Folate blood levels in North America have dramatically increased over the past decade owing to folic acid (FA) fortification and widespread supplement use. Furthermore, over 50% of newly diagnosed colorectal cancer (CRC) patients use vitamin supplements containing FA while receiving chemotherapy whose mechanisms of action are based on interruption of folate metabolism. This study therefore investigated whether FA supplementation can affect chemosensitivity of human colon cancer cells to 5FU, the cornerstone of CRC treatment, using a xenograft model. FA supplementation was associated with a non-dose dependent decrease in chemosensitivity, where mice receiving 8 mg FA did not respond to 5FU and had greater tumor growth with treatment, compared to 2 (control) or 25 mg FA. Results of this study pose concern given the drastically increased intake of FA, particularly among recently diagnosed CRC patients, and from mandatory fortification. Further studies are warranted to confirm our findings and to elucidate underlying mechanisms.

Folate is an essential cofactor in one-carbon transfer reactions including nucleotide biosynthesis, thereby playing an important role in DNA replication and repair. In cancer cells, folate depletion interrupts DNA synthesis, thereby causing cancer cell death. This has been the basis for chemotherapy using antifolates and 5-fluorouracil (5FU). We determined the effect of folate deficiency on the sensitivity of colon cancer cells to 5FU in a well established in vitro model of folate deficiency. Folate deficient cells had lower intracellular folate concentrations, had functional evidence of intracellular folate depletion, proliferated less, and had increased chemosensitivity to 5FU with and without Leucovorin. These data suggest that folate deficiency significantly enhances the sensitivity of colon cancer cells to 5FU based chemotherapy via changes in intracellular folate. Dietary or other strategies to deplete intracellular folate concentrations in colon cancer cells to enhance chemosensitivity to 5FU are worthy of further investigation.

The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells. <p> This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.

The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells. <p> This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.

Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.

To understand the mechanisms of transcriptional regulation of PXR, we performed yeast two-hybrid screenings to search for PXR-interacting proteins in a human liver cDNA library using the PXR ligand binding domain as the bait. More than one million independent clones were screened. One positive clone was a partial cDNA of CNOT2 (amino acid 183-540). CNOT2 is a component of CCR4-NOT that is a multi-subunit protein complex highly conserved from yeast to humans. Using a mammalian two-hybrid system in CV-1 cells and GST-pull down assays, we confirmed the direct interaction between PXR and CNOT2 and mapped the specific domains of association. In HepG2 cells, over expression of CNOT2 suppressed the PXR-regulated luciferase reporter gene activity. siRNA knockdown of CNOT2 potentiated PXR-transcriptional activity. These results strongly suggest that the CCR4-NOT complex is significantly involved in transcriptional regulation of PXR. The immuno-precipitated CNOT2 complex contained deadenylase activity as determined by an in vitro RNA decay assay. The presence of transfected PXR inhibited the cNOT2-associated deadenylase activity, as demonstrated by poly(A) tail PCR. Cellular localization of PXR and cNOT2 by immuno-fluorescence microscopy indicates that the interaction might occur within Cajal Bodies. Taken together, these results suggest that PXR regulates the mRNA turnover through direct interaction with the NOT2 component of the CCR4-NOT complex. PXR is also involved in colon cancer progression. Our results indicate that the evolutionarily conserved PXR protects organisms from carcinogenesis by inhibiting tumor growth as well as eliminating carcinogenic substances. Our laboratory proposes that pregnane X receptor has an important role in maintaining the balance of cells progressing through the cell cycle. In vitro and in vivo experiments demonstrate expression of PXR in colon cancer cells slows the progression of tumor formation. Colony growth of the PXR-transfected HT29 cells was suppressed in soft agar assay. In the xenograft assay, the tumor size formed in nude mice was significantly suppressed in HT29 cells stably transfected with PXR (310 mg /- 6.2 vs. 120 mg±6, p<0.01). The number of Ki-67 positive cells were significantly decreased in PXR-transfected HT29 xenograft tumor tissue compared vector-transfected HT29 controls (p<0.01) as determined by immuno-histochemistry suggesting that PXR inhibits proliferation of colon cancer cells. Results of flow cytometry analysis indicated that PXR-transfection in HT29 cells caused G0/G1 arrest. The growth inhibitory effects of PXR are likely mediated through the E2F/Rb-regulated check point since E2F1 nuclear expression was significantly inhibited by PXR over expression.

This study examines the carcinogenic effect of HZE radiation and protective effects of different types of diets against colon carcinogenesis in a rat model. The effect of HZE radiation on health state and colon cancer development was evaluated. HZE radiation was found to suppress food consumption (P<0.0001) leading to lower body weight gain of irradiated rats when compared to the non-irradiated rats (P<0.05). The animals exposed to HZE radiation were found to start dying and/or getting pathologies 11 weeks earlier and at the end of the study had morbidity/mortality rate 14.2% higher (P=0.0005) than non-irradiated rats. There was no significant effect of HZE radiation on colon cancer incidence. The effects of dietary fibers and oils on health state and colon carcinogenesis were evaluated. Morbidity/mortality was found to be delayed in rats fed with pectinbased diets when compared to cellulose-based diet, regardless of radiation treatment. Similarly, fish oil was found to beneficially affect health of the experimental animals when compared to corn oil. Ten- and twenty-week delayed morbidity/mortality for irradiated and non-irradiated groups, respectively, was observed for rats fed with fish oil-based diets when compared to corn oil-based diets. Fish oil was also found to significantly reduce colon tumor incidence and multiplicity in non-irradiated rats (P<0.05). A similar trend was observed for the irradiated animals. No significant effect of fiber on colon cancer incidence was found. Finally, the effect of diets on general health and colon cancer development was investigated. Rats fed with corn oil/cellulose diet started dying and/or getting a disease earlier than rats fed with other diets, regardless of radiation treatment. The effect of diet on colon cancer development was found to depend on radiation treatment. Thus, in the absence of radiation treatment fish oil/cellulose was found to significantly reduce tumor incidence and multiplicity when compared to corn oil/pectin diet (P<0.05). In the presence of radiation treatment fish oil/pectin was found to lower the values of tumor incidence and tumor multiplicity, though the data obtained were not significant.

Consumption of whole grains has been linked to reduced risk of colon cancer. This study determined estrogenic activity of sorghum phenolic extracts of different phenolic profiles and identified possible estrogenic compounds in sorghum in vitro, as well as evaluated the potential of estrogenic sorghum phenolic extracts to prevent colon carcinogenesis in vivo. The thermal stability of sorghum 3-deoxyanthocyanins was also studied, to determine their suitability as functional food colorants. White and TX430 (black) sorghum extracts showed estrogenic activity in cell models predominantly expressing estrogen receptor-α (ERα) or ERβ at 5 and 10 µg/mL, respectively. The same treatments led to induction of apoptosis in cells expressing ERβ. The red TX2911 sorghum did not possess these activities. Compositional analysis revealed differences in flavones and flavanones. Flavones with estrogen-like properties, i.e. luteolin and apigenin, were detected in White and TX430 (black) sorghum extracts, but not in red TX2911 extract. Naringenin, a flavanone known to antagonize ERα signalling, was only detected in the red TX2911 extract. Additional experiments with sorghum extracts of distinct flavones/flavanone ratio, as well as with pure apigenin and naringenin, suggested that flavones are the more potent ERβ agonists in sorghum. On the other hand, 3-deoxyanthocyanins were probably not estrogenic. Estrogenic white and black sorghum phenolic extracts (fed at 1% level in the diet) reduced the number of azoxymethane induced colon premalignant lesion (aberrant crypt foci) by 39.3% and 14.7%, respectively, in ovariectomized mice. Further studies are needed to elucidate the protective mechanisms induced by these sorghum extracts. Sorghum 3-deoxyanthocyanins retained good color stability after 30 minutes of heat treatment at 121 °C under pressure: More than 80% of color retained in pH 1 and 2 HCl and citric acid solutions, and 39-84% retained from pHs 3-7. Formic acid negatively affected the color stability at pH 1 and pH 2 due to its reducing capacity. Methoxylation decreased the thermal stability of 3-deoxyanthocyanins. The heat stability of 3-deoxyanthocyanins indicates good potential for food use. Overall, the inherent estrogenic activity of specific sorghum phenolic extracts is a likely mechanism for colon cancer prevention. Further studies are needed to assess physiologically relevant dietary level of sorghum phenolics for prevention of colon cancer, and effect of food processing on the activity and bioavailability of the chemopreventive components.

The acute cellular responses to DNA damaging agents are critical in determining the long term outcome of disease. A cell’s susceptibility to damage, or its capacity to remove or repair this damage, all contributes to the eventual health or disease of tissues. This process is especially crucial in colonic epithelial cells and in the development of colorectal oncogenesis. The colonic lumen is constantly subjected to different environmental compounds that may have genotoxic properties that can initiate mutational events and possibly carcinogenesis. Therefore, the study of a regulatory dietary agent that improves the colonic cells ability to withstand damage, improve repair and retain its general health is a significant and practical tool in the fight against colorectal cancer. The health benefits of fish oil, including its potential chemopreventative properties, have been reported in numerous studies. However, the mechanism by which this protective effect occurs remains unclear. A gap in current literature exists that fails to explore the effect of fish oil on the early cellular responses to carcinogenic agents. Therefore, this thesis aims to firstly, better understand the specific host responses to an insult of carcinogen in vivo; secondly, to determine if regulation of these responses can be achieved by dietary fish oil; and lastly, to explore the potential consequences of this regulation for colorectal oncogenesis. All experimental work was carried out using a rat – azoxymethane (AOM) animal model of colorectal carcinogenesis. The key host responses to the carcinogen that were measured included the formation of acute O6methyldeoxyGuanosine (O6medG) DNA damage, the acute apoptotic response to genotoxic carcinogen (AARGC) and cell proliferation rates. A novel immunochemical assay was designed to detect both the levels and distribution of O6medG in colonic cells. With this established, a pattern of these host responses were mapped out over time. A dietary intervention study trialling a range of fish oil diets containing different doses and forms was then carried out to determine if modulation of responses occurred. This study was then followed on by a longer term study that explored the consequences of regulation by fish oil on pre-neoplastic lesions in the colon. The acute host responses to an insult of AOM showed that colonic O6medG formation began 2h post AOM administration and peaked at 6h. The AARGC response followed the pattern of O6medG by a 2h delay, peaking at 8h post AOM administration, while cell proliferation rates decreased significantly after 6h. The inclusion of tuna oil in the diet did not affect either the AARGC or cell proliferation rates when given in any form or at any dose. Animals fed a diet with 15% free tuna oil and 7% encapsulated tuna oil did however have significantly reduced levels of O6medG DNA damage in the distal colon. This reduction in O6medG levels did not translate into a reduction of ACF lesion, with a protective effect against ACF lesions only being observed in animals fed the high dose fish oil groups. Analysis of the data suggest that the acute host responses to an insult of DNA damaging agent appear to be closely related, all reaching their peak level of response 6-8h after the insult. The short time frame between both O6medG and apoptosis also did not support the current popular theory which explains O6medG mediated apoptosis. An alternate hypothesised BER mediated apoptotic pathway was also not supported. Regulation of the acute apoptotic response or the cell proliferation rate was not achieved by dietary fish oil. However, a high dose fish oil diet did regulate the level of O6medG in colonic epithelial cells by significantly reducing the total O6medG DNA damage load. This reduction of O6medG by a high fish oil diet however, was not translated into a protective effect against the formation of pre-neoplastic lesions. These data suggests that regulation of the acute O6medG response to a damaging agent does not necessarily imply protection for longer term colorectal oncogenesis. Additional studies exploring both the effect of fish oil on AOM metabolising enzymes and also a longer term cancer study may help to answer some pertinent questions evolving from this thesis.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A incidÃncia e mortalidade por cÃncer colorretal apresentam tendÃncia ao crescimento. O cÃncer colorretal à a quinta neoplasia mais incidente no Brasil. A etiologia està relacionada com hereditariedade e modificaÃÃes no estilo de vida. A lesÃo prÃ-neoplÃsica mais precoce com presenÃa de displasia à o foco de cripta aberrante, estando relacionada como lesÃo precursora de adenomas colorretais e cÃncer em humanos. Entender a natureza destas lesÃes contribui na pesquisa de agentes preventivos eficazes no cÃncer colorretal. O objetivo foi verificar o potencial quimiopreventivo da soluÃÃo aquosa do lÃtex de Euphorbia tirucalli L. quanto a formaÃÃo de focos de cripta aberrante (FCA) em ratos induzidos com azoximetano (AOM). Foram usados 32 ratos da linhagem Wistar, machos, com peso mÃdio estimado de 100g -200g (4 â 6 semanas). Foram distribuÃdos aleatoriamente em 04 grupos contendo 08 animais, denominados: GRUPO 01- grupo estudo com ratos induzidos com AOM e tratados com extrato aquoso do lÃtex da E. tirucalli L..GRUPO 02 - grupo estudo controle com ratos induzidos com AOM sem tratamento com extrato aquoso do lÃtex da E tirucalli L..GRUPO 03 - grupo estudo controle sem induÃÃo com AOM e tratados com extrato aquoso do lÃtex da E. tirucalli L.. GRUPO 04 - grupo estudo controle sem induÃÃo com AOM e sem tratamento com extrato aquoso do lÃtex da E. tirucalli L..Os animais dos Grupos 01 e 02 receberam injeÃÃo de AOM 12 mg/kg, intraperitoneal (IP), uma vez por semana, por 02 semanas. Uma semana antes do inÃcio da administraÃÃo do carcinÃgeno, foi administrada diariamente soluÃÃo por gavagem, respectivamente, de extrato aquoso do lÃtex da E. tirucalli L. 400mg/Kg e soluÃÃo fisiolÃgica a 0,9% em uma administraÃÃo diÃria. Os Grupos 03 e 04 nÃo foram induzidos com AOM e receberam soluÃÃo por gavagem, respectivamente, de extrato aquoso do lÃtex da E. tirucalli L. 400mg/Kg e soluÃÃo fisiolÃgica a 0,9%. Todos os grupos continuaram recebendo a soluÃÃo por gavagem diÃria atà o dia estabelecido para eutanÃsia. Foram mortos na 15 semana, apÃs a induÃÃo com carcinÃgeno ou administraÃÃo IP de soluÃÃo estÃril para injeÃÃo. Os animais foram avaliados quanto ao peso, alteraÃÃo clÃnica, presenÃa de adenomas ou tumores cÃlicos, e quanto à presenÃa de FCA e o nÃmero de criptas por cada foco (multiplicidade) de acordo com a localizaÃÃo cÃlica, definidas regiÃo distal, medial e proximal. Verificamos no presente estudo que o extrato aquoso da E tirucalli L. (400mg/kg) apresentou uma diminuiÃÃo significante do nÃmero total de FCA do grupo 01 em relaÃÃo ao grupo 02 (p<0,001), adicionalmente a multiplicidade nestes grupos apresentou diferenÃa significante (p<0,0001) quanto a presenÃa de &#8804; 5 criptas por foco em todo cÃlon examinado. Este estudo sugere que extrato aquoso de E. tirucalli L tem potencial quimiopreventivo em relaÃÃo a carcinogÃnese cÃlica, inibindo a formaÃÃo de FCA em ratos induzidos com AOM. / Colorectal cancer is the fifth most frequently diagnosed malignancy in Brazil. The etiology may be related with inherited and life-style that can be modified. Aberrant crypt foci have been recognized as early preneoplastic lesions and being related with colorectal adenoma and precursors of cancer in humans. Undertanding the nature of early appearing lesions efforts to find effective agents in colorrectal cancer. The aim of this study was verify the potential chemopreventive of aqueous solution of the latex of Euphorbia tirucalli L. in aberrant crypt foci (ACF) in rats induced with azoxymethane (AOM). Thirty-two Wistar male rats were used, average weight 100g -200g (4 - 6 weeks). They were randomly divided into 04 groups of 08 animals each. Group 01 with rats induced with AOM and treated with aqueous extract of the latex of E. tirucalli L., group 02 with rats induced with AOM without treatment with aqueous extract of the latex of E. tirucalli L., group 03 with rats without AOM induction and treated with aqueous extract of the latex of E. tirucalli L. and group 04 with rats without induction with AOM and without treatment with aqueous extract of the latex of E. tirucalli L.. The animals of the groups 01 and 02 were injected intraperitoneallly (IP), with AOM once a weekly for 02 weeks at a dose level of 12 mg/kg body weight. One week before the beginning of the administration of the carcinogen, it was administered solution daily by intragastric gavage, respectively, aqueous extract of latex of E. tirucalli L. 400mg/Kg and physiologic solution to 0,9%. The groups 03 and 04 were not induced with AOM and they received daily solution by gavage, respectively, aqueous extract of the latex of E. tirucalli L. 400mg/Kg and physiologic solution to 0,9%. All groups continued receiving daily solutions by intragastric gavage until the day established for euthanasia. All animals were sacrificed by diethyl ether inhalation 15th week, after AOM initiation or sterile solution injected IP. The animals were evaluated concerning to the weight, clinical alteration, presence of adenomas or colic tumors, and the presence of ACF and the number of crypts for each focus (multiplicity) in agreement with colic location, defined area distal, medial and proximal. We verified in the present study that the aqueous extract of E. tirucalli L. (400mg/kg) presented a significant decrease of the global number of ACF group 01 compared to the group 02 (p<0,001). Additionally, the multiplicity in these groups showed significant decrease (p<0,0001) in the number of ACF with 5 crypts/focus in the whole large intestine. This study suggests the aqueous extract of E. tirucalli L. has potential chemopreventive against colon carcinogenesis with inhibition of the formation of ACF in rats induced with AOM .