7 days of recovery only, there was no significant difference in mucosal damage. Genetic damage, as measured by the comet assay, was 2 fold higher in rats fed high protein diet compared with those fed a low protein diet. Concurrent feeding of high protein and either HAMS or HAMSB resulted in significantly less genetic damage. Genetic damage in rats fed 20% HAMSB was half the levels of the 20% HAMS group, and was the same as the low protein diet. Conclusions: Butyrylated starch delivered butyrate to the large bowel in rats effectively, was less susceptible to small intestinal digestion and had greater stability following cooking than the unmodified base starch. Increased digesta butyrate did not affect large bowel function or colonocyte proliferation in the normal rat; the effects on mucosal damage in the DSS rat model of ulcerative colitis were inconclusive. Increased luminal butyrate prevented high-protein induced colonocyte genetic damage. Butyrylated starches have potential to assist with the maintenance of bowel health and to contribute to reduced risk of colonic disease in the community. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1320754 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

One of the most consistent biochemical changes associated with colon cancer progression is the altered expression of cell-associated carbohydrates. For example, the elevated expression of β1-6 branched N-linked oligosaccharides correlates with the presence of metastatic disease in colon cancer patients. Thus, it has become desirable to identify glycoproteins that are modified by these cancer-associated carbohydrates. Previous work in our laboratory identified the tumor-associated antigen 90 kDa (TAA90K) as a carrier of these cancer-associated carbohydrates. Since TAA90K has been previously implicated in cancer progression and metastasis, we examined its expression and function in human colon tumors. Immunohistochemical analysis revealed elevated expression of TAA90K in all tumor samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we performed protein binding and cellular assays with TAA90K purified from HT-29 human colon cancer cells infected with recombinant vaccinia virus expressing TAA90K. Purified TAA90K bound to ECM proteins including fibronectin, collagen IV, laminins-1, -5 and -10 and galectin-3. Unlike TAA90K isolated from other cell types, TAA90K isolated from HT-29 cells failed to mediate adhesion of colon cancer and normal cell lines, due in part to cell-specific glycosylation differences. Although TAA90K did not directly mediate cellular adhesion, it did modulate galectin-3-dependent adhesion of HT-29 cells. In addition, TAA90K bound to and was a substrate for MMP-7, a matrix metalloproteinase previously implicated in colon cancer progression. MMP-7-cleavage of TAA90K had little effect on its binding to pro- and active MMP-7, laminin-1 and galectin-3, but reduced significantly its binding to fibronectin and laminin-10. In addition, treatment of cells with MMP-7-cleaved TAA90K resulted in lower levels of proMMP-7 in the conditioned medium than cells treated with intact TAA90K. This may be mediated by the reduced binding of MMP-7-cleaved TAA90K to IL-6 and IL-1β, cytokines previously implicated in enhanced proMMP-7 expression in prostate cancer cells. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins and extracellular matrix remodeling through interactions with MMP-7 and galectin-3.

I-Abstract I.1-Objectives- To grossly evaluate colonic abnormalities within the Manitoba population by determining prevalence according to; age, gender, body mass index, body weight, body length, colon length, and fecal weight. I.2-Methods- A population study of 67 medico-legal autopsies from two major teaching hospitals was performed, examining the colon for abnormalities. The colon was resected just proximal to the ileocecal valve and just distal to the recto-sigmoid junction. Once the specimen was detached it was weighed with the autopsy scale opened and the feces removed. The specimen was patted dry and re-weighed. The specimen was laid on a blue specimen photograph board where the length was measured from the cecum to terminal sigmoid/rectum and photographed digitally. The specimen was then evaluated for any abnormalities. The specimen was then placed back into the body. I.3-Results Of the 67 colons assessed, 66% had an abnormality, of which 37.3% had diverticular disease and 24% had polyps, the two most common diseases. Age was the only significant factor (p=0.004) in this study affecting prevalence. The prevalence of multiple polyps was 63% with colon length being the only significant factor (p=0.0265) in this study affecting prevalence. I.4-Conclusions A progressive risk of increased abnormality formation is noted with age. Diverticular disease and polyps have similar prediction factors and disease prevalence. Many factors are suggested in the literature to influence the prediction of abnormalities, however only age was determined to be significant in this study. Multiplicity of polyps in the colon is significantly related to colon length. / October 2007

One of the most consistent biochemical changes associated with colon cancer progression is the altered expression of cell-associated carbohydrates. For example, the elevated expression of β1-6 branched N-linked oligosaccharides correlates with the presence of metastatic disease in colon cancer patients. Thus, it has become desirable to identify glycoproteins that are modified by these cancer-associated carbohydrates. Previous work in our laboratory identified the tumor-associated antigen 90 kDa (TAA90K) as a carrier of these cancer-associated carbohydrates. Since TAA90K has been previously implicated in cancer progression and metastasis, we examined its expression and function in human colon tumors. Immunohistochemical analysis revealed elevated expression of TAA90K in all tumor samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we performed protein binding and cellular assays with TAA90K purified from HT-29 human colon cancer cells infected with recombinant vaccinia virus expressing TAA90K. Purified TAA90K bound to ECM proteins including fibronectin, collagen IV, laminins-1, -5 and -10 and galectin-3. Unlike TAA90K isolated from other cell types, TAA90K isolated from HT-29 cells failed to mediate adhesion of colon cancer and normal cell lines, due in part to cell-specific glycosylation differences. Although TAA90K did not directly mediate cellular adhesion, it did modulate galectin-3-dependent adhesion of HT-29 cells. In addition, TAA90K bound to and was a substrate for MMP-7, a matrix metalloproteinase previously implicated in colon cancer progression. MMP-7-cleavage of TAA90K had little effect on its binding to pro- and active MMP-7, laminin-1 and galectin-3, but reduced significantly its binding to fibronectin and laminin-10. In addition, treatment of cells with MMP-7-cleaved TAA90K resulted in lower levels of proMMP-7 in the conditioned medium than cells treated with intact TAA90K. This may be mediated by the reduced binding of MMP-7-cleaved TAA90K to IL-6 and IL-1β, cytokines previously implicated in enhanced proMMP-7 expression in prostate cancer cells. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins and extracellular matrix remodeling through interactions with MMP-7 and galectin-3.

Thesis (M.P.H.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 33-41).

Thesis--PlanA (M.S.)--University of Wisconsin--Stout, 2002. / Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 169-186). Also available in print.