Overwhelming evidence indicates that aspirin (ASA) and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity against colorectal cancer (CRC). Although the underlying mechanisms have yet to be fully elucidated, the host laboratory have shown that nucleolar sequestration of the NF-κB component RelA is critical. In the course of these studies, it was noted that alongside effects on the NF- κB pathway, ASA has a profound effect on nucleoli, including a dramatic increase in nucleolar size. These data were particularly interesting as, in addition to its role in ribosome biogenesis, the nucleolus is known to act as a stress sensor and play a key role in the regulation of cell growth and apoptosis. Indeed, this organelle has been identified as a potential target for anti-tumour agents. However, how stress causes changes to nucleolar function, and how these are translated into changes in cell phenotype, remain unclear. Therefore, the aim of my thesis was to fully characterise ASA effects on nucleoli and to determine whether these effects contribute to the anti-tumour activity of this agent. I found that ASA induced an atypical form of nucleolar stress that was associated with enlargement of the organelle, relocalisation of nucleolar markers to the periphery, depletion of the critical component of the Pol I transcription factor complex, TIF-IA, and inhibition of rRNA transcription. These effects were independent of the p38 and JNK2 MAP kinase pathways. However, they were mimicked by inhibition of CDK4, which had previously been shown to lie upstream of ASA effects on the NF-κB pathway. These data describe a novel mechanism by which ASA, and CDK4 inhibition, may inhibit the growth of colon cancer cells. In addition to this candidate approach, I used Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based quantitative proteomics to obtain a global overview of ASA effects on nucleoli of colon cancer cells. Firstly, a protocol was successfully developed to isolate pure nucleoli from SW480 CRC cell lines. This protocol was then applied to SILAC labelled cells treated with ASA for three time-points (0, 6, 10 h). In collaboration with R.T Hay and M. Tatham (University of Dundee), proteomic analysis was then carried out by tandem-mass spectrometry. These data confirmed that ASA has a significant effect on the nucleolar proteome. They also revealed that ASA induces a distinct type of nucleolar stress that is associated with the accumulation of chaperones, translational regulators and members of the ubiquitin-proteasome system (UPS) in this organelle. These data were reminiscent of studies previously published on the effect of proteasome inhibition on nucleoli. I therefore used SILAC-based proteomics to compare ASA effects on nucleoli to those induced by the proteasome inhibitor, MG132. I found that similar sub-groups of proteins accumulate in nucleoli in response to both agents and that ASA induced proteotoxic stress in a similar manner to MG132. Fluorescence correlation spectroscopy in collaboration with R. Duncan and K. Martin (Heriot-Watt University) demonstrated the relative reduction in mobility of nucleolar DsRed-RelA, indicating that, similar to MG132, ASA induces formation of nucleolar aggresomes. Mechanistic studies suggested that blocking ASA-mediated proteotoxic stress blocked the apoptotic effects of the agent. Taken together, these data define a distinct type of nucleolar stress that may be involved in the cells response to proteotoxic stress and be required for the anti-tumour activity of ASA.

Aim: To assess SELDI-TOF MS technology as a tool for biomarker discovery in the stool and serum of colorectal cancer patients. Materials and Methods: 1.Initially a technique of analysis was developed and optimised using tumour samples and matched normal mucosa, obtained from the Tayside Tissue Bank. These samples were then analysed using SELDI on a PBS II Protein Chip Mass Spectrometer to identify abundant proteins. 2. A technique of stool preparation and subsequent SELDI analysis was developed and then optimised (CM10 chip at pH4) to allow comparison of faecal samples from cancer and controls. Faecal samples were then collected from cancer patients and controls and analysed. In addition, FOB testing was carried out on all stool samples from cancer and controls and subgroup analysis of spectras was performed controlling for FOB status. 3. A test set of cancer and normal serum samples was used to optimise the method of analysis using 4 different chip surfaces at differing pH. Serum samples were collected from cancer patients and normal controls and were analysed on the H50 chip. Serum was then depleted of major proteins in an attempt to improve the detection of peaks. The mass spectra from each sample type were compared to identify any common protein peaks. Results: 1. Tumour analysis methods were optimised using an initial 4 samples of tumour and normal mucosa. Analysis of 8 further paired samples showed protein peaks at 2826, 3374, 3444, 3489 and 10854 Da which were abundant in tumour and reduced in the normal mucosa. 2. In serum analysis the initial experiment of 10 cancer versus 10 normal revealed 4 peaks on the H50 chip (3479, 3364, 3434, 3700 Da) that had significantly higher mass to charge ratios in cancer. The experiment was repeated on the H50 chip using 92 cancers and 92 controls and 5 different peaks were identified (7901, 8124, 8566, 8799 and 17 409Da) as being significant but these had higher mass to charge ratios in the controls. After depletion of the serum samples of albumin, transferrin, haptoglobin, anti-trypsin, IgG and IgA SELDI-TOF analysis showed a greatly reduced profile that yielded no meaningful spectra. 3. Stool analysis revealed 5 protein peaks (4633, 16511, 33423, 37087 and 47026 Da) in colorectal cancer patients, which were absent in stools from controls with a sensitivity of 83% when using all 5 peaks. Degradation of the spectra was observed after prolonged storage of stool samples. Conclusions: A method of stool analysis has been developed that yielded valid peaks differentiating between cancer and normal, which warrant further research through protein identification. Serum analysis was not reproducible across experiments and depletion of major proteins failed to reveal the sub-proteome raising doubts about whether discovery-based serum proteomics can accurately detect cancer. SELDI-TOF was not able to demonstrate that any of the peaks present in the tumour analysis were present in the stool or the serum samples.

Colorectal cancer (CRC) is a primary cause of cancer-related mortality in the United States. Asian Americans have the highest CRC mortality rates. CRC screening tests can reduce CRC incidence, yet Asian Americans, specifically the subgroup of Vietnamese Americans, underuse CRC screening. The purpose of this phenomenological study was to understand why Vietnamese Americans, ages 50 to 75, underuse CRC screening. The health belief model constructs of susceptibility, severity, benefits, barriers, and self-efficacy were the framework for understanding this population's health-related behaviors. Three research questions focused on how knowledge, language, and cultural beliefs and perceptions affect Vietnamese Americans' CRC screening decisions. Interviews were conducted with 11 participants, and transcribed interview responses were input into NVivo 11 software to maintain a reliable database and to identify emerging themes. Key study findings revealed knowledge and English language gaps as well as adverse cultural perceptions of fear and doubt that influenced CRC screening choices among these 11 Vietnamese Americans. Future researchers might focus on cultural-tailored strategies to minimize these barriers for Vietnamese Americans. An understanding of this study population's perspectives offers the promise of positive social change for health services and public health administrations to develop cultural-tailored interventions that promote healthy lifestyles, prevention, early CRC detection and, consequently, reduce mortality rates and associated health care costs.

Colorectal cancer (CRC) is one of the most common causes of death from malignant disease. Nevertheless, no ideal screening method exists and there is a lack of prognostic and predictive factors to support clinical decisions and to aid the development of a more individualized treatment for patients with CRC. The aim of this thesis was to investigate early detection, prognostic and predictive factors of CRC. In the first paper, a novel method to collect cells for DNA quantification from the rectal mucosa was investigated. The sensitivity and specificity of this test to detect CRC or any pathology in colon and rectum were ultimately too low to be acceptable. In the second paper, the prognostic value of growth differentiation factor 15 (GDF 15) was evaluated in patients curatively operated for colorectal cancer. GDF 15 expression was demonstrated to be associated with a negative prognosis in patients with stages I-III and III disease. In the third paper, the prognostic value of BRAF, PIK3CA KRAS and MSI was evaluated in a cohort of patients with CRC stratified by disease and recurrence. The results indicated that patients with CRC stage III without recurrence have a higher frequency of BRAF mutation compared to stage III patients with recurrence. In the fourth paper, histopathological predictors of pathologic complete response (pCR) as well as the association between pre-treatment carcinoembryonic antigen (CEA) levels and pCR in non-smoking and smoking patients receiving preoperative chemo-radiotherapy for rectal cancer were evaluated. Only in non-smokers was a low CEA level significantly associated with pCR, suggesting that the predictive value of CEA for pCR in rectal cancer in smokers can be limited. In sum, this research has investigated a new method for CRC detection and further evaluated the clinical use of prognostic and predictive markers in CRC.

This study evaluates a new serum-based biomarker for colorectal cancer (CRC) screening and diagnosis. The biomarker (GTA-446) is a member of hydroxy -polyunsaturated ultra-long chain fatty acids and was found to be reduced in CRC patients compared to CRC-free subjects. Diagnostic test performance characteristics were used to identify the effectiveness of the test. Methods: Serum levels of GTA-446 were measured in 4924 subjects who underwent colonoscopy for any reason, pathology results and clinical data were also collected. Two sets of age-matched control subjects were used; First were the lab controls (number=383) which were serum samples collected from Saskatchewan Disease Control Laboratory along with age and gender data. Second, were the endoscopy controls (number=762) which were obtained from the colonoscopy population after being determined to be cancer-free. Cut-off values were calculated using Receiver Operating Characteristic (ROC) curve. Results: Serum GTA-446 was found to be reduced in 87% of CRC patients. Compared to lab controls, the GTA-446 biomarker has a sensitivity of 87%, specificity of 75%, positive likelihood ratio of 3.6, and negative likelihood ratio of 0.16. Using endoscopy controls to calculate test performance characteristics, the biomarker has a sensitivity of 87%, specificity of 50%, positive likelihood ratio of 1.74, and negative likelihood ratio of 0.24. Also, the level of GTA-446 was found to significantly decline with age (r=-0.20, p<0.01). Conclusion: Serum GTA-446 is a potential biomarker for minimally invasive detection of colorectal cancer that compares favorably to other serum-based biomarkers.

Abstract The homeobox gene prox1 is a transcription factor related to the Drosophila gene Prospero. It play an essential role in the development of central nervous system, lens, liver and pancreas. In addition, prox1 is a master gene controlling the early development of the lymphatic vasculature. In tumorigenesis, prox1 has been shown to function as a tumor suppressor gene in hepatocellular carcinoma and breast cancer. Conversely, prox1 is over-expressed in the majority of colorectal cancer (CRC) and it promotes dysplasia, tumor growth and malignant progression. I report the findings here and show that ectopic expression of prox1 in prox1-null DLD-1 colon cancer cells will increase cell invasion but decrease cell adhesion. In addition, prox1 may induce epithelia- mesenchymal transition (EMT) by attenuating E-cadherin expression and up-regulating other EMT markers. On the contrary, knockdown of prox1 increases E-cadherin expression in SW620 cells; reduction of prox1 increases cell adhesion but decreases invasion. In short, the transcription factor prox1 plays an oncogenic role and promotes cancer metastasis in CRC.

Colorectal cancer (CRC) is the 3rd common cancer in the world. Because the five-year survival rate is below 60 % in the patients with CRC, two important respects in CRC researches are early diagnosis and more effective chemotherapeutic drugs. In fact, the studies on molecular pathology of CRC can resolve these two problems. Insulin has a role in the carcinogenesis and developments of CRC, and 5-FU is a standard chemotherapeutic drug for the patients with stage III CRC. As a human securin, PTTG has a major role in many functions. In our studies of 2-D proteomics, PTTG correlates with the invasiveness of CRC. Besides, it is found to be highly expressed in many types of cancer, but the expression of PTTG is, however, low or undetectable in normal tissues. This character of tumor-specific expression is suitable for drug and target therapy. Therefore, we use cell line HT-29 to study the effects of 5-FU and insulin on the expression of PTTG. We have found that insulin in the physiologic level up-regulates PTTG. In normal physiologic level, insulin up-regulates PTTG, in a dose-dependent manner. On the other hand, the induction of PTTG by insulin more than normal physiologic level is decreased. In the studies with 5-FU, PTTG has a higher level after treatment, but not in dose-dependent manner. The up-regulation of PTTG by 5-FU is decreased in a higher dose. In cancer cells, insulin regulated pathways may contribute to the growth, proliferation, and apoptosis of tumors by activating oncogenic molecular targets such as PTTG. We have showed that insulin of bio-physiologic level can up-regulate PTTG in colon cancer HT-29 cell lines. Induction of PTTG by insulin suggests a mechanism by which insulin may contribute to the development and/or progression of colon cancers. To tumor- specific expression of PTTG, induction of PTTG by insulin, consequently, may be a target of colon cancer treatment. In the studies of 5-FU and PTTG, we have found that 5-FU in HT-29 cells can induce PTTG, with a peak effect in a dose around IC50. Interestingly, the induction of PTTG is decreased in a higher dose of 5-FU. This is a new finding in the effect of 5-FU on PTTG. Accordingly, we can realize why PTTG in some studies is suppressed; the others have a higher level. More importantly, the connection of PTTG expression between sister chromatic separation and DNA repair after DNA damage is more reasonable. Based on this finding, we propose that PTTG connects DNA damage response pathways, sister chromatic separation and apoptosis after DNA damage. Therefore, PTTG has a key role after DNA damage, deciding cells to have DNA repair/cell cycle arrest or to progress to apoptosis.

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. Today, P. longifolia var. pendula is in large-scale cultivation in south Taiwan as a landscape plant. A new compound, clerodane diterpenoid 16-hydroxycleroda-3,13-dien-15, 16 -olide¡]CD¡^, was isolated from the bark of Polyalthia longifolia var. pendula. The compound was shown with cell growth inhibitory property at first screen in KMU¡]Kaohsiung Medical University¡^. In our study, antiproliferative activity of CD on human colorectal adenocarcinoma cells¡]HT29 and HCT116¡^were tested and the inhibitory concertration of 50¢Mcell viability¡]IC50¡^is 5 £gg/ml¡]HCT116¡^ and 10 £gg/ml¡]HT29¡^determined by MTT(3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The caspase 3 and PARP cleavage experiment results indicated that CD induced apoptosis is dose dependent manner. We found that CD induced sub-G1 accumulation and reactive oxygen species(ROS) release by flow cytometry analysis. Pretreatment of N-Acetyl-L-cystine(NAC), an antioxidant agent, can reverse the anti-proliferation effect by CD. We found that CD induced loss of mitochondria membrane potentiol¡]£G£Zm¡^. We also analysis differentially expressed proteins by 2-D electrophoresis and find drug response proteins, e.g. HSP10, Profilin-1, Peroxindoxin-1. RT-PCR and western blot was performed to confirm the protein expression changes. It is interesting to reveal the role of these proteins in the colorectal cancinogenesis and anti-tumor drug response.

In Taiwan, colorectal cancer incidence rate and mortality rate had been increased remarkably. Colorectal cancer was the third leading cause of death in all cancers in Taiwan, which had cause serious threaten on the health condition of people. The CRC screening rate is relatively low in Taiwan, from the statistic information in Bureau of Health Promotion shows that less than 20 % of colorectal cancer cases were detected on stage 0 and stage £L in Taiwan. The early detection rate was relatively low as comparing with colorectal cancer in the United States. However, if colorectal cancer is vital for the optimal treatment to obtain a correct diagnosis promptly, the long-term survival rate can reach as high as 90% for the early staged CRC. As we know, regular screening examination for colorectal cancer was the first step to treat colorectal cancer. The key point for promoting the colorectal cancer screening rate was to realize the factors which affecting the willingness of receiving the screening examination among older adults. Due to that, this study is aimed to identify the attitude toward screening and health-belief recognition among older adults above 40 years old within the community screening program, in order to realize factors influence the older adults¡¦ willingness of the colorectal cancer screening and Colonoscopy. The study design was a cross-sectional co-relational community-based survey. The data collection period was from August 2007 to May 2008. Data were collected from older adults which above 40 years old in Taipei and Kaohsiung. There were 462 samples which included 279 samples were having an experience of CRC screening before, and 183 samples were never had such screening before. The data was analyzed with descriptive statistics and inferred statistics such as Chi-square, Independent T-Test, Logistic regression analysis, Path analysis. The Logistic regression analysis was used to determine the related factors which might affecting the willingness of CRC screening among the older adults in community, including the factors applying from Health Belief Model such as Perceived threat, Perceived benefits of taking actions, Perceived barriers of taking actions, Cues to actions, Self-Efficacy, Health Motivation. Also addressed the factors related to attitudes such as fear about cancer, worry about accurate or safety of screening method, optimistic. The results of the study were as following. (1)In health belief related sectors, Perceived threat, Barriers, Cues to actions were significant with both the willingness of receiving Colonoscopy and FOBT screening. Except for those three factors that mentioned above, Health motivation would influence the willingness of FOBT screening, instead of Colonoscopy. (2) In attitude related sectors, fear about cancer, worry about accurate or safety of screening method were significant with both the willingness of receiving Colonoscopy and FOBT screening. (3) In health status sectors,bowel symptoms were significant with the willingness of receiving FOBT screening. Health behavior would only significant with the willingness of receiving Colonoscopy. (4) Age, social activity, screening experience would affect the willingness of receiving FOBT screening. On the other hand, the willingness of receiving Colonoscopy was no significant with demographic characteristic. Besides, the results of the path analysis were shown as below. (1)Optimistic would affect Cues to actions factor, and health behavior may affect Health motivation factor, under such relation, optimistic and health behavior would indirect affect the willingness of receiving FOBT.(2)Bowel symptoms would affect Perceived threat which may indirect affect the willingness of receiving Colonoscopy. (3)The knowledge of CRC and CRC screening would also indirect affect the willingness of receiving FOBT and Colonoscopy. The major attribution of this study was to realize the factors influence the willingness of accepting CRC screening, which could provide for health promotion policy makers to develop suitable policy for CRC health protect planning. The study may be implicated in the following aspects¡G(1) Not only settle the goals of screening, and also suggest policy makers to promote the awareness and health belief toward colon cancer to target groups which the study result had shown. (2)With the union of the community and medical, physician counseling may enhance the CRC screening rate, especially Colonoscopy. (3)Fulfill the knowledge of detect colon cancer and CRC screening among older adults, make themselves know how to decrease the threat of the disease, in order to achieve the goal of improving their health and saving the expense of the injure.

Background: Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer death in Arizona. Given that by the year 2030 Arizona is expected to be the second most populated state in the U.S., it is imperative to evaluate whether this state has the colorectal screening capacity to accommodate the growing population.Methods: 338 members of the American College of Gastroenterology were invited to participate. Information for the total number of colonoscopies and sigmoidoscopies performed during an average week was ascertained by analyzing 105 surveys. We estimated the current and potential volume of screening procedures.Results: Physicians reported performing 8,717 endoscopic procedures weekly (7,990 colonoscopies and 727 sigmoidoscopies). They reported being able to increase their capacity by an additional 3,183 (36.5%) procedures a week (2,347 colonoscopies and 836 flexible sigmoidoscopies).Conclusions: Our findings suggest that Arizona has the ability to significantly expand its endoscopic capacity.