Le cancer colorectal est la deuxième cause de mortalité due au cancer en Europe et aux États-Unis. Etant donné l’efficacité limitée et la toxicité élevée des agents de chimiothérapie, de nouvelles approches sont nécessaires. Le flavanolignane silybine représente le principal constituant actif du chardon-marie (Silybum marianum). Les mécanismes moléculaires des propriétés anticancéreuses de la silybine ont été étudiés dans un modèle cellulaire de progression du cancer colorectal humain : les cellules SW480 issues d’un adénocarcinome, et leurs dérivées métastatiques les cellules SW620. Les effets chimiopréventifs de la silybine ont été étudiés dans un modèle de cancérogenèse colique induite par l’azoxyméthane chez le rat. La silybine induit une mort apoptotique avec activation de la caspase-3 dans les deux lignées. L’expression des récepteurs de mort TRAIL est augmentée, et la caspase-8 activée. Le potentiel mitochondrial est perturbé provoquant une libération du cytochrome c et une activation de la caspase-9. En plus de l’activation des voies apoptotiques extrinsèque et intrinsèque la silybine induit une réponse autophagique. La combinaison de la silybine et de TRAIL, un agent anti-cancéreux prometteur, provoque une mort cellulaire synergique dans les deux lignées. Un effet synergique est aussi observé avec la combinaison de la silybine et des inhibiteurs des histones déacétylases (HDAC) : TSA et SAHA. Dans le modèle chez le rat, la silybine réduit de 50% le nombre des lésions prénéoplasiques. En conclusion, la silybine est un agent naturel intéressant pour la prévention du cancer colorectal et dans le cadre d’une combinaison avec TRAIL/des inhibiteurs d’HDACs. / Colorectal cancer (CRC) is the second most common cause for cancer-related deaths in Europe and in the USA. Because of the limited efficacy and considerable toxicity of chemotherapeutic agents, new approaches are needed. The hepatoprotective flavonolignan silibinin is the major biologically active compound of the milk thistle (Silybum marianum).The molecular mechanisms of the anticancer properties of silibinin in CRC were studied in an in vitro model of cancer progression consisting of the adenocarcinoma cell line SW480 and its derived metastatic cell line SW620. Its chemopreventive effects were assessed in an in vivo model of azoxymethane-induced colon carcinogenesis in the rat. Silibinin induced apoptotic cell death with activation of caspase-3 in both cell lines. The expression of death receptors was upregulated, and caspase-8 was activated. The potential of the mitochondrial membrane was perturbed permitting the release of cytochrome c and the activation of caspase-9. Besides the activation of the extrinsic and the intrinsic apoptotic pathway, silibinin induced an autophagic response. Combination of silibinin and TRAIL, a promising anticancer agent selectively inducing apoptosis in cancer cells, induced synergistic cell death in both cell lines. Synergy in cell death induction was also observed by the combination of silibinin and the histone deacetylase (HDAC) inhibitors TSA and SAHA. In the preclinical model in the rat, silibinin administration was able to reduce by half the number of preneoplastic lesions present in the colon. In conclusion, silibinin is a promising natural agent for colon cancer chemoprevention and for combination therapy with TRAIL/HDAC inhibitors.

Doctor of Philosophy / Department of Human Nutrition / Weiqun Wang / Anthocyanins are flavonoid pigments that account for the purple color in many plant foods. It has been investigated that anthocyanins’ predominant occurrences in human diet and their health beneficial activities such as antioxidant, anti-inflammatory, and anti-carcinogenetic effects. Based on those scientific evidences, anthocyanins are now recognized as potential therapeutic compounds. Particularly, the chemopreventive effect of anthocyanins has been widely studied by many researchers in nutrition. However, their bioactivities are diverse due to different chemical structures of anthocyanins from different sources. In this study, we discuss the chemopreventive activity of anthocyanins from purple sweet potato. Previously, we selected a purple-fleshed sweetpotato clone, P40, crossbred seeds obtained from the International Potato Center in Lima, Peru. We hypothesized that anthocyanins enriched P40 may provide health beneficial activities in cancer prevention. For the first part of this study, we analyzed nutrient compositions, dietary fiber content, anthocyanins contents, total phenolics contents and total antioxidant activity. Even thought P40 presents similar composition and amount of nutrients with the control cultivars, white-fleshed O’Henry and yellow-fleshed NC Japanese, HPLC-MS analysis confirmed that it possesses much higher anthocyanin content even up to 7.5g/kg dry matter. Also, dietary fiber, particularly soluble dietary fiber content, total phenolics content, and total antioxidant capacity of P40 were significantly higher. For the second part of the study, we tested the potential anticancer characteristic of P40 cultivar in human colonic SW480 cancer cells and in azoxymethane-induced aberrant crypt foci in mice. Treatment with 0 – 40 μM of peonidin-3-glucoside or P40 extract containing corresponding amount of anthocyanins resulted in inhibition of cell growth in a dose-dependent manner. Interestingly, even though the patterns of growth inhibition were similar in the two treatment groups, the cells treated with P40 extract tend to survive significantly less than those treated with peonidin-3-glucoside. Cell cycle analysis confirmed that the growth inhibition was not due to cytotoxicity, but cytostatic mechanism with increased number at the G1 phase of the cell cycle. The cell cycle arrest was also significantly correlated with the anthocyanin contents in P40 cultivar when compared with the white-fleshed O’Henry and yellow-fleshed NC Japanese controls. After Azoxymethane (AOM) or saline injected mice were fed basal AIN-93M diet or diets containing 10~30% of P40, 20% O’Henry or 20% NC Japanese for 6 weeks, aberrant crypt foci (ACF) multiplicity was significantly inhibited by 10~30% P40 diet. Imunohistochemistry results of colonic mucosa showed that the expression level of apoptosis marker, caspase-3, was significantly induced in the mice treated with 10~20% P40 diet. Also, PCNA expression level, which is proliferation marker, was significantly inhibited by the 30% P40 diet. These findings indicated that consuming a purple sweet potato, P40, may prevent colon cancer by modulating antioxidant status, inducing apoptosis, and reducing cell proliferation.

We gathered normal and colorectal cancer tissues from 18 patients after tumor surgery. The tumor tissues represent caner stages from T1 to T4 (TNM system).Two-dimensional electrophoresis and MALDI-TOF techniques were utilized to identify the differentially expressed proteins. Our studies showed that there are about 18 differentially expressed proteins in normal and tumor tissues (p<0.05) which 13 proteins increased in tumor were Keratin 8, Protein disuflde isomerase A3 precursor, Keratin 18, Fractalkine precursor, LDH-B, Tropomyosin alpha 4 chain, Tropomyosin alpha 3 chain, chloride intracellular channel protein 1, PTTG1(Pituitary tumor-transforming protein 1),

Folic acid is a B vitamin involved in DNA CpG methylation. Mandated dietary fortification has led to a subsequent increase in blood folate concentration which has been correlated to a simultaneous spike in colorectal cancer incidence in Canada and the US. Several human colorectal cancer cell lines were cultivated under low (0 mg/L), standard (4 mg/L), and high (16 mg/L) folate conditions for seven days, then assessed for DNA methyltransferase1 protein expression, changes in DNA methylation, and ability to generate colonospheres in culture. Low folic acid levels generally led to reduced DNMT1 protein expression, CpG hypomethylation, and reduced colonosphere yield. High folic acid levels led to increased DNMT1 protein expression, CpG hypermethylation, and maintained colonosphere yield. This data demonstrates that varying levels of folic acid in vitro can influence the methylation status and cancer stem cell self-renewal ability of human colorectal cancer cells. / Canadian Cancer Society

Colorectal cancer (CRC), which includes cancer of the large bowel and rectum is the third most common cancer in men and the second in women and there is a poorer survival rate in less developed regions of the world such as West Africa mainly due to the ‘out of reach’ costs of chemotherapy. Evidence suggests that aspirin, a non-steroidal anti-inflammatory drug (NSAID) has the potential to decrease incidence of, or mortality from, a number of cancers including CRC through several mechanisms of action. However, this evidence is dampened by aspirin’s gastrointestinal (GI) toxicity, which have been found to be mostly age-dependent. The search for potential aspirin-related compounds with the same or better cytotoxic effects against cancer cells accompanied by a safer toxicity profile has been ongoing over the years and led to us to synthesise a number of novel aspirin analogues. One of the mechanisms of action suggested for the anticancer property of aspirin is the COX-dependent pathway. In this thesis SW480 cell line, a CRC cell line that is COX-2 negative and mismatch repair (MMR) proficient was used to study the possible COX-independent mechanism of action for aspirin, its analogues and diflunisal at 0.5 mM. Diflunisal was included in this study because it is also a salicylate with reports of having cytotoxic effects. OE33 and FLO1 oesophageal cancer cells were also employed in the epidermal growth factor receptor (EGFR) and synergy experiments to show effects were not just specific to SW480 cells alone. These aspirin analogues were synthesised, identified using nuclear magnetic resonance (NMR) and infra-red (IR) spectroscopy, and tested for purity using thin layer chromatography (TLC) and melting point. The findings of this study suggest that these compounds breakdown into salicylates and perturb epidermal growth factor (EGF) internalization with PN517 (fumaryldiaspirin) and PN590 (ortho-thioaspirin) also driving EGF co-localization with early-endosome antigen-1 (EEA1). The perturbation of the internalization of EGF by aspirin and PN517 was also observed by a time-lapse assay using live confocal imaging. These compounds also had specific effects on different tyrosine phosphorylation sites of the EGFR, with none but PN590 inhibiting 4 phosphorylation at Y1068, and all but PN502 (ortho-aspirin), PN548 (meta-aspirin) and PN549 (para-aspirin) inhibiting phosphorylation at Y1045 and Y1173. Given that the EGF internalization assay involved the cells being treated with compounds for 2 h, cells were also treated for this same time period and probed with pEGFR 1045, which resulted in the compounds having no significant effect on phosphorylation at that site which is responsible for the ubiquitination of the EGFR. Most of these compounds were apoptotic with some showing a combination of apoptosis and necrosis. Aspirin and its isomers drove apoptotic cell death in SW480 cells via the BCL2-BAX pathway while the thioaspirins appear to follow the p21 pathway by decreasing the expression of the protein. In addition, it was shown that PN502 (aspirin), PN517 and PN590 had synergistic effects when used in combination with oxaliplatin at ED50, ED75 and ED90 in SW480 CRC cells. The cytotoxicity of these compounds individually or in combination was determined using MTT assay followed by the use of the CompuSyn and CalcuSyn software to calculate combination index (CI), which indicated whether a drug combination was synergistic, antagonistic or additive. PN517 and PN524 were synergistic when used in combination with cisplatin in OE33 oesophageal cancer cells. Effect of these compounds on the EGFR indicates a delay or disruption of the signalling pathway involved in the proliferation of cancer cells, thus, translating into protection against tumour formation or progression while the synergistic effects of these compounds when used in combination with platinum compounds can provide patients with less toxic chemotherapeutic regimen especially in patients with CRC tumours that harbour mutant TP53 gene and normally resistant to oxaliplatin. It is therefore proposed that the perturbation of EGF internalization is a novel mechanism of action for aspirin and its analogues in cancer therapy. These positive findings shed light on the understanding of the possible mechanism of action for aspirins and gives hope for a more affordable, less toxic therapy for the prevention, treatment and management of cancer.

Doctor of Philosophy / Food Science Institute / Channa B. Rajashekar / Weiqun Wang / As health-promoting agents, phytochemicals are biosynthesized in the plants that typically respond to environmental stresses. This study focused on the analysis of phytochemical contents in vegetables in response to environmental changes of high tunnel and light spectra. A potential anticancer activity was further studied by developing a novel 3D cell culture model. Three specific studies were conducted as follows. Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific. Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species. Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 μM. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies. Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application. Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific. Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species. Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 M. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies. Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application.

Le cancer colorectal est la deuxième cause de mortalité due au cancer en Europe et aux États-Unis. Etant donné l'efficacité limitée et la toxicité élevée des agents de chimiothérapie, de nouvelles approches sont nécessaires. Le flavanolignane silybine représente le principal constituant actif du chardon-marie (Silybum marianum). Les mécanismes moléculaires des propriétés anticancéreuses de la silybine ont été étudiés dans un modèle cellulaire de progression du cancer colorectal humain : les cellules SW480 issues d'un adénocarcinome, et leurs dérivées métastatiques les cellules SW620. Les effets chimiopréventifs de la silybine ont été étudiés dans un modèle de cancérogenèse colique induite par l'azoxyméthane chez le rat. La silybine induit une mort apoptotique avec activation de la caspase-3 dans les deux lignées. L'expression des récepteurs de mort TRAIL est augmentée, et la caspase-8 activée. Le potentiel mitochondrial est perturbé provoquant une libération du cytochrome c et une activation de la caspase-9. En plus de l'activation des voies apoptotiques extrinsèque et intrinsèque la silybine induit une réponse autophagique. La combinaison de la silybine et de TRAIL, un agent anti-cancéreux prometteur, provoque une mort cellulaire synergique dans les deux lignées. Un effet synergique est aussi observé avec la combinaison de la silybine et des inhibiteurs des histones déacétylases (HDAC) : TSA et SAHA. Dans le modèle chez le rat, la silybine réduit de 50% le nombre des lésions prénéoplasiques. En conclusion, la silybine est un agent naturel intéressant pour la prévention du cancer colorectal et dans le cadre d'une combinaison avec TRAIL/des inhibiteurs d'HDACs.