Progress 07/01/10 to 06/30/15
Outputs
OUTPUTS: This project had two research components: Project A hypothesized that dietary sources of nitrate and nitrite may be associated with the risk of colorectal cancer progression. Previous data and research work provided additional support for that hypothesis. The work found that dietary patterns high in nitrate and nitrite from fruits and vegetables may be responsible for part of the hypotensive effect of the Dietary Approaches to Stop Hypertension diet. Results were extended by applying nitrite to colon carcinoma cells at different stages of transformation to ascertain potential preventive or promotional risks. Studies were also implemented to determine if sodium nitrite in drinking water can affect colitis severity in a murine model of colitis called the SMAD3 knock out mouse. The preliminary data indicated that low (50 mg/liter) and high (2 grams/liter) concentrations of sodium nitrite dose-dependently decrease colitis scores compared to non-nitrite fed control animals. Project B hypothesized that an energy restricted ketogenic diet affects the growth of gioblastoma in humans. It was titled "Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (OXCT1) and Glial Fibrillary Acidic Protein (GFAP) Content in Human Astrocytoma and Gliosarcoma: Implication for Ketone Utilization in Cancer" Results from project B demonstrated that astrocytomas and gliosarcomas express low levels of OXCT1 and BDH1 in human GBM tumors while BDH2 and ACAT1 are expressed in these tumors. This limited data supported the hypothesis that astrocytomas may express low leve3ls of the enzymes necessary to utilize ketones for energy and growth. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Project A : Overall, the data indicates that nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at high concentrations in cells representing stage 4 colon carcinomas. Project B: Preliminary results from human GBM indicates that an energy restricted ketogenic diet is a biologically plausible therapy for GBM.
Publications
- Jiang H, Tang Y, Garg HK, Parthasarathy DK, Torregrossa AC, Hord NG, Bryan NS. 2012. Concentration- and stage-specific effects of nitrite on colon cancer cell lines. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society. 26(4):267-73.
- Hord NG. 2011. Dietary nitrates, nitrites, and cardiovascular disease.. Current atherosclerosis reports. 13(6):484-92.
- Hord NG, Ghannam JS, Garg HK, Berens PD, Bryan NS. 2011. Nitrate and nitrite content of human, formula, bovine, and soy milks: implications for dietary nitrite and nitrate recommendations. Breastfeeding medicine : the official journal of the Academy of Breastfeeding Medicine. 6(6):393-9.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Project A findings provide additional support for our hypothesis that dietary sources of nitrate and nitrite may be associated with health benefits. Our published results and work described below support our hypothesis that nitrite may be associated with risk of colorectal cancer progression dependent upon stage of colon carcinoma. There is growing evidence revealing that nitrate and nitrite metabolism occurs in blood and tissue to form NO and other bioactive nitrogen oxides, representing an alternative to classical L-arginine-NO synthase -NO signaling pathway. NO is involved in neurotransmission, vasodilation, inflammation, and immunity and is also believed to play roles in multiple stages of various cancers. Our collaborative group has reported that dietary patterns high in nitrate and nitrite from vegetables and fruits may be responsible for part of the hypotensive effect of the Dietary Approaches to Stop Hypertension diet. These data have been presented at invited seminars at the University of Arizona's Nutrition and Health meeting as well as local and state dietetic association meetings. This year we have extended our results by applying nitrite to colon carcinoma cells at different stages of transformation to ascertain potential preventive or promotional risks. This work is "in press" for the journal Nitric Oxide. We have also implemented studies to determine if sodium nitrite in drinking water can affect colitis severity in a murine model of colitis called the SMAD3 knock out mouse. This model initiates colitis with a bacterium called H. hepaticus and colitis development is measured using colitis score by Dr. Inge Langhor, our collaborating pathologist. Our preliminary data indicate that low (50 mg/liter) and high (2 grams/liter) concentrations of sodium nitrite dose-dependently decrease colitis score compared to non-nitrite fed control animals. Project B was developed using funds from the American Cancer Society, in collaboration with Drs. L. Karl Olson and Howard Chang, to determine if ketone metabolizing enzymes in brain cancer tissues are expressed, as hypothesized, at low levels. This work, titled "Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (OXCT1) and Glial Fibrillary Acidic Protein (GFAP) Content in Human Astrocytoma and Gliosarcoma: Implications for Ketone Utilization in Cancer" indicate that the expression of OXCT1 and BDH1 is negative in human GBM tumors while BDH2 and ACAT1 are expressed in these tumors. Quantitation and publication of these data will occur upon completion of data analysis and manuscript preparation. The human study protocol to implement the energy restricted ketogenic diet for GBM patients has received approved from appropriate IRB committees at MSU and has received funding for patient accrual. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Scientists and Registered Dietitians providing dietary advice for the prevention of chronic diseases by dietary means will benefit from this research. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The data from Project 1 indicates that nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas. These data may lend biological plausibility to the heterogeneous risk profiles of dietary nitrite exposure and risk of certain cancers in humans and support context-specific recommendations for consumption of dietary nitrite related to cancer risk. The manuscript describing these data are in press at Nitric Oxide. Our results from project B demonstrate that astrocytomas and gliosarcomas express low levels of OXCT1 and BDH1 in human GBM tumors while BDH2 and ACAT1 are expressed in these tumors. These limited data support our hypothesis that astrocytomas may express low levels of the enzymes necessary to utlize ketones for energy. Our preliminary results from human GBM indicates that an energy restricted ketogenic diet is a biologically plausible therapy for GBM.
Publications
- Hord, NG (2011) Regulation of dietary nitrate and nitrite: balancing essential physiological roles with potential health risks. In: "Nitrate and Nitrite in Human Health and Disease." ed: NS Bryan, J Loscalzo. Humana Press.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Project A has built upon previous data and supports our hypothesis that dietary sources of nitrate and nitrite may be associated with health benefits. Our published results and work described below support our hypothesis that nitrite may be associated with risk of colorectal cancer progression dependent upon stage of colon carcinoma. There is growing evidence revealing that nitrate and nitrite metabolism occurs in blood and tissue to form NO and other bioactive nitrogen oxides, representing an alternative to classical L-arginine-NO synthase -NO signaling pathway. NO is involved in neurotransmission, vasodilation, inflammation, and immunity and is also believed to play roles in multiple stages of various cancers. PARTICIPANTS: Worked with collaborators, Drs. L. Karl Olson and Howard Chang, to determine if ketone metabolizing enzymes in brain cancer tissues are expressed, as hypothesized, at low levels. This work, titled "Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (OXCT1) and Glial Fibrillary Acidic Protein (GFAP) Content in Human Astrocytoma and Gliosarcoma: Implications for Ketone Utilization in Cancer" was presented in August, 2010 by Ms. Courtney Meredith at an MSU undergraduate research forum. Our collaborative group, Drs. N. Hord, A. Al-Janadi, K. Berger, N. Bryan, J. Fenton and I. Langhor, has reported that dietary patterns high in nitrate and nitrite from vegetables and fruits may be responsible for part of the hypotensive effect of the Dietary Approaches to Stop Hypertension diet. These data have been presented at invited seminars at the University of Florida and Johns Hopkins University Bloomberg School of Public Health as well as the American Institute for Cancer Research Annual Meeting. TARGET AUDIENCES: Examine dietary nitrite exposure and risk of certain cancers in humans and support context-specific recommendations for consumption of dietary nitrite related to cancer risk. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our collaborative group has reported that dietary patterns high in nitrate and nitrite from vegetables and fruits may be responsible for part of the hypotensive effect of the Dietary Approaches to Stop Hypertension diet. These data have been presented at invited seminars at the University of Florida and Johns Hopkins University Bloomberg School of Public Health as well as the American Institute for Cancer Research Annual Meeting. This year we have extended our results by applying nitrite to colon carcinoma cells at different stages of transformation to ascertain potential preventive or promotional risks. Project B was developed using funds from the American Cancer Society, in collaboration with Drs. L. Karl Olson and Howard Chang, to determine if ketone metabolizing enzymes in brain cancer tissues are expressed, as hypothesized, at low levels. This work, titled "Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (OXCT1) and Glial Fibrillary Acidic Protein (GFAP) Content in Human Astrocytoma and Gliosarcoma: Implications for Ketone Utilization in Cancer" was presented in August, 2010 by Ms. Courtney Meredith at an MSU undergraduate research forum.
Publications
- Hord NG, Ghannam JS, Garg HK, Berens PD, Bryan NS. (2010) Nitrate and Nitrite Content of Human, Formula, Bovine, and Soy Milks: Implications for Dietary Nitrite and Nitrate Recommendations. Breastfeed Med. 2010 Oct 19. [Epub ahead of print]
- Hord, NG. (2010) Regulation of dietary nitrate and nitrite: balancing essential physiological roles with potential health risks. In: Nitrates and Nitrites in Human Health and Disease; Springer/Humana Press, Editors: Joseph Loscalzo, M.D., Ph.D (Harvard University), Nathan Bryan (University of Texas).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This progress report details effort on two projects involving the potential of two types of dietary constituents to modulate biological processes related to colon cancer prevention. The second project also has relevance for cardiovascular health. The first project has established a role for body composition in determining the concentration of inflammatory mediators in a mouse model. The first project (with Dr. Jenifer Fenton) reports data associating body composition in control, lean and obese mice with the presence of disparate patterns of inflammatory mediators in serum. These data have implications for colon cancer risk because obesity in humans is influenced by adiposity. Six-week-old female C57BL/6N mice (n = 12 per group) were randomized to one of three diets: control (fed ad libitum); lean (30% calorie-restricted regimen relative to control) and diet-induced obese (DIO; high calorie diet, fed ad libitum). After 10 weeks on the diets, blood samples were collected, and adipokine/cytokine/chemokine serum profiles were measured by antibody array. Lean mice, relative to the control group, displayed increased concentrations of insulin-like growth factor (IGF) binding protein-3, -5 and -6 and adiponectin and decreased IGF-1. These mice also showed increased concentrations of interleukin (IL)-10, IL-12 p40/p70, eotaxin, monocyte chemoattractant protein-5 and SDF-1. In contrast, DIO mice displayed increased leptin, IL-6 and LPS-induced chemokine and decreased concentrations of all chemokines/cytokines measured relative to control mice. The second project quantifies nitrate and nitrite from dietary sources in order to highlight the irrational basis of regulations that limit nitrate and nitrite consumption from food sources. Nitrates and nitrites serve as substrates for production of nitric oxide, a free radical that is required for cardiovascular function. Approximately 80% of dietary nitrates are derived from vegetableconsumption; sources of nitrites include vegetables, fruit, and processed meats. Nitrites are produced endogenously through the oxidation of nitric oxide and through a reduction of nitrate by commensal bacteria in the mouth and gastrointestinal tract. As such, the dietary provision of nitrates and nitrites from vegetables and fruit may contribute to the blood pressure-lowering effects of the Dietary Approaches to Stop Hypertension (DASH) diet. We quantified nitrate and nitrite concentrations by HPLC in a convenience sample of foods. Incorporating these values into 2 hypothetical dietary patterns that emphasize high-nitrate or low-nitrate vegetable and fruit choices based on the DASH diet, we found that nitrate concentrations in these 2 patterns vary from 174 to 1222 mg. The hypothetical high-nitrate DASH diet pattern exceeds theWorld Health Organization's Acceptable Daily Intake for nitrate by 550% for a 60-kg adult. PARTICIPANTS: Project one collaborators include individuals from Michigan State University (J. Fenton), the Department of Nutrition, University of Texas at Austin (S. Hursting, N. Nunez and S. Perkins) and the Department of Endocrinology, Mt. Sinai School of Medicine, New York, NY (S. Yakar). Project two collaborators are from the University the Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center, Houston, TX (N. Bryan and Y. Tang). TARGET AUDIENCES: The target audience for project one are basic science and applied health researchers working in the biological effects of obesity. The target audience for project two are governmental regulators and scientists interested in the relationship between nitrate/nitrite consumption and health. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Results from project one demonstrate that differential adipokine, cytokine and chemokine protein profiles in control, lean and DIO mice may have implications for immune responsiveness and risk of disease. In the context of a hypothetical obese individual, higher levels of dipokine, cytokine and chemokine proteins may represent evidence of inflammation-related processes that could increase risk of colon cancer. Result from project two are consistent with the hypotheses that vegetables and fruit can serve as dietary sources of nitrate and nitrite. These data call into question the rationale for recommendations to limit nitrate and nitrite consumption from plant foods; a comprehensive reevaluation of the health effects of food sources of nitrates and nitrites is appropriate. The strength of the evidence linking the consumption of nitrate- and nitrite-containing plant foods to beneficial health effects supports the consideration of these compounds as nutrients.
Publications
- Fenton JI, Nunez NP, Yakar S, Perkins SN, Hord NG, Hursting SD. (2009) Diet-induced adiposity alters the serum profile of inflammation in C57BL/6N mice as measured by antibody array. Diabetes Obes Metab.11(4):343-54.
- Hord, NG, Tang, YP, Bryan, NS (2009) Food sources of nitrates and nitrites: the physiological context for potential health benefits, American Journal of Clinical Nutrition. 90: 1-10.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This progress report details effort on two projects involving the potential of two types of dietary constituents to modulate biological processes related to colon cancer prevention: probiotic bacteria and nitrate/nitrites. The first project has established, for the first time, the role of probiotic bacteria in colonic epithelial cell signaling. We extend these observations in this progress report to include a publication detailing the effects of prokaryotes, including probiotic bacteria, on physiologic sensing mechanisms and potential health outcomes due to eukaryotic-microbiota interaction. The effects of adipocyte-specific cytokines and growth facters on colon epithelial cell gene expression and phenotype are detailed in two publications below. The second project relates to the potential health benefits of dietary nitrates and nitrites, food constituents that have their origin in the food cycle due to the actions of soil microbiota. The presence of nitrate/nitrite in the diet has been demonstrated to modulate the effects of myocardial ischemia and ischemia/reperfusion injury as well as have implications for gastrointestinal cancer. This project has demonstrated that these dietary constituents, heretofore regulated based on potential toxicities, exceed regulatory limits at normal dietary intake levels. Taken together, these data suggest a reexamination of the scientific basis for potential benefits and risks of dietary nitrate/nitrite. Together with collaborator, Dr. Nathan Bryan of the Center for Cell Signaling at the University of Texas, we have our first manuscript accepted in the American Journal of Clinical Nutrition. PARTICIPANTS: Nathan Bryan, PhD (University of Texas Health Science Center, Houston) Jenifer Fenton, PhD (Michigan STate University) Stephen Hursting (University of Texas, Austin) TARGET AUDIENCES: Target audiences for the results of these projects include scientists, policy makers and the public. Scientists and policy makers will appreciate the exposition of the physiologic contexts in which dietary factors exert their biological actions that modulate processes related to the promotion of colon carcinogenesis. PROJECT MODIFICATIONS: The addition of the work related to nitrates/nitrites to this project with Dr. Bryan are important due to their association with gastrointestinal cancer risk. Future studies and grant proposals will be developed to address these associations in animal models used by Drs. Hursting and Fenton.
Impacts
The impact of our work on probiotics and adipocytokines in colon epithelial cells has been to demonstrate the context-specific physiology of promotional influences on the process of colon carcinogenesis. The impact of our work on dietary nitrates/nitrites, when published, will influence scientists and regulators to examine the potential health effects of nitrates/nitrites. These effects may be a significant portion of the hypotensive effects of vegetables observed in the Dietary Approaches to Stop Hypertension studies.
Publications
- Hord NG (2008) Eukaryotic-microbiota crosstalk: potential mechanisms for health benefits of prebiotics and probiotics. Annu Rev Nutr.;28:215-31.
- Fenton JI, Birmingham JM, Hursting SD, Hord NG. (2008) Adiponectin blocks multiple signaling cascades associated with leptin-induced cell proliferation in Apc Min/+ colon epithelial cells. Int J Cancer. Jun 1;122(11):2437-45.
- Fenton JI, Lavigne JA, Perkins SN, Liu H, Chandramouli GV, Shih JH, Hord NG, Hursting SD. (2008) Microarray analysis reveals that leptin induces autocrine/paracrine cascades to promote survival and proliferation of colon epithelial cells in an Apc genotype-dependent fashion. Mol Carcinog. Jan;47(1):9-21.
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Progress 01/01/07 to 12/31/07
Outputs
Inflammatory bowel diseases increase colon cancer risk. Bacteria may influence inflammation in the colon by inducing toll-like receptor (TLR)-dependent increase in epithelial production of inflammatory mediators and subsequently activate immune cells implicated in carcinogenesis. This progress report details effort on this project involving the potential of probiotic bacteria to modulate biological processes caused by a bacterial pathogen in a cell culture model system. Non-tumorigenic murine colon epithelial cell lines with distinct Apc genotypes, YAMC (Apc+/+) and IMCE (ApcMin/+) cells) were used to identify the signaling pathways that probiotic bacteria utilize to mitigate the production of proinflammatory mediators and cytokines caused by exposure to the bacterial pathogen. We hypothesized that probiotic bacteria, such as Lactobacillus rhamnosus GG, Bifidobacterium breve, Lactobacillus salivarius, and Bifidobacterium lactis would decrease Escherichia coli O157: H7
(EC) induced production of nitric oxide (NO) and interleukin-6 (IL-6) and macrophage activation and chemotaxis in a genus- and species-specific manner. EC induced NO and IL-6 production in colon epithelial cells compared to untreated cells (p-value <0.001). Probiotic bacteria induced IL-6 production, but not NO production, with a greater amount in IMCE cells (p-value <0.05). IMCE and YAMC cells co-treated with EC and probiotic bacteria showed a probiotic genus- and species-specific decrease in NO and IL-6 when compared to EC (p-value <0.001). Neutralizing antibodies against TLR-2 and -4, as well as a chemical inhibitor of TLR-4 (polymyxin B) reduced NO and IL-6 production by epithelial cells when compared to EC (p-value <0.05). Conditioned media collected from IMCE and YAMC cells treated with EC exposed to macrophages induced IL-6 (p<0.001) but not NO macrophage production. Conditioned media collected from IMCE and YAMC cells co-treated with EC and probiotic bacteria exposed to
macrophages caused genus- and species-specific decrease in macrophage production of IL-6 compared to EC (p-value <0.001). Only exposure to conditioned media from EC-treated IMCE cells resulted in macrophage chemotaxis (p <0.01). These results suggest that EC-induced inflammatory mediator production occurs, in part, by activating TLR-2 and 4. The mechanisms by which probiotic bacteria ameliorate these EC-induced TLR-mediated events will require further research.
Impacts
These provide the first evidence that probiotic bacteria inhibit induction of inflammatory mediators by bacterial pathogen E.coli:0157:H7 in non-tumorigenic colonic epithelial cells via TLR-dependent mechanisms. If these data are confirmed, it would suggest a new dietary strategy for the prevention of intestinal inflammation caused by enteric bacterial pathogens by probiotic bacteria.
Publications
- Fenton, JI , Hord, NG, Hursting SD et al. (2008) Microarray analysis reveals that leptin induces autocrine/paracrine cascades to promote survival and proliferation of colon epithelial cells in an Apc genotype-dependent fashion., Molecular Carcinogenesis, 47(1)9.
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Progress 01/01/06 to 12/31/06
Outputs
This progress report details effort on two projects involving the potential of two types of dietary constituents to modulate processes related to colon cancer prevention in a cell culture model system. The first project has established a role of the spice component, curcumin, and flavonoids in causing cell migration in colonic epithelial cells. These data extend these observations by employing flavonoids, like ECGC, to determine if these compounds, associated with a preventive effect on colon cancer, can induce migration in colon epithelial cells through a specific mechanism. This migratory phenotype is dependent upon wild-type adenomatous polyposis coli (Apc) expression. Non-tumorigenic murine colon epithelial cell lines with distinct Apc genotypes, YAMC (Apc+/+) and IMCE (ApcMin/+) cells) were used to assess the association between Apc genotype, flavonoid-induced cell motility and the flavonoid-induced activity of mitogen-activated protein kinase (MAPK) activity in
these cells. A secondary goal was to identify whether the 67 kDa laminin receptor (LR), the putative receptor for ECGC, mediates ECGC-induced migration in these cell types. YAMC and IMCE cells were treated with ECGC (ECGC; 1 microM) or hepatocyte growth factor (as positive control HGF; 25 ng/ml). HGF (25 ng/ml) induced a greater migratory response in YAMC compared to IMCE cells after 24 hours (p<0.05). Treatment with ECGC (1 microM) induced an equivalent or greater migratory response in IMCE than YAMC cells. When migrating cells were treated with inhibitors of MAPKs, migration was inhibited in both cell types. The LR was identified by western blot in both cell types. Antibodies against LR blocked ECGC-mediated cell migration. These data provide the first evidence that LR-mediated ECGC signaling involving MAPKs induce a migratory phenotype in colon epithelial cells. The second project in which significant progress has been made utilizes the same cell culture models described above.
These studies have identified specific lactic acid bacteria (LAB) and bifidobacteria donated by Danisco, Inc. which can decrease the production of proinflammatory mediators caused by exposure of these cells to bacterial pathogen, E. coli 0157:H7 (EC). LAB bacteria may inhibit inflammatory processes resulting from exposure to EC by blocking nuclear factor kappa B (NF-kB) nuclear translocation and DNA binding as well as inducible nitric oxide (NO) synthase and NO production. The objective of this study was to assess the ability of Lactobacillus casei (LC), Lactobacillus reuteri (LR) and Bifidobacterium brevis (BB), to block EC-induced NF-kB translocation and NO production in a non-tumorogenic murine colon epithelial cell line. Nuclear translocation of NF-kB, was assessed by immunocytochemistry and western blotting. NF-kB DNA binding by competitive ELISA. NO was assessed in culture supernatants using the Greiss reaction. LC, LR and BB differentially inhibited nuclear translocation and
DNA binding of NF-kB and NO production induced by EC. Western blots from nuclear extracts confirmed that LR was more effective than LC in blocking EC-induced NF-kB translocation and DNA binding.
Impacts
Data from Project One suggests specific signaling pathways initiated by specific flavonoids (e.g., NAR, API, HES and ECGC) may allow cells heterozygous for Apc (IMCE) to overcome defective cell migration and experience cell differentiation and apoptosis. These dietary factors could, therefore, decrease the number of preneoplastic cells at risk for transformation into tumor cells. These data identify the 67 Kda laminin receptor as the mediator for the flavonoid-induced migratory effect. This is a new potential mechanism by which dietary factors may decrease risk of colorectal cancer. Grant funding has being utilized to definitively determine the specific intracellular signaling machinery and their associated transcription factor targets that these flavonoids activate in order to produce the migratory phenotype. Data from Project Two provide the first evidence that probiotic bacteria inhibit induction of inflammatory mediators by bacterial pathogen E.coli:0157:H7 in
non-tumorigenic colonic epithelial cells. We have identified intracellular signaling pathways activated by probiotic bacteria which may mediate these anti-inflammatory effects. If these data are confirmed, it would suggest a new dietary strategy for the prevention of intestinal inflammation caused by enteric bacterial pathogens by probiotic bacteria.
Publications
- Collaborative Work from our lab: Fenton, JI, Hursting SD, Perkins S, Hord NG (2006) Interleukin-6 production induced by leptin treatment promotes cell proliferation in an Apc(Min/+) cell line, Carcinogenesis, 27(7)1507-15.
- Fenton JI, Hursting SD, Perkins SN, Hord NG. (2006) Leptin induces an Apc genotype-associated colon epithelial cell chemokine production pattern associated with macrophage chemotaxis and activation. Carcinogenesis. 2006 Aug 4; [Epub ahead of print].
- Hord NG and Fenton JI. (2007) Context is everything: mining the normal and preneoplastic microenvironment for insights into the diet and cancer risk conundrum. Molecular Food and Nutrition Research, Jan 51 (1), 100-106
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Progress 01/01/05 to 12/31/05
Outputs
This progress report details effort on two projects involving the potential of two types of dietary constituents to modulate biological processes related to colon cancer prevention in a cell culture model system. The first project has established, for the first time, the role of the spice component, curcumin, in causing cell migration in colonic epithelial cells. We extend these observations in this progress report to include the specific cell signaling mechanisms initiated by the dietary flavonoids naringin (NAR), apigenin (API), hesperidin (HES) and epigallocatechin (ECGC). Colonic epithelial cell migration is required for movement up the crypt-villus axis and hence, normal differentiated cell function. This migratory phenotype is dependent upon wild-type adenomatous polyposis coli (Apc) expression. Non-tumorigenic murine colon epithelial cell lines with distinct Apc genotypes, YAMC (Apc+/+) and IMCE (ApcMin/+) cells) were used to assess the association between Apc
genotype, cell motility and matrix metalloproteinase (MMP) activity in these cells. The migration-inducing flavonoids (NAR, API, HES and ECGC) induced migration in IMCE cells at 1 (p<0.05) and 50 micromolar (p<0.01) We have extended these findings by demonstrating that flavonoids utilize specific cell signaling pathways, namely, p38 and MEK1/2 mitogen-activated protein kinases, JNK, nuclear factor kappa B (NF-kB) and NAPDH oxidase, but not estrogen receptor-mediated signaling pathways, to elicit migration. induced by flavonoids that may contribute to the migratory phenotype. These have been assessed using enzymatic inhibitors of these pathways. Activation of these pathways by NAR, API and ECGC, but not HES, is associated with nuclear translocation of the redox-sensitive transcription factor called Nrf2. Nrf2 is a transcription factor which regulates antioxidant response element-containing genes. These data highlight a new and potentially important mechanism by which flavonoids may
induce Nrf2-mediated transcription to accomplish cell migration. Work is ongoing, using transcription factor-DNA binding assays, to identify other transcription factor networks activated by flavonoids associated with cell migration.. The second project has not progressed significantly due to the lack of personnel support. A new graduate student is pursuing follow-up studies in this area. This project utilizes the same cell culture models described above to identify the signaling pathways that probiotic bacteria utilize to mitigate the production of proinflammatory mediators and cytokines. These data indicate that IMCE cells may enhance the chemotaxis of immune cells after exposure to E.coli 0157:H7 by producing proinflammatory cytokines. These data provide first evidence that probiotic (Lactobacillus) bacterial strains may modulate the inflammatory immune response in colon epithelial cells exposed to EC.
Impacts
Data from Project One suggests specific signaling pathways initiated by specific flavonoids (e.g., NAR, API, HES and ECGC) may allow cells heterozygous for Apc (IMCE) to overcome defective cell migration and experience cell differentiation and apoptosis. These dietary factors could, therefore, decrease the number of preneoplastic cells at risk for transformation into tumor cells. These data offer a new potential mechanism by which dietary factors may decrease risk of colorectal cancer. Grant funding is being utilized to definitively determine the specific intracellular signaling machinery and their associated transcription factor targets that these flavonoids activate in order to produce the migratory phenotype. Data from Project Two provide the first evidence that probiotic bacteria inhibit induction of inflammatory mediators by bacterial pathogen E.coli:0157:H7 in non-tumorigenic colonic epithelial cells. We have identified intracellular signaling pathways activated
by probiotic bacteria which may mediate these anti-inflammatory effects. If these data are confirmed, it would suggest a new dietary strategy for the prevention of intestinal inflammation caused by enteric bacterial pathogens by probiotic bacteria.
Publications
- Davis, C.D. and Hord, N.G. (2005) Nutritional "Omics" Technologies for Elucidating the Role(s) of Bioactive Food Components in Colon Cancer Prevention, Journal of Nutrition, 135, 2694-2697.
- Fenton JI, Hord NG, Lavigne JA, Perkins SN, Hursting SD. (2005) Leptin, insulin-like growth factor-1, and insulin-like growth factor-2 are mitogens in ApcMin/+ but not Apc+/+ colonic epithelial cell lines, Cancer Epidemiology Biomarkers and Prevention, 14 (7), 1646-1652
- Clark C.A., Gardiner, J., McBurney, M.I., Weatherspoon, L.J., Henry, D.N., Hord, N.G. (2006) Effects of breakfast meal composition on second meal metabolic responses in adults with Type 2 diabetes mellitus. European Journal of Clinical Nutrition (in press, 2006).
- Fenton, J.I. and Hord, N.G. STAGE MATTERS: Employing in vitro models useful for diet and cancer chemoprevention prevention research . Carcinogenesis (in press, 2006)
- Hord, N.G. (2005) How Are Dietary Signals (Probiotics and Prebiotics) Processed by GI Cells to Effect Measurable Changes in Immune Parameters Systemically?, Journal of Nutrition, 135, 2914-2915.
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Progress 01/01/04 to 12/31/04
Outputs
This progress report details effort on two projects involving the potential of two types of dietary constituents to modulate biological processes related to colon cancer prevention in a cell culture model system. The first project has established, for the first time, the role of the spice component, curcumin, in causing cell migration in colonic epithelial cells. We extend these observations in this progress report to include specific dietary flavonoids. Colonic epithelial cell migration is required for movement up the crypt-villus axis and hence, normal differentiated cell function. This migratory phenotype is dependent upon wild-type adenomatous polyposis coli (Apc) expression. Non-tumorigenic murine colon epithelial cell lines with distinct Apc genotypes, YAMC (Apc+/+) and IMCE (ApcMin/+) cells) were used to assess the association between Apc genotype, cell motility and matrix metalloproteinase (MMP) activity in these cells. The glycosylated flavanones, naringin
and hesperidin, induced migration in differentially in YAMC and IMCE cells. Apigenin induced migration in IMCE cells at 1 (p<0.05) and 50 microM (p<0.01) and did not induce migration in the YAMC cells. Epigallocatechin gallate induced migration at 1 microM only in IMCE cells. We have extended these findings to identify specific cell signaling pathways induced by flavonoids that may contribute to the migratory phenotype. Using enzymatic inhibitors of intracellular signaling, we have established that p38 and p42/44 (MEK1) mitogen-activated protein kinases (MAPKs) mediate the migration-inducing effect of the flavonoids tested. The second project utilizes the same cell culture models described above to identify the signaling pathways that probiotic bacteria utilize to mitigate the production of proinflammatory mediators and cytokines. We hypothesized that probiotic bacteria, Lactobacillus casei (LC) and Lactobacillus reuteri (LR) would decrease production of proinflammatory mediators
(e.g. nitric oxide [NO], chemotactic cytokines TNF-alpha and MIP-2 by ELISA) in response to exposure to bacterial pathogen E.coli:0157:H7 (EC). Two murine colon epithelial cell lines (i.e. Young Adult Mouse Colon [YAMC]; Immortomouse/Min Colon Epithelial [IMCE], were used to assess the production of NO and cytokines when treated with EC, LC, LR or both. EC caused a dose-dependent increase in NO production in both cell types (YAMC > IMCE; p< 0.001). There was a differential effect of LC and LR on EC induced NO, TNF-alpha and MIP-2. LC and LR caused a greater decrease in NO (p < 0.01) and increase in TNF-alpha (p < 0.001) in IMCE cells compared to YAMC cells. These data indicate that IMCE may enhance the chemotaxis of immune cells after exposure to E.coli 0157:H7. These data provide first evidence that probiotic bacterial strains LC and LR may modulate the inflammatory immune response in colon epithelial cells exposed to EC.
Impacts
Data from Project One suggests specific signaling pathways initiated by specific flavonoids (e.g., naringin, hesperidin, epigallocatechin gallate) may allow cells heterozygous for Apc (IMCE) to overcome defective cell migration and experience cell differentiation and apoptosis. These dietary factors could, therefore, decrease the number of preneoplastic cells at risk for transformation into tumor cells. These data offer a new potential mechanism by which dietary factors may decrease risk of colorectal cancer. Grant funding is being utilized to definitively determine the specific intracellular signaling machinery that these flavonoids activate in order to produce the migratory phenotype. Data from Project Two provide the first evidence that probiotic bacteria inhibit induction of inflammatory mediators by bacterial pathogen E.coli:0157:H7 in non-tumorigenic colonic epithelial cells. We have identified intracellular signaling pathways activated by probiotic bacteria
which may mediate these anti-inflammatory effects. If these data are confirmed, it would suggest a new dietary strategy for the prevention of intestinal inflammation caused by enteric bacterial pathogens by probiotic bacteria.
Publications
- Fenton, J.I. and Hord, N.G. (2004) Flavonoids alter cell migration in non-tumorigenic colon epithelial cells differing in Apc genotype. Nutr. Cancer 48: 2
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Progress 01/01/03 to 12/31/03
Outputs
The first project has established, for the first time, the role of the spice component, curcumin, in causing cell migration in colonic epithelial cells. We extend these observations to include specific dietary flavonoids. Colonic epithelial cell migration is required for movement up the crypt-villus axis and hence, normal differentiated cell function. Murine colon epithelial cell lines with distinct Apc (adenomatous polyposis coli) genotypes, YAMC (Apc+/+) and IMCE (ApcMin/+) cells) were used to assess the association between Apc genotype and cell motility in these cells. The citrus flavanones naringenin and hesperetin did not induce cell migration comparable to HGF in either cell type. However, the glycosylated forms of these flavanones, naringin and hesperidin, induced migration in differentially in YAMC and IMCE cells. Specifically, naringin and hesperidin induced the greatest migratory response in IMCE cells at 1 microM (p<0.01) and induced migration greater than
untreated control cells (p<0.05) but equal to HGF-treated cells. In YAMC cells, hesperidin did not induce migration except at the 100 microM concentration. Apigenin induced migration in IMCE cells at 1 (p<0.05) and 50 microM (p<0.01) and did not induce migration in the YAMC cells. Catechin induced migration at the highest concentration (300 microM) only in the IMCE cells while epicatechin induced migration at the lowest concentration only (1 microM) in IMCE cells. Overall, the glycosylated citrus flavanones induced the greatest migratory response at the lowest concentration in IMCE cells. Co-treatment of IMCE cells with MMP inhibitor Ilomastat in the presence of naringin, hesperidin or apigenin demonstrated that flavonoid-induced migration was dependent on MMP activity. The second project in which significant progress has been made utilizes the same cell culture models described above. These studies have identified specific lactic acid bacteria (LAB) which can decrease the production
of proinflammatory mediators caused by exposure of these cells to bacterial pathogen, E. coli: 0157:H7. The balance of commensal, probiotic and pathogenic bacteria in the colon may influence colon carcinogenesis by modulating the inflammatory immune response. We hypothesized that probiotic bacteria, Lactobacillus casei (LC) and Lactobacillus reuteri (LR) would decrease production of proinflammatory mediators (e.g. nitric oxide [NO], chemotactic cytokines TNFa and MIP-2 by ELISA) in response to exposure to bacterial pathogen E.coli:0157:H7 (EC). Two murine colon epithelial cell lines (i.e. Young Adult Mouse Colon [YAMC]; Immortomouse/Min Colon Epithelial [IMCE], were used to assess the production of NO and cytokines when treated with EC, LC, LR or both. EC caused a dose-dependent increase in NO production in both cell types (YAMC > IMCE; p< 0.001). There was a differential effect of LC and LR on EC induced NO, TNF-alpha and MIP-2. LC and LR caused a greater decrease in NO (p < 0.01)
and increase in TNF-alpha (p < 0.001) in IMCE cells compared to YAMC cells. These data indicate that IMCE may enhance the chemotaxis of immune cells after exposure to E.coli 0157:H7.
Impacts
Data from Project One suggests a potential mechanism by which curcumin and specific may allow cells heterozygous for Apc (IMCE) to overcome defective cell migration and experience cell differentiation and apoptosis. By causing these events, these dietary factors could decrease the number of preneoplastic cells at risk for transformation into tumor cells. These data offer a new potential mechanism by which dietary factors may decrease risk of colorectal cancer. Grant funding is being sought to determine the cell signaling machinery that curcumin activates in order to produce the migratory phenotype. Data from Project Two provide the first evidence that probiotic bacteria inhibit induction of inflammatory mediators by bacterial pathogen E.coli:0157:H7 in non-tumorigenic colonic epithelial cells. These data provide first evidence that probiotic bacterial strains LC and LR may modulate the inflammatory immune response in colon epithelial cells exposed to EC. If these data
are confirmed, it would suggest a new dietary strategy for the prevention of intestinal inflammation caused by enteric bacterial pathogens by probiotic bacteria.
Publications
- Fenton, J.I. and Hord, N.G. (2004) Flavonoids Promote Matrix Metalloproteinase-dependent Cell Migration in Non-Tumorigenic Colon Epithelial Cells Differing in Apc Genotype. Nutrition and Cancer (in press).
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Progress 01/01/02 to 12/31/02
Outputs
This progress report details effort on two projects involving the potential of two types of dietary constituents to modulate biological processes related to colon cancer prevention in a cell culture model system. The first project has established, for the first time, the role of the spice component, curcumin, in causing cell migration in colonic epithelial cells. Colonic epithelial cell migration is required for movement up the crypt-villus axis and hence, normal differentiated cell function. This migratory phenotype is dependent upon wild-type adenomatous polyposis coli (Apc) expression. Non-tumorigenic murine colon epithelial cell lines with distinct Apc genotypes, YAMC (Apc+/+) and IMCE (ApcMin/+) cells) were used to assess the association between Apc genotype, cell motility and matrix metalloproteinase (MMP) activity in these cells. YAMC and IMCE cells were treated with epidermal growth factor (EGF; 1, 10 and 25 ng/ml) or hepatocyte growth factor (HGF; 1, 10 and
25 ng/ml) and/or curcumin (0.1-100 microM). EGF (25 ng/ml) and HGF (25 ng/ml) induced a greater migratory response in YAMC compared to IMCE cells after 24 hours (p<0.05). Treatment with curcumin induced an equivalent or greater migratory response in IMCE than YAMC cells. When migrating cells were treated with an inhibitor of matrix metalloproteinases (Ilomastatr?; I), migration was inhibited in both cell types. Curcumin-induced migration was inhibited by I in both cell types at the highest dose (50 microM), while the lower doses (10 and 25 microM) had minimal effects. These data provide the first evidence that colon epithelial cells heterozygous for Apc are less migratory than cells containing wild-type Apc. This migration is dependent on MMP activity and likely due specifically to changes in membrane type-1-MMP. The second project in which significant progress has been made utilizes the same cell culture models described above. These studies have identified specific lactic acid
bacteria (LAB) which can decrease the production of proinflammatory mediators caused by exposure of these cells to bacterial pathogen, Salmonella dublin. Probiotic bacteria may inhibit inflammatory processes resulting from exposure to bacterial pathogens by blocking nuclear factor kappa B (NF-kB) nuclear translocation and nitric oxide (NO) production. The objective of this study was to assess the ability of two probiotic bacteria, Lactobacillus casei (LC) and Lactobacillus reuteri (LR), to block Salmonella dublin-induced NF-kB translocation and NO production in a non-tumorogenic murine colon epithelial cell line. Nuclear translocation of NF-kB, was assessed by immunocytochemistry and western blotting; NO was assessed in culture supernatants using the Greiss reaction. LC and LR differentially inhibited nuclear translocation of NF-kB and NO production induced by S. dublin. Western blots from nuclear extracts confirmed that LR was more effective than LC in blocking S. dublin-induced
NF-kB translocation.
Impacts
Data from Project One suggests a potential mechanism by which curcumin may allow cells heterozygous for Apc (preneoplastic cells) to overcome defective cell migration and experience cell differentiation and apoptosis. By causing these events, curcumin could decrease the number of preneoplastic cells at risk for transformation into tumor cells. These data offer a new potential mechanism by which dietary factors may decrease risk of colorectal cancer. Data from Project Two provide the first evidence that probiotic bacteria inhibit induction of inflammatory mediators by S. Dublin in non-tumorigenic colonic epithelial cells. If these data are confirmed, it would suggest a new dietary strategy for the prevention of intestinal inflammation caused by enteric bacterial pathogens.
Publications
- Fenton, J.I., Wolff M.S., Orth, M.I. and Hord, N.G. (2002) Membrane-type matrix metalloproteinases mediate curcumin -induced cell migration in non-tumorigenic colon epithelial cells differing in Apc genotype. Carcinogenesis 23 (6): 1065-1070.
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Progress 01/01/01 to 12/31/01
Outputs
Project A (Cooperators: Dr. Jenifer I. Fenton) Current Progress: Results of this research project support a role for dietary compounds in eliciting phenotypes associated with differentiation of non-tumorigenic colonic epithelial cells. Our data indicate that curcumin, a common component of the spice, tumeric, can induce cell motility at concentrations achievable by dietary, rather than pharmacologic, means. By inducing the motile phenotype in these cells, it is plausible that such dietary interventions may reduce risk of colorectal cancer. We have extended these observations to include specific flavonoid compounds. Two manuscripts have been submitted for publication, including one to Carcinogenesis (Fenton, J.I., Wolff M.S., Orth, M.I. and Hord, N.G. Membrane-type matrix metalloproteinases mediate curcumin -induced cell migration in non-tumorigenic colon epithelial cells differing in Apc genotype) and one to Nutrition and Cancer (Fenton, J.I. and Hord, N.G. Flavonoids
alter cell migration in non-tumorigenic colon epithelial cells differing in Apc genotype). Project C : (Cooperators: Drs. Lorraine Weatherspoon (FSHN), Douglas Henry (Physiology)) This study is testing the hypothesis that psyllium fiber will improve metabolic control in individuals with Type 2 diabetes. Forty-five subjects completed the three treatment crossover study to determine the effect of soluble fiber on glycemic and lipemic indices of metabolic control. Primary data analyses have been completed, resulting in the submission of an abstract titled, "Implications of Breakfast on Midday Meal Metabolic Responses in Individuals with Type 2 Diabetes Mellitus" by authors Clark, CA, Weatherspoon, LJ, McBurney, MI, Henry, DN and Hord, NG for the 2002 American Diabetes Association Annual Meeting.
Impacts
Understanding the mechanism by which diet-derived compounds may overcome the genetic predisposition to impaired cell migration associated with colorectal cancer may contribute to our ability to make specific dietary recommendations to prevent colorectal cancer. it also has the potential to yield benefits for those at risk for colorectal cancer and inflammatory boil diseases. Additionally, if such data could be leveraged to establish efficacy in ill-designed clinical trials, the plant products that serve as sources for these compounds could prove beneficial for U.S. agriculture.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
One aspect of this research project provides insight into the hypothetical mechanism(s) of action of several dietary components linked to colon cancer risk and prevention. Results of this research project support a role for dietary compounds in eliciting anticarcinogenic events in non-tumorigenic colonic epithelial cells. Our data indicate that curcumin, a common component of the spice, tumeric, can induce cell motility at concentrations achievable by dietary, rather than pharmacologic, means. By inducing the motile phenotype in these cells, it is plausible that such dietary interventions may reduce risk of colorectal cancer. Another aspect of this project assessed several dietary fibers. The substrates used in this study were arabinogalactan, high viscosity beta-glucan, medium viscosity eta-glucan, low viscosity beta-glucan, a mixture of inulin and fructooligosaccharide, a mixed fiber source, oat flour, oat groats, and Solka-Floc (crystalline cellulose). The
production of total short chain fatty acids ranged from 5.30 millimoles per gram original organic matter (high viscosity beta-glucan) to 0.92 millimoles per gram original organic matter (Solka-Floc). Acetate was the most abundant short chain fatty acid produced in all substrates. Substrate organic matter disappearance ranged from -1.79% (Solka-Floc) to 83.84% (mixed fiber). Potential water holding capacity was inversely correlated to organic matter disappearance (r = -.91). There were significant differences among donors with respect to the above indices of colonic fermentation. Overall , these results reflect the ability of certain soluble dietary fibers to exert prebiotic effects in this in vitro model of colonic fermentation. This final aspect of this project is testing the hypothesis that psyllium fiber will improve metabolic control in individuals with Type 2 diabetes. Five subjects have been enrolled and two subjects have completed the study. Recruitment and analysis of blood
and serum samples is progressing well.
Impacts
The study of the mechanisms and role of dietary components and their use and effect on disease risk factors, control and prevention, specifically diabetes and colon cancer.
Publications
- Mei, J.M., Hord, N.G., Winterstein, D., Donald, S. and Phang, J.M. 2000. Differential Formation of beta-catenin/LEF-1 DNA Binding Complex Induced by Nitric Oxide in Mouse Colonic Epithelial Cells Differing in Apc Genotype. Cancer Research 60: 3379 3383.
- Mei, J.M., Hord, N.G., Winterstein, D., Donald, S. and Phang, J.M. 2000. Expression of prostaglandin endoperoxide H synthase-2 and formation of beta-catenin/LEF-1 DNA binding complexes induced by nitric oxide in conditionally immortalized murine colonic epithelial cells. FASEB J. 14: 1188-1201.
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