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.

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic and intestinal anti-inflammatory hormone. Hence, I hypothesized that treatment with degradation-resistant hGly2GLP-2 increases, while blocking endogenous GLP-2 decreases colorectal cancer (CRC) in rodents. In mice, treatment with dextran sodium sulphate (DSS) and azoxymethane (AOM) induced colitis-associated CRC, which was further increased by treatment with hGly2GLP-2 and reduced by blocking endogenous GLP-2 with the antagonist hGLP-23-33. Moreover, while colonic damage score (CDS) was not altered by hGly2GLP-2 or hGLP-23-33 treatment, hGly2GLP-2 increased small intestinal growth and hGLP-23-33 reduced jejunal crypt cell proliferation. In rats fed with of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and high fat (HF) diet for aberrant crypt foci (ACF) induction, treatment with hGly2GLP-2 increased small intestinal growth and ACF occurrence. Moreover, in rats fed with PhIP-HF diet for tumour induction, early treatment with hGly2GLP-2 appears to increase the occurrence of intestinal tumours. Collectively, these findings indicate a pro-carcinogenic role for both exogenous and endogenous GLP-2.

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.

While assessment of folate requirements has been based only on dietary intakes, folate produced by the colonic microflora can exceed amounts consumed in food. Bacterially synthesized folate is absorbed across the rat and piglet colon. In vitro studies suggest, but direct evidence is lacking that folate is absorbed across the intact human colon. If indeed folate is absorbed, the amount synthesized may be susceptible to manipulation by fibre and prebiotics intake. We therefore performed two studies to investigate folate absorption across the colon. To confirm absorption across the intact human colon, in our first study, 684 nmol (320 µg) 13C5-glutamyl-[6S]-5-formyltetrahydrofolate was infused into the cecum of six adults and blood samples were collected. Tandem mass spectrometry confirmed folate absorption across the colon by appearance in plasma of 13C5-[6S]-5-methyltetrahydrofolate, at a rate of 0.6±0.2 nmol/h versus 7±1.2 nmol/h after intravenous injection of 172 nmol 13C5-5-formyltetrahydrofolate. Since bifidobacteria are potent folate producers, in our second study we evaluated the influence of bifidogenic oligosaccharides on colonic folate production and host folate status, using a piglet animal model. Piglets (n=12) were randomly assigned a milk-based formula with 5g/L inulin + 5g/L galactooligosaccharides, or 5g/L maltodextrin (control). After 28 days, the weights of colon contents (178 %) and colon tissue (37.9 %) of piglets fed oligosaccharides were greater than controls (P=0.0003, P=0.0044, respectively). The bacterial load and folate contents in the colons of piglets fed oligosaccharides were greater than controls (P=0.0022, P=0.0218, respectively). Body weights, blood folate status and liver and kidney folate concentrations did not differ. In conclusion, folate is absorbed across the human colon. Supplementation of the piglet diet with 5g/L inulin and 5g/L galactooligosaccharides increased the amounts of microbial folate, and the weights of colon tissue and contents, but folate concentrations in colon contents, blood and organs were not affected.

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.

Introducción: Existe controversia en la literatura sobre la utilidad del ganglio centinela (GC) en cáncer de colon.Objetivo: Detección de micrometástasis en los GC que permita una mayor precisión en la estadificación del cáncer de colon.Metodología: Estudio prospectivo controlado con grupos apareados, de 60 pacientes con cáncer de colon izquierdo en estadio preoperatorio 0, I y II. Se utiliza Azul de Metileno o Nanocoll® para identificar los GC. Comparamos la estadificación linfática convencional mediante hematoxilina-eosina (H-E) y la estadificación obtenida añadiendo al estudio convencional el análisis inmunohistoquímico con Citoqueratina CAM 5.2 de los GC. Resultados: Identificación de GC: 100%, 2,4 GC por paciente. Con el estudio patológico convencional, el 31,7% de los pacientes (19/60) presentaban afectación neoplásica linfática (N+) y el 68,3% (41/60) no presentaban afectación linfática (N0). De los pacientes estadificados N0 por estudio convencional se objetivó afectación micrometastásica en los GC en el 9,7 % (4/41). Además, en el grupo de los pacientes N0 fueron objetivados grupos celulares < 0,2 mm y/o >10 células aisladas cytoketatina positiva en los GC en el 17,7% (7/41) y fueron considerados como pacientes con "alto riesgo de progresión neoplásica". Con la estadificación combinada el número de pacientes N+ fue del 38,3% (23 pacientes), supraestadificación respecto al estudio convencional del 6,6% (p= ns). La estadificación podría ascender hasta el 18,3% (p=0,001) si son considerados como N+ los pacientes con "alto riesgo de progresión neoplásica".Conclusiones: El estudio linfático combinado (Convencional + GC) incrementa en un 6,6% la estadificación en nuestra serie y además permite identificar un 17% de pacientes N0 con "alto riesgo de progresión neoplásica". / Background: The use of the sentinel node lymph (SNL) in connection with colorectal cancer is a subject in which there has been some controversy. Purpose: To research the hidden micrometastases in the SNL in order to improve the accuracy of colon cancer staging. Patients and Methods: Prospective control trial of 60 patients with left colon cancer stage 0, I and II. Methylene Blue® or Nanocoll® had been used to identify the SNL. The standard pathologic examination with hematoxylin and eosin (H&E) had been compared with the study of SNL with H&E and Cytokeratine CAM 5.2. Results: Identification of the SNL: 100%, 2.4 SNL detected in each patient. Nodal disease (N+) was found in 19 patients (31.7%), and 41 patients (68.3%) were classified as node-negative (N0). Hidden micrometastasis in the SNL were found in 9.7% (4/41) patients considered as N0 by conventional histopathology. However, in 17.7% (7/41) patients considered as N0 we found large cell clusters, contatining up to 10 tumor cells, falling short of the AJCC on cancer criteria, and were considered to have high-risk disease. Having applied both the SNL study and the standard pathologic examination the number of patients N+ has increased to 38.3% (23 patients), ultrastaging: 6.6% (p= ns). If the patients with large cell clusters in the SNL had been considered as N+ the number of N+ patients would have increased to 50%(30 patients), ultrastaging 18,3%(p=0,001). Conclusions: The combined nodal study (H&E + SNL) increased 6.6% the staging in our group, and it let to identify a group of patients N0 (17%) with high-risk tumor progression.

La barrera mucosa gastrointestinal, formada por surfactante, moco y epitelio mucoso, separa la luz del tubo digestivo del medio interno. El surfactante, compuesto de fosfolípidos, condiciona propiedades de superficie a la mucosa. Estudios sobre esta barrera se han llevado a cabo en estómago de animales. Objetivo. Estudiar los fenómenos de superficie en la mucosa del colon humano. Material y método. Muestras de mucosa de colon y recto humano obtenidas de piezas quirúrgicas se han procesado en solución fisiológica modificada (SF), con factor de crecimiento epidérmico (EGF), prostaglandina E2 (PGE2), diclofenaco (45 mg y 90 mg) e indometacina (mismas dosis) determinándose con un goniómetro el ángulo de contacto (q) como medida de la hidrofobicidad. Desde un homogeneizado de mucosa determinamos las proporciones de fosfolípidos. Análisis estadístico: U de Mann-Whitney, análisis de la varianza de Kruskal-Wallis, coeficiente de correlación de Spearman, t de Student y ANOVA. Nivel de significación: 5%. Resultados. De 74 pacientes se obtuvieron 134 muestras de mucosa. El q en el grupo control fue de 39.52; en el grupo procesado con SF 42.29 (p<0.0001). Las muestras se dividieron en las de pacientes con antecedentes patológicos (n=47, q=39.34) y las de pacientes sin antecedentes (n=87, q=39.64) (p=0.01). Este grupo se dividió en: dislipemia ( q=40), broncopatía ( q=39.58), cardiopatía ( q=39.30), diabetes mellitus ( q=39) e HTA ( q=39) (p=0.01). El proceso con EGF (q=49.44) y PGE2 (q=47.32) determinó diferencias (p<0.001) y frente al grupo SF (p<0.0001). El proceso con diclofenaco (q=35.43) e indometacina (q=35.97) determinó diferencias (p<0.001) y frente al grupo SF (p<0.0001); con ambos la duplicación de la dosis llevó un decremento (q=35º) y (q=34.53) (p<0.0001). La división del colon en 7 segmentos: ciego (q=39.26), c.ascendente (q=39.53), c. transverso proximal (q=39.26), c. transverso distal (q=39.46), c. descendente (q=39.52), sigma (q=39.70) y recto (q=39.6), no llevó diferencias (p=0.1). En cuatro sectores: c. derecho, c. transverso, c. izquierdo y recto, tampoco (p>0.1).El coef. de correlación entre el q y la proporción de surfactante (mM de fosfolípidos / g de tejido): ciego (200.3), c.ascendente (203.1), c. transverso proximal (210.4), c. transverso distal (212), c. descendente (217.5), sigma (223.4) y recto (226.2), fue r=0.7207 (p=0.08). Conclusión. La hidrofobicidad en la superficie mucosa del colon humano se ve disminuida por la acción de los AINE y en diferentes enfermedades. Dicha propiedad se ve incrementada por la acción de citoprotectores. La hidrofobicidad es menor en tramos proximales del colon y mayor en tramos distales. Esto se correlaciona directamente con las proporciones de fosofolípidos que se aíslan en cada segmento del colon así como con la función, de absorción de agua en tramos proximales y de conducción de las heces en tramos distales, que caracterizan a dichos tramos. / Gastrointestinal mucosal barrier, formed by surfactant, mucus and epithelium, separates the digestive tract lumen from the internal milieu. The surfactant, made up of phospholipids, establishes mucosal surface properties. Previous studies in this barrier have been carried out in animal stomachs. Aims. Study the surface properties in the human colonic mucosal. Materials and Methods. Samples of human colonic and rectal mucosal obtained from surgical pieces have been processed in modified physiologic solution (SF), with epidermal growth factor (EGF), prostaglandin E2 (PGE2), diclofenac (45 mg and 90 mg) and indomethacin (same dose) being determined with the goniometer the contact angle (q) to measure the hydrophobicity. Phospholipid proportions are determined from mucosal homogenized. Statistical analysis: Mann-Whitney test, Kruskal-Wallis test, Spearman correlation coefficient, Student test and ANOVA. Values of p<0.05 were considered statistically significant. Results. 134 mucosal samples were obtained from 74 patients. In the group control q was 39.52º; in the group processed with SF, 42.29º (p < 0.0001). The samples were divided in those of patients with pathological antecedents (n=47, q=39.34) and those of patients without antecedents (n=87, q=39.64º) (p=0.01). This second group was divided in: disorders of lipid metabolism (40º), chronic bronchitis (39.58º), heart disease (39.30º), diabetes mellitus (39º) and hypertension (39º) (p=0.01). The process with EGF (49.44º) and PGE2 (47.32º) determined differences (p < 0.001) and also the group SF (p < 0.0001). The process with diclofenac (35.43º) and indomethacin (35.97º) determined differences (p < 0.001) and also the group SF (p < 0.0001); in both of them, the duplication of the dose increases the reduction (35º) and (34.53º) (p < 0.0001). Colonic division in 7 segments: cecum (39.26º), ascending (39.53º), proximal transverse (39.26º), distal transverse (39.46ª), descending (39.52º), sigmoid (39.70º) and rectum (39.6º), didn't set differences (p=0.1) . In four sectors: right, transverse, left and rectum, neither (p>0.1). Correlation coefficient between q and surfactant proportion (phospholipids mM / tissue g): cecum (200.3), ascending (203.1), transverse proximal (210.4), transverse distal (212), descending (217.5), sigmoid (223.4) and rectum (226.2), was r=0.7207 (p=0.08). Conclusion. Hydrophobicity in the human colonic mucosal surface decreases by the action of the NSAID. This property increases by the action of the cytoprotectors. In different diseases the hydrophobicity is decreased. Hydrophobicity is smaller in colonic proximal tracts and bigger in distals. The correlation between this property and the proportion of phosopholipids is direct and it is related with the function in each segment of the colon: water absorption in proximal tracts and fecal conduction in distal tracts.