Browsing by Subject "colon cancer"
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Item Apigenin and Naringenin Increase Apoptosis and Decrease Proliferation via Transcriptional Regulation(2014-11-26) Daniels, Wesley DaniellePrevious studies have shown that apigenin and naringenin (flavonoids) suppress colon carcinogenesis by inducing apoptosis and suppressing proliferation in rats. The goal of this thesis was to test the hypothesis that apigenin and naringenin affect colonocyte proliferation and apoptosis by regulating expression of genes involved in microbial recognition (TLR-2, TLR-4), short chain fatty acid transport (MCT-1), cell cycle (p21), and apoptosis (Bax, Bcl-2, Fas, Noxa) as well as to determine if the mechanism of action was p53 dependent. Scraped mucosa was obtained from rats which received diets (0.02% naringenin, 0.1% apigenin, or basal) for 10 wks and were treated with AOM. YAMC and mp53 YAMC cells were treated with apigenin (0.1, 1, and 10 ?M), naringenin (0.1, 1, 10, 25, and 50 ?M), or estradiol (1 nM, positive control) for 96 h at non-permissive conditions. In vivo, apigenin suppressed MCT-1 (p<0.03), Bax (p=0.05), and Fas (p<0.05) expression compared to the control diet; and both flavonoids suppressed p21 (p<0.02) and TLR-4 (p<0.01) expression. Diet did not affect expression of Bcl-2 or TLR-2. 1 ?M or greater apigenin or naringenin treatment exhibited dose-dependent decreases (p<0.005) in cell numbers compared to vehicle in YAMCs, while no differences were identified in the mp53 YAMCs except with the highest treatment concentrations (p< 0.0001). No differences in proliferation were observed with apigenin or naringenin in either cell line, except with 20 ?M apigenin treatment (p< 0.0001). Apoptosis and gene expression data were inconclusive in vitro due to a lack of response in the positive control. Considering MCT-1 is a butyrate transporter and butyrate induces colonocyte p21 expression, the suppression of p21 expression may be a MCT-1 mediated effect. Reduction of MCT-1, p21, and TLR-4 expression by apigenin and naringenin suggests that these flavonoids may be able to reduce colon carcinogenesis through their influence on expression of genes involved in multiple pathways. The dose-dependent reduction in cell number induced by apigenin and naringenin is in part p53-mediated; however, the reduction in mp53 YAMC cells resulting from the greatest concentrations suggests alternate pathways can be induced. These reductions in cell number were not related to changes in proliferation.Item Effect of HZE radiation and diets rich in fiber and n-3 poly unsaturated fatty acids (n-3 PUFA) on colon cancer in rats(Texas A&M University, 2006-08-16) Glagolenko, Anna AnatolievnaThis 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.Item Effects of bran from sorghum grains containing different classes and levels of bioactive compounds in colon carcinogenesis(2009-05-15) Lewis, Jayme BethIn order to test the dietary effects of bioactive compounds present in whole grains, we decided to observe the effect of varying types of sorghum bran on colon cancer promotion. We used 40 rats consuming diets containing 6% fiber from either cellulose or bran from white (contains phenolic acids), brown (contains tannins), or black (contains anthocyanins) sorghum (n=10). Diets were fed for 10 wk, during which two azoxymethane (AOM) injections (15 mg/kg BW) were administered in wk 3 and 4. We observed that the total number of aberrant crypts (AC) and high multiplicity aberrant crypt foci (HMACF) were lower in rats consuming black (p < 0.04) and brown (p < 0.006) sorghum diets when compared to the cellulose diet, and that these decreases were an inverse function of diet antioxidant activity (ABTS). These observations led us to evaluate the effect of these diets on endogenous enzymatic activities (superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GPx), redox status as measured by reduced and oxidized glutathione, and cell cycle processes, proliferation and apoptosis, in the rat colon. Total SOD activity was higher (p < 0.04) in rats consuming black sorghum when compared to all other diets. A similar, but not significant, trend occurred in mitochondrial SOD. The white sorghum diet had enhanced (p < 0.02) CAT activity compared to the cellulose diet, but the black and brown sorghum diets were intermediate. Finally, all sorghum diets suppressed GPx activity relative to cellulose (p < 0.04). However, no changes were seen in levels of reduced and oxidized glutathione or the ratio of the two. The black sorghum fed rats had a lower proliferative index (p < 0.01) and zone (p < 0.04) compared to cellulose; brown and white sorghum rats were intermediate. Apoptotic index was highest in brown sorghum rats compared to cellulose (p < 0.03), while other sorghum diets were intermediate. These data suggest that the suppression of AC and HMACF formation in rats consuming sorghum bran may have resulted through the differential actions of the sorghum brans on endogenous antioxidant enzymes, which may affect colonocyte proliferation and apoptosis.Item Effects of dietary fat and fiber on the oxidative status of the small intestine and colon of rats(Texas A&M University, 2006-08-16) Sanders, Lisa MerleColon cancer is one of the most commonly diagnosed cancers in the US, yet small intestine cancer is a rare event. While there are many similarities between these two tissues, inherent differences such as redox status, may contribute to the variation in cancer occurrence. We examined the difference in reactive oxygen species (ROS) generation, antioxidant enzyme activity and oxidative DNA damage in the small and large intestine of rats under normal conditions and following exposure to exogenous oxidative stress. Basal ROS and antioxidant enzyme activities were greater in the colon than the small intestine, and the balance of ROS to antioxidant enzymes in the colon was more pro-oxidant than in the small intestine. During oxidative stress, ROS and oxidative DNA damage were greater in the colon than the small intestine. Thus the colon responds to oxidative stress less effectively than the small intestine, possibly contributing to increased cancer incidence at this site. We next wanted to understand how diets containing a combination of fish or corn oil and pectin or cellulose may alter the redox environment of the colon. ROS, oxidative DNA damage, antioxidant enzyme activity and apoptosis were measured in colonocytes of rats fed one of four diets containing either corn oil or fish oil and cellulose or pectin. Measurements were madein rats untreated with carcinogen and rats exposed to a chemical carcinogen and radiation. In rats not treated with a carcinogen, fish oil enhanced ROS, and fish oil/pectin suppressed antioxidant enzymes as compared to corn oil/cellulose. Oxidative DNA damage was inversely related to ROS in the fish oil/pectin diet and apoptosis was enhanced relative to other diets. In carcinogen treated and irradiated rats, a similar protective effect was seen with fish oil/pectin as evidenced by a reduction in oxidative DNA damage and enhancement of apoptosis. This suggests that a diet containing fish oil/pectin may protect against colon carcinogenesis by modulation of the redox environment to promote apoptosis and minimize oxidative DNA damage.Item Effects of fish oil and butyrate on diet-mediated apoptosis at the promotion stage of colon carcinogenesis(Texas A&M University, 2005-11-01) Newton, Anne HenryWe have previously shown that dietary fish oil and the fiber pectin protect against colon cancer in rats by increasing apoptosis induced by reactive oxygen species (ROS) at the initiation stage of tumorigenesis. We hypothesized that fish oil would incorporate into the cardiolipin of colonic mitochondrial membranes, creating an environment in which butyrate, a fermentation product of pectin, would also increase ROS and lead to apoptosis, as evidenced by decreased mitochondrial membrane potential (MMP), enhanced caspase-3 activity and cytochrome c translocation from the mitochondria, thus protecting against colon cancer by removing DNA damaged cells at the promotion stage of carcinogenesis. Sixty rats were provided a diet containing 15% corn or fish oil for 11 wk and injected with azoxymethane (AOM) or saline at wk 3 and 4. At wk 11, colonocytes were exposed to +/- butyrate ex vivo for 30 or 60 min. ROS and MMP were measured using fluorescence microscopy, and cytochrome c concentration and caspase-3 activity were measured using ELISA assays. Cardiolipin fatty acid enrichment was measured via TLC and GC. Butyrate increased ROS (p<0.0001) regardless of diet or treatment group. In colonic crypts from fish oilconsuming rats, butyrate reduced MMP (p=0.05). However, butyrate had no effect on MMP if the rats were consuming corn oil. In colonocytes from rats consuming fish oil, butyrate decreased mitochondrial cytochrome c (11%; p=0.02) concomitant with an increase in caspase-3 activity (17%; p=0.04) in the distal colon. In fish oil-fed animals, the n-3 fatty acids DHA and EPA were incorporated into cardiolipin at the expense of n-6 fatty acids. Regression analysis revealed a positive relationship between DHA (R=0.49, p=0.03) and EPA (R=0.59, p=0.02) and cytosolic cytochrome c content. As the percentage of DHA and EPA in the cardiolipin increased, the level of cytochrome c in the cytosol increased. These relationships were not seen in rats consuming corn oil and suggest that these results, induced only by the combination of butyrate with fish oil, may lead to increased apoptosis at the promotion stage of colon carcinogenesis via a mitochondria-mediated mechanism.Item Estrogen Receptor Beta and p53 Play Integral Roles in Estradiol Mediated Protection against Colon Tumor Development(2012-10-19) Weige, CharlesHormone replacement therapy and estrogen replacement therapy have shown the ability to reduce risk of colon cancer development in clinical and animal studies, but in vitro research has been unable to reproduce an estradiol (E2) induced response in colon cancer cell lines. We demonstrated that young adult mouse colonocytes (YAMC, non-malignant colonocytes) exhibit an anti-proliferative response to E2 treatment. These cells demonstrate reduced cell culture growth and increased apoptosis in response to E2. YAMC cells containing an activated Ras mutation are considered to be malignantly transformed, and lose the ability to respond to E2 treatment. Fulvestrant (ICI) was used as an estrogen receptor antagonist to determine that these results were estrogen receptor mediated. Furthermore, this effect was demonstrated to require the presence of ER? through the use of a transgenic ERbeta knockout mouse. In these mice, the presence of E2 significantly reduced the formation of azoxymethane induced premalignant lesions. Since YAMC cells exhibit an anti-proliferative response to E2 treatment, we utilized isogenic YAMC cell lines with and without a dominant negative p53 mutation to demonstrate that this E2 induced action involves p53 activity. E2 treatment results in increased p53 transcriptional activity and a pro-apoptotic change in expression of p53 downstream targets. Presence of the dominant negative p53 mutant nullifies these effects of E2 treatment. The involvement of p53 in the previously described protection against AOM induced premalignant lesions, was investigated using wild type and heterozygous p53 knockout (Het p53KO) mice. The reduction in p53 protein corresponded to reduced effectiveness of E2 treatment on the prevention of premalignant lesion formation in Het p53KO mice. In summary, our data indicate that E2 treatment induces anti-proliferative responses in non-malignant colonocytes and protects against the formation of carcinogen-induced premalignant lesions. These effects require the presence of functional ER? and p53. Further studies are required to more thoroughly elucidate the specific interactions and downstream effects of ER? and p53 in response to E2 stimulation.Item Estrogenic Properties of Sorghum Phenolics: Possible Role in Colon Cancer Prevention(2013-07-03) Yang, LiyiConsumption 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-? (ER?) or ER? at 5 and 10 ?g/mL, respectively. The same treatments led to induction of apoptosis in cells expressing ER?. 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 ER? 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 ER? 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.Item Mixture Modeling and Outlier Detection in Microarray Data Analysis(2010-01-16) George, Nysia I.Microarray technology has become a dynamic tool in gene expression analysis because it allows for the simultaneous measurement of thousands of gene expressions. Uniqueness in experimental units and microarray data platforms, coupled with how gene expressions are obtained, make the field open for interesting research questions. In this dissertation, we present our investigations of two independent studies related to microarray data analysis. First, we study a recent platform in biology and bioinformatics that compares the quality of genetic information from exfoliated colonocytes in fecal matter with genetic material from mucosa cells within the colon. Using the intraclass correlation coe?cient (ICC) as a measure of reproducibility, we assess the reliability of density estimation obtained from preliminary analysis of fecal and mucosa data sets. Numerical findings clearly show that the distribution is comprised of two components. For measurements between 0 and 1, it is natural to assume that the data points are from a beta-mixture distribution. We explore whether ICC values should be modeled with a beta mixture or transformed first and fit with a normal mixture. We find that the use of mixture of normals in the inverse-probit transformed scale is less sensitive toward model mis-specification; otherwise a biased conclusion could be reached. By using the normal mixture approach to compare the ICC distributions of fecal and mucosa samples, we observe the quality of reproducible genes in fecal array data to be comparable with that in mucosa arrays. For microarray data, within-gene variance estimation is often challenging due to the high frequency of low replication studies. Several methodologies have been developed to strengthen variance terms by borrowing information across genes. However, even with such accommodations, variance may be initiated by the presence of outliers. For our second study, we propose a robust modification of optimal shrinkage variance estimation to improve outlier detection. In order to increase power, we suggest grouping standardized data so that information shared across genes is similar in distribution. Simulation studies and analysis of real colon cancer microarray data reveal that our methodology provides a technique which is insensitive to outliers, free of distributional assumptions, effective for small sample size, and data adaptive.Item Modulation of Intestinal Micrornas by a Chemoprotective Diet(2012-12-05) Shah, Manasvi Shailesh 1984-We have hypothesized that dietary modulation of intestinal miRNA expression may contribute to the chemoprotective effects of nutritional bioactives (fish oil and pectin). Using a rat colon carcinogen model, we determined miRNAs-let-7d, miR-15b, miR-107, miR-191 and miR-324-5p were modulated by fish oil + pectin. We also demonstrated that BACE1 and PTEN are targets of miR-107 and miR-21, respectively. To further elucidate the biological effects of diet and carcinogen on miRNAs, we integrated global miRNAs, total and polysomal gene expression datasets obtained from the above mentioned study and used four computational approaches. We demonstrated that polysomal profiling is tightly related to microRNA changes when compared with total mRNA profiling. In addition, diet and carcinogen exposure modulated a number of microRNAs and complementary gene expression analyses showed that oncogenic PTK2B, PDE4B, and TCF4 were suppressed by the chemoprotective diet at both the mRNA and protein levels. To determine the function of select diet and colon carcinogen modulated miRNAs and to validate their targets, we carried out a series of loss and gain of function experiments along with luciferase reporter assays. We verified that PDE4B and TCF4 are direct targets of miR-26b and miR-203, respectively. PTK2B was determined to be an indirect target of miR-19b. In addition, microRNA physiological function was assessed by examining effects on apoptosis and cell proliferation. To better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we investigated the chemoprotective effects of dietary agents on miRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creERT2 knock in mice injected with AOM. We demonstrated that based on relative expression of miR-125a-5p, miR-190b and miR-191 in stem cells vs. daughter cells and differentiated cells, these miRNAs may be stem cell specific miRNAs. We also identified miR-21 to be significantly reduced in stem cells compared to differentiated cells and selectively modulated by these dietary agents in stem cells. In summary, our results indicate for the first time that fish oil plus pectin protect against colon tumorigenesis in part by modulating a subset of miRNAs and their target genes (mRNAs) implicated in the regulation of the colon stem cell niche and tumor development.Item Molecular mechanisms of Kruppel-like Factor 4(2008-06-18) Paul Michael Evans; Dr. Cornelis Elferink, Ph.D.; Dr. Jie Du, Ph.D.; Dr. Chunming Liu, Ph.D.; Dr. Binhua P. Zhou, M.D., Ph.D.; Dr. B. Mark Evers, M.D.The Wnt/beta-catenin pathway is a key pathway involved in the regulation of proliferation and differentiation within many tissues, including the epithelium of the intestine and of the skin. Wnt signaling is implicated in stem cell renewal. Deregulation of Wnt/beta-catenin signaling is crucial early event in colorectal tumorigenesis. Earlier work in our lab demonstrated that Kruppel-like factor 4 (KLF4) interacts with beta-catenin in vivo, repressing Wnt signaling and inhibiting tumor growth. KLF4 is an anti-proliferative transcription factor expressed in differentiated epithelial cells in the intestine. Previous studies clearly establish KLF4 as a tumor suppressor in colorectal cancer. Expression of KLF4 is downregulated in colorectal tumors, and heterozygous deletion of the Klf4 allele in a mouse model of colorectal cancer results in the formation of approximately 50% more tumors. In addition, KLF4 is important in stem cell programming. KLF4, in combination with three additional transcription factors, is sufficient to reprogram differentiated fibroblasts into embryonic stem cells. This dissertation focuses on the molecular mechanisms of KLF4-mediated transcription, both in the context of KLF4-mediated activation and KLF4-mediated repression. I demonstrate that the N-terminal transactivation domain of KLF4 recruits the co-activator p300/CBP to the promoter of the positively-regulated gene IAP, resulting in increased histone acetylation. On the negatively regulated gene, Cyclin B1, I demonstrate that KLF4 recruits HDAC3, decreasing histone acetylation. In addition, I show that KLF4 is acetylated by p300/CBP, and that acetylation of KLF is important in the activation of target genes as well as its ability to inhibit proliferation. I demonstrate that KLF4 inhibits Wnt/â-catenin signaling by blocking beta-catenin-mediated recruitment of p300/CBP to Wnt-regulated genes. Finally, I show that acetylation of beta-catenin is important in its ability to interact with p300/CBP and that KLF4 inhibits beta-catenin acetylation. These studies provide significant insight into the molecular mechanisms of KLF4-mediated transcription, and will prove useful in the development of targeted therapies for colorectal cancer as well as providing a deeper understanding of the mechanisms behind stem cell reprogramming.Item Novel Functions for the Pregnane X Receptor include Regulation of mRNA Turnover and Involvement in Colon Cancer Progression(2011-10-21) Eagleton, Navada LorraineTo 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.Item PI Control of Gene Expression in Tumorous Cell Lines(2010-01-16) Mendonca, Rouella J.Recent experiments are bringing to the fore more and more information about the effects of different treatments on the gene expression of different genes. The results obtained from these experiments show that some definite trends are observed in different genes in the Human Embryonic Kidney and Human Colon Adenocarcinoma Grade II cell lines. The difference in the gene expressions of the two cell lines motivates the problem in this thesis. The thesis provided intervention methods to make the colon cancer cell line genes behave more like their Human Embryonic Kidney cell line counterparts. Two methods of intervention were introduced. The first method was the simpler on-off control intervention while the second method used a more advanced proportional integral control to meet the goal. A comparison of these two intervention methods showed the clear implementational advantages of proportional integral control over on-off control.Item Postharvest irradiation treatment effect on grapefruit functional components and their role in prevention of colon cancer(Texas A&M University, 2005-11-01) Vanamala, Jairam Krishna PrasadThis dissertation examines the effects of postharvest treatment and processing on biologically active compounds of orange juice, and ??Rio Red?? grapefruit and their ability to prevent chemically induced colon cancer in rat model. The first study evaluated the differences in flavonoid content of commercial ??made from concentrate?? (MFC) orange juices and ??not from concentrate?? (NFC) orange and grapefruit juices. Total flavonoid content of MFC orange juices (53 mg/100 mL; n = 12) was significantly (P ≤ 0.05) higher than NFC orange juices (36.5 mg/100 mL; n = 14). The second study investigated the ionizing radiation and storage effects on bioactive compounds and quality of ??Rio Red?? grapefruit. Results showed that storage and irradiation significantly (P ≤ 0.05) affected the bioactive compounds in grapefruit, however, the effect of storage was prominent. The third study examined the influence of irradiation and freeze drying on bioactive compounds of grapefruit. Irradiation of grapefruit prior to freeze drying resulted in enhanced (P ≤ 0.05) flavonoid content (naringin and narirutin). Freeze drying markedly reduced (P ≤ 0.05) lycopene content. Freeze drying and irradiation reduced (P ≤ 0.05) volatile compounds (d-limonene and myrcene), with the exception of ethanol. In the fourth study suppression of colon cancer development in Sprague Dawley rats by natural and irradiated grapefruits and their functional compounds, naringin and limonin, were evaluated.The total number of aberrant crypts (AC; P = 0.02), number of high multiplicity AC foci (ACF; P = 0.01), and proliferative index (P = 0.02) were lower and apoptosis (P = 0.02) was higher in azoxymethane (AOM) injected rats on experimental diets. However, only natural grapefruit and limonin only suppressed AOM induced expansion (P = 0.008) of proliferative zone and also enhanced apoptosis more effectively than other experimental diets indicating that natural grapefruit and limonin may serve as better chemopreventive agents compared to IGFPP and naringin. The present study indicates that postharvest quarantine doses of irradiation slightly alter composition of bioactive compounds and in turn marginally reduce the chemopreventive ability of grapefruit against the promotion stage of colon cancer. These results warrant the necessity of testing the impact of post harvest treatments on fruits and vegetables chemopreventive ability.Item PTEN loss induces epithelial-mesenchymal transition in human colon cancer cells(2009-06-16) Kanika Alake Bowen; B. Mark Evers; Tien Ko; Sarita Sastry; Peter Zhou; Dai ChungBackground: Epithelial-mesenchymal transition is a critical early event in the invasion and metastasis of many cancers, including colorectal cancer. Chronic inflammation is an inducer of several cancers and inflammatory cytokines have been implicated in tumor invasion. Materials and Methods: Human colon cancer cell lines HCT116 and SW480 were transfected with PTEN siRNA or non-targeting control. Invasiveness was measured using a modified Boyden chamber assay and migration was assessed using a scratch assay. Results. PTEN knockdown increases the invasion and migration of CRC cells and the addition of medium containing TNF-á further enhanced the migration and invasion. PTEN knockdown resulted in nuclear â-catenin accumulation and increased expression of downstream proteins c-Myc and cyclin D1. Conclusions. Our study supports clinical studies identifying an association of PTEN loss with late stage cancer. Cellular factors secreted from the surrounding tumor milieu likely act in concert with genetic changes in the tumor cells and contribute to enhanced tumor invasion.Item The Protective Effects of Estradiol on Sporadic and Inflammation-associated Colon Cancer(2013-11-22) Armstrong, Cameron MichelleEpidemiological studies suggest pre-menopausal women have a reduced risk for sporadic and inflammation-associated colon cancer compared to post-menopausal women and men. The studies presented herein aim to determine the protective mechanisms of estradiol (E2) during sporadic and inflammation-associated colonic carcinogenesis. When investigating the role of E2 and fish oil at the earliest stage of sporadic colon cancer development, E2 had no effect on DNA adduct formation while dietary fish oil significantly reduced DNA adduct formation. Contrarily, E2 significantly induced apoptosis of damaged colonocytes while fish oil was not protective. In an in vivo model of inflammation-associated colon carcinogenesis with E2 administered following induction of DNA damage and initiation of inflammation, E2 treatment was associated with decreased colon tumor size and number in wild type (WT) but not estrogen receptor (ER) ? knockout (ER?KO) mice. Interestingly, apoptosis was reduced and proliferation increased by E2 in these tumors in WT mice. This may be due to the altered ER expression in these tissues as the tumors developed, with ER??expression decreasing concomitantly with ER? expression increasing. Contrary to the protective effect of E2 on inflammation-associated colon tumor formation, which was dependent on ER?, during acute inflammation in the colon E2 was protective against inflammation in both WT and ER?KO mice and injury in ER?KO mice. The protection against inflammation is likely due to the reduction of pro-inflammatory cytokine expression by E2. Apoptosis and proliferation were decreased and increased in the proximal and distal colon respectively in ER?KO mice. In vitro studies further elucidated the roles of ER? and ER? in colonocytes. E2 and ER?, but not ER?, specific agonists reduced cell number and induce apoptosis in nonmalignant colonocytes. This effect was lost in the presence of mutated p53. In ER? overexpressed nonmalignant colonocytes, E2 had no effect on cell number while ER? agonist and ER? agonists decreased and increased cell number respectively. These studies suggest that E2 is protective in the colon and ER? is required for protection against carcinogenesis but not protection against inflammation. Additionally, the protection against colon carcinogenesis is likely p53 mediated.Item The Role of Docosahexaenoic Acid in Regulation of Epidermal Growth Factor Receptor Activation and Function(2012-08-30) Turk, Harmony 1985-The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase integral in regulating cell growth, survival, and migration. EGFR signaling, which is dependent on localization of the receptor within lipid rafts, is often hijacked during colon tumorigenesis. Previous work has found that docosahexaenoic acid (DHA) is protective against colon cancer. This fatty acid is proposed to function in part by perturbing lipid rafts and thereby altering cell signaling. The overall objective of this work was to determine whether DHA alters EGFR function and signaling. We assessed EGFR localization and ligand-induced phosphorylation in YAMC cells treated with fatty acids. We found that DHA reduced the localization of EGFR to lipid rafts. Concomitant with altering receptor localization, DHA was found to increase EGFR phosphorylation. However, DHA paradoxically suppressed EGFR signal transduction. We found that DHA uniquely altered EGFR activity, and other long chain polyunsaturated fatty acid did not exert the same effect. We additionally observed similar effects on EGFR activation and signaling by feeding mice a diet enriched in fish oil (high in DHA), and this was attendant with reduced colon tumorigenesis. We next probed the mechanism by which DHA enhances EGFR phosphorylation. We found that DHA facilitates receptor dimerization to increase phosphorylation. We additionally identified Ras activation as the site of perturbation of signal transduction. DHA suppressed signal transduction by both changing the localization of EGFR within the plasma membrane and increasing receptor endocytosis and degradation. Lastly, we extended our observations into a wounding model. Although DHA uniquely altered ligand-stimulated EGFR activity, both DHA and EPA altered EGFR transactivation and signaling upon injury. This culminated in reduced wound healing in DHA and EPA treated cells. In an animal model, we found that diets enriched in either DHA or EPA altered EGFR signaling in the colonocytes of wounded animals. Overall, we found that DHA modifies EGFR signaling, which can be beneficial or detrimental for health depending on the disease state of an individual. These data help elucidate a mechanism by which DHA protects against colon cancer, as well as indicating a potential downside of n-3 PUFA therapy.