In vivo regulation of colonic cell proliferation, differentiation, apoptosis, and P27Kip1 by dietary fish oil and butyrate in rats.

We have shown that dietary fish oil is protective against experimentally induced colon cancer, and the protective effect is enhanced by coadministration of pectin. However, the underlying mechanisms have not been fully elucidated. We hypothesized that fish oil with butyrate, a pectin fermentation product, protects against colon cancer initiation by decreasing cell proliferation and increasing differentiation and apoptosis through a p27(Kip1)-mediated mechanism. Rats were provided diets of corn or fish oil, with/without butyrate, and terminated 12, 24, or 48 hours after azoxymethane (AOM) injection. Proliferation (Ki-67), differentiation (Dolichos Biflorus Agglutinin), apoptosis (TUNEL), and p27(Kip1) (cell-cycle mediator) were measured in the same cell within crypts in order to examine the coordination of cell cycle as a function of diet. DNA damage (N(7)-methylguanine) was determined by quantitative IHC analysis. Dietary fish oil decreased DNA damage by 19% (P = 0.001) and proliferation by 50% (P = 0.003) and increased differentiation by 56% (P = 0.039) compared with corn oil. When combined with butyrate, fish oil enhanced apoptosis 24 hours after AOM injection compared with a corn oil/butyrate diet (P = 0.039). There was an inverse relationship between crypt height and apoptosis in the fish oil/butyrate group (r = -0.53, P = 0.040). The corn oil/butyrate group showed a positive correlation between p27(Kip1) expression and proliferation (r = 0.61, P = 0.035). These results indicate the in vivo effect of butyrate on apoptosis and proliferation is dependent on dietary lipid source. These results demonstrate the presence of an early coordinated colonocyte response by which fish oil and butyrate protects against colon tumorigenesis.

Exploring the benefits and challenges of establishing a DRI-like process for bioactives.

Bioactives can be defined as: "Constituents in foods or dietary supplements, other than those needed to meet basic human nutritional needs, which are responsible for changes in health status" (Office of Disease Prevention and Health Promotion, Office of Public Health and Science, Department of Health and Human Services in Fed Reg 69:55821-55822, 2004). Although traditional nutrients, such as vitamins, minerals, protein, essential fatty acids and essential amino acids, have dietary reference intake (DRI) values, there is no such evaluative process for bioactives. For certain classes of bioactives, substantial scientific evidence exists to validate a relationship between their intake and enhanced health conditions or reduced risk of disease. In addition, the study of bioactives and their relationship to disease risk is a growing area of research supported by government, academic institutions, and food and supplement manufacturers. Importantly, consumers are purchasing foods containing bioactives, yet there is no evaluative process in place to let the public know how strong the science is behind the benefits or the quantitative amounts needed to achieve these beneficial health effects. This conference, Bioactives: Qualitative Nutrient Reference Values for Life-stage Groups?, explored why it is important to have a DRI-like process for bioactives and challenges for establishing such a process.

Colon cancer cell apoptosis is induced by combined exposure to the n-3 fatty acid docosahexaenoic acid and butyrate through promoter methylation.

DNA methylation and histone acetylation contribute to the transcriptional regulation of genes involved in apoptosis. We have demonstrated that docosahexaenoic acid (DHA, 22:6 n-3) and butyrate enhance colonocyte apoptosis. To determine if DHA and/or butyrate elevate apoptosis through epigenetic mechanisms thereby restoring the transcription of apoptosis-related genes, we examined global methylation; gene-specific promoter methylation of 24 apoptosis-related genes; transcription levels of Cideb, Dapk1, and Tnfrsf25; and global histone acetylation in the HCT-116 colon cancer cell line. Cells were treated with combinations of (50 µM) DHA or linoleic acid (18:2 n-6), (5 mM) butyrate or an inhibitor of DNA methyltransferases, and 5-aza-2'-deoxycytidine (5-Aza-dC, 2 µM). Among highly methylated genes, the combination of DHA and butyrate significantly reduced methylation of the proapoptotic Bcl2l11, Cideb, Dapk1, Ltbr, and Tnfrsf25 genes compared to untreated control cells. DHA treatment reduced the methylation of Cideb, Dapk1, and Tnfrsf25. These data suggest that the induction of apoptosis by DHA and butyrate is mediated, in part, through changes in the methylation state of apoptosis-related genes.

A chemoprotective fish oil/pectin diet enhances apoptosis via Bcl-2 promoter methylation in rat azoxymethane-induced carcinomas.

We have demonstrated that diets containing fish oil and pectin (FO/P) reduce colon tumor incidence relative to control (corn oil and cellulose [CO/C]) in part by inducing apoptosis of DNA-damaged colon cells. Relative to FO/P, CO/C promotes colonocyte expression of the antiapoptotic modulator, Bcl-2, and Bcl-2 promoter methylation is altered in colon cancer. To determine if FO/P, compared with CO/C, limits Bcl-2 expression by enhancing promoter methylation in colon tumors, we examined Bcl-2 promoter methylation, mRNA levels, colonocyte apoptosis and colon tumor incidence in azoxymethane (AOM)-injected rats. Rats were provided diets containing FO/P or CO/C, and were terminated 16 and 34 weeks after AOM injection. DNA isolated from paraformaldehyde-fixed colon tumors and uninvolved tissue was bisulfite modified and amplified by quantitative reverese transcriptase-polymerase chain reaction to assess DNA methylation in Bcl-2 cytosine-guanosine islands. FO/P increased Bcl-2 promoter methylation (P = 0.009) in tumor tissues and colonocyte apoptosis (P = 0.020) relative to CO/C. An inverse correlation between Bcl-2 DNA methylation and Bcl-2 mRNA levels was observed in the tumors. We conclude that dietary FO/P promotes apoptosis in part by enhancing Bcl-2 promoter methylation. These Bcl-2 promoter methylation responses, measured in vivo, contribute to our understanding of the mechanisms involved in chemoprevention of colon cancer by diets containing FO/P.

Interactive effects of fatty acid and butyrate-induced mitochondrial Ca²âº loading and apoptosis in colonocytes.

BACKGROUND: The combination of fish oil-derived docosahexaenoic acid (DHA) (22:6; omega 3 [n-3]) and butyrate (4:0), a fiber fermentation product, synergized to enhance colonocyte apoptosis by inducing a p53-independent, oxidation sensitive, mitochondrial Ca(2+) -dependent (intrinsic) pathway. METHODS: In this study, the authors probed the specificity of n-6 and n-3 polyunsaturated fatty acid induction of Ca(2+) -dependent proapoptotic events in immortalized young adult mouse colonocytes and determined whether combinations of polyunsaturated fatty acid and butyrate could trigger endoplasmic reticulum (ER) stress conditions, thereby promoting mitochondrial Ca(2+) overload. Cultures were treated with 0 µM to 50 µM of DHA (22:6; n-3), EPA (20:5; n-3), arachidoinic acid (AA) (20:4; n-6), linoleic acid (18:2; n-6), or oleic acid (OA) (18:1; n-9) for a total of 72 hours with or without RU-360 (to inhibit the mitochondrial Ca(2+) uniporter) for 30 minutes before cotreatment with butyrate (0 mM or 5 mM). RESULTS: Combined DHA and butyrate maximally induced apoptosis and mitochondrial-to-cytosolic Ca(2+) levels. By comparison, EPA, a precursor to DHA, was minimally effective. Similarly, AA and OA in combination with butyrate had no effect on mitochondrial Ca(2+) or apoptosis compared with butyrate alone. DHA with or without butyrate cotreatment minimally altered the ER stress-regulated genes DNA damage-inducible transcript 3, the CCAAT enhancer binding protein (C/EBP) homologous protein (CHOP), and eukaryotic initiation factor 2α. CONCLUSIONS: The current data indicated that butyrate and DHA, but not EPA, worked in a coordinated fashion to trigger an ER-independent, Ca(2+) -dependent, intrinsic mitochondrial-mediated apoptotic pathway in colonocytes.

Dietary fish oil promotes colonic apoptosis and mitochondrial proton leak in oxidatively stressed mice.

An alteration of mitochondrial function can result in disruption of redox homeostasis and is associated with abnormal cancer cell growth. Manganese superoxide dismutase (SOD2) and glutathione peroxidase 4 (Gpx4) are two of the most important antioxidant defense enzymes that protect cells against oxidative stress. We had previously shown that n-3 polyunsaturated fatty acids (PUFA) promote colonocyte apoptosis, a marker of colon cancer risk, in part by enhancing phospholipid oxidation. To elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, we fed heterozygous SOD2(Het), Gpx4(Het), and transgenic Gpx4(Tg) mice diets containing either 15% corn oil by weight (CO, enriched in n-6 PUFA) or 3.5% CO + 11.5% fish oil (FO, enriched in n-3 PUFA) for 4 weeks. Our data showed that (i) genetic predeposition to oxidative stress facilitates apoptosis in the mouse colon (Gpx4(Het) > SOD2(Het) > Wt > Gpx4(Tg)), (ii) dietary n-3 PUFA have an additive effect on the induction of apoptosis in Gpx4(Het) and SOD2(Het) mice; and (iii) dietary n-3 PUFA reverse the phenotype in oxidatively protected Gpx4(Tg) mice by elevating apoptosis to a level observed in wild-type (Wt; control) animals. Complimentary experiments examining colonic mitochondrial bioenergetic profiles indicate that FO-fed mice exhibit a significantly (P < 0.05) increased respiration-induced proton leak relative to control CO treatment. This finding was consistent with a loss of membrane potential in response to chronic oxidative stress and supports the contention that n-3 PUFA alter mitochondrial metabolic activity, thereby enhancing apoptosis and reducing colon cancer risk.

A chemoprotective fish oil- and pectin-containing diet temporally alters gene expression profiles in exfoliated rat colonocytes throughout oncogenesis.

We have demonstrated that fish oil- and pectin-containing (FO/P) diets protect against colon cancer compared with corn oil and cellulose (CO/C) by upregulating apoptosis and suppressing proliferation. To elucidate the mechanisms whereby FO/P diets induce apoptosis and suppress proliferation during the tumorigenic process, we analyzed the temporal gene expression profiles from exfoliated rat colonocytes. Rats consumed diets containing FO/P or CO/C and were injected with azoxymethane (AOM; 2 times, 15 mg/kg body weight, subcutaneously). Feces collected at initiation (24 h after AOM injection) and at aberrant crypt foci (ACF) (7 wk postinjection) and tumor (28 wk postinjection) stages of colon cancer were used for poly (A)+ RNA extraction. Gene expression signatures were determined using Codelink arrays. Changes in phenotypes (ACF, apoptosis, proliferation, and tumor incidence) were measured to establish the regulatory controls contributing to the chemoprotective effects of FO/P. At initiation, FO/P downregulated the expression of 3 genes involved with cell adhesion and enhanced apoptosis compared with CO/C. At the ACF stage, the expression of genes involved in cell cycle regulation was modulated by FO/P and the zone of proliferation was reduced in FO/P rats compared with CO/C rats. FO/P also increased apoptosis and the expression of genes that promote apoptosis at the tumor endpoint compared with CO/C. We conclude that the effects of chemotherapeutic diets on epithelial cell gene expression can be monitored noninvasively throughout the tumorigenic process and that a FO/P diet is chemoprotective in part due to its ability to affect expression of genes involved in apoptosis and cell cycle regulation throughout all stages of tumorigenesis.

Dietary fish oil and curcumin combine to modulate colonic cytokinetics and gene expression in dextran sodium sulphate-treated mice.

Both fish oil (FO) and curcumin have potential as anti-tumour and anti-inflammatory agents. To further explore their combined effects on dextran sodium sulphate (DSS)-induced colitis, C57BL/6 mice were randomised to four diets (2 × 2 design) differing in fatty acid content with or without curcumin supplementation (FO, FO+2 % curcumin, maize oil (control, MO) or MO+2 % curcumin). Mice were exposed to one or two cycles of DSS in the drinking-water to induce either acute or chronic intestinal inflammation, respectively. FO-fed mice exposed to the single-cycle DSS treatment exhibited the highest mortality (40 %, seventeen of forty-three) compared with MO with the lowest mortality (3 %, one of twenty-nine) (P = 0·0008). Addition of curcumin to MO increased (P = 0·003) mortality to 37 % compared with the control. Consistent with animal survival data, following the one- or two-cycle DSS treatment, both dietary FO and curcumin promoted mucosal injury/ulceration compared with MO. In contrast, compared with other diets, combined FO and curcumin feeding enhanced the resolution of chronic inflammation and suppressed (P < 0·05) a key inflammatory mediator, NF-κB, in the colon mucosa. Mucosal microarray analysis revealed that dietary FO, curcumin and FO plus curcumin combination differentially modulated the expression of genes induced by DSS treatment. These results suggest that dietary lipids and curcumin interact to regulate mucosal homeostasis and the resolution of chronic inflammation in the colon.

Integrated microRNA and mRNA expression profiling in a rat colon carcinogenesis model: effect of a chemo-protective diet.

We have recently demonstrated that nutritional bioactives (fish oil and pectin) modulate microRNA molecular switches in the colon. Since integrated analysis of microRNA and mRNA expression at an early stage of colon cancer development is lacking, in this study, four computational approaches were utilized to test the hypothesis that microRNAs and their posttranscriptionally regulated mRNA targets, i.e., both total mRNAs and actively translated mRNA transcripts, are differentially modulated by carcinogen and diet treatment. Sprague-Dawley rats were fed diets containing corn oil ± fish oil with pectin ± cellulose and injected with azoxymethane or saline (control). Colonic mucosa was assayed at an early time of cancer progression, and global gene set enrichment analysis was used to obtain those microRNAs significantly enriched by the change in expression of their putative target genes. In addition, cumulative distribution function plots and functional network analyses were used to evaluate the impact of diet and carcinogen combination on mRNA levels induced via microRNA alterations. Finally, linear discriminant analysis was used to identify the best single-, two-, and three-microRNA combinations for classifying dietary effects and colon tumor development. We demonstrate 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 (miR-16, miR-19b, miR-21, miR26b, miR27b, miR-93, and miR-203) linked to canonical oncogenic signaling pathways. Complementary gene expression analyses showed that oncogenic PTK2B, PDE4B, and TCF4 were suppressed by the chemoprotective diet at both the mRNA and protein levels.

Classification of diet-modulated gene signatures at the colon cancer initiation and progression stages.

BACKGROUND: The effects of dietary polyunsaturated (PUFAs) and monounsaturated fatty acids (MUFAs) on intestinal cytokinetics within the context of colon cancer initiation and progression have been extensively studied. n-3 PUFAs have received the most attention due to their potential protective role. However, further investigation of the epigenetic perturbations caused by fatty acids in the context of colon cancer development is needed. METHODS: We used DNA microarrays to identify discriminative gene signatures (gene combinations) for the purpose of classifying n-3 PUFA-fed, carcinogen-injected, Sprague-Dawley rats at the initiation and progression stages. Animals were assigned to three dietary treatments differing only in the type of fat (corn oil/n-6 PUFA, fish oil/n-3 PUFA, or olive oil/n-9 monounsaturated fatty acid). RESULTS: The effects of diet on colonic mucosal gene expression signatures during tumor initiation and progression were subsequently compared (12 h and 10 weeks after azoxymethane injection). Microarray analysis revealed that the number of differentially expressed (DE) genes in each of the three diet comparisons increased with the progression of colon cancer. Each dietary lipid source exhibited its own unique transcriptional profile, as assessed by linear discriminant analysis. Applying this novel approach, we identified the single genes and the two- to three-gene combinations that best distinguished the dietary treatment groups. For the chemoprotective (fish oil) diet, mediators of stem cell homeostasis, e.g., ephrin B1 and bone morphogenic protein 4, were the top-performing gene classifiers. CONCLUSIONS: These results suggest that dietary chemoprotective n-3 PUFA impact genes that regulate the colon stem cell niche and tumor evolution.

Linoleic acid and butyrate synergize to increase Bcl-2 levels in colonocytes.

The biological properties of polyunsaturated fatty acid (PUFA) classes have been the source of much contention. For example, n-3 PUFA are chemoprotective, whereas n-6 PUFA may promote tumor development. Since dietary components can have combinatorial effects, we further examined the apoptotic properties of n-3 or n-6 fatty acids when combined with different fiber sources. Mice were fed diets supplemented with either fish oil (FO; enriched in n-3 PUFA) or corn oil (CO; enriched in n-6 PUFA) and nonfermentable (cellulose) or fermentable (pectin) fiber sources. In complementary experiments, immortalized young adult mouse colonic (YAMC) cells were treated with docosahexaenoic acid (DHA; 22:6n-3) or linoleic acid (LA; 18:2n-6) with or without butyrate. Mice fed a FO and pectin diet had significantly (p < 0.05) increased levels of apoptosis in colonocytes compared to all other diets. Similarly, apoptosis was highly induced in DHA and butyrate cotreated YAMC cells. In contrast, in both YAMC and mouse models, LA/CO with butyrate/pectin treatment reduced apoptosis and enhanced expression of bcl-2. The LA and butyrate induced antiapoptotic phenotype was reversed by knocking down bcl-2 using targeted siRNA. In comparison, overexpression of bcl-2 blocked the proapoptotic effect of DHA and butyrate. These data provide new mechanistic insights into the regulation of apoptosis by dietary PUFA and fiber.

Recommending flavanols and procyanidins for cardiovascular health: current knowledge and future needs.

Data on the potential health benefits of dietary flavanols and procyanidins, especially in the context of cardiovascular health, are considerable and continue to accumulate. Significant progress has been made in flavanol analytics and the creation of phytonutrient-content food databases, and novel data emanated from epidemiological investigations as well as dietary intervention studies. However, a comprehensive understanding of the pharmacological properties of flavanols and procyanidins, including their precise mechanisms of action in vivo, and a conclusive, consensus-based accreditation of a causal relationship between intake and health benefits in the context of primary and secondary cardiovascular disease prevention is still outstanding. Thus, the objective of this review is to identify and discuss key questions and gaps that will need to be addressed in order to conclusively demonstrate whether or not dietary flavanols and procyanidins have a role in preventing, delaying the onset of, or treating cardiovascular diseases, and thus improving human life expectancy and quality of life.

Apigenin and naringenin suppress colon carcinogenesis through the aberrant crypt stage in azoxymethane-treated rats.

Epidemiological evidence suggests that a diet abundant in fruits and vegetables may protect against colon cancer. Bioactive compounds, including flavonoids and limonoids, have been shown to possess antiproliferative and antitumorigenic effects in various cancer models. This experiment investigated the effects of four citrus flavonoids and one limonoid mixture at the promotion stage of chemically induced colon cancer in rats. Male Sprague-Dawley rats (n = 10 rats/group) were randomly allocated to one of six diets formulated to contain 0.1% apigenin, 0.02% naringenin, 0.1% hesperidin, 0.01% nobiletin, 0.035% limonin glucoside/obacunone glucoside mixture or a control diet (0% flavonoid/limonoid). Rats received experimental diets for 10 weeks and were injected with azoxymethane (15 mg/kg) at weeks 3 and 4. Excised colons were evaluated for aberrant crypt foci (ACF) formation, colonocyte proliferation (proliferating cell nuclear antigen assay), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling assay) and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (immunoblotting). When compared with the control diet, apigenin lowered the number of high multiplicity ACF (HMACF >4 aberrant crypts/focus) by 57% (P < 0.05), while naringenin lowered both the number of HMACF by 51% (P < 0.05) and the proliferative index by 32% (P < 0.05). Both apigenin and naringenin increased apoptosis of luminal surface colonocytes (78% and 97%, respectively; P < 0.05) when compared with the control diet. Hesperidin, nobiletin and the limonin glucoside/obacunone glucoside mixture did not affect these variables. The colonic mucosal protein levels of iNOS or COX-2 were not different among the six diet groups. The ability of dietary apigenin and naringenin to reduce HMACF, lower proliferation (naringenin only) and increase apoptosis may contribute toward colon cancer prevention. However, these effects were not due to mitigation of iNOS and COX-2 protein levels at the ACF stage of colon cancer.

Noninvasive stool-based detection of infant gastrointestinal development using gene expression profiles from exfoliated epithelial cells.

We have developed a novel molecular methodology that utilizes stool samples containing intact sloughed epithelial cells to quantify intestinal gene expression profiles in the developing human neonate. Since nutrition exerts a major role in regulating neonatal intestinal development and function, our goal was to identify gene sets (combinations) that are differentially regulated in response to infant feeding. For this purpose, fecal mRNA was isolated from exclusively breast-fed (n = 12) and formula-fed (n = 10) infants at 3 mo of age. Linear discriminant analysis was successfully used to identify the single genes and the two- to three-gene combinations that best distinguish the feeding groups. In addition, putative "master" regulatory genes were identified using coefficient of determination analysis. These results support our premise that mRNA isolated from stool has value in terms of characterizing the epigenetic mechanisms underlying the developmentally regulated transcriptional activation/repression of genes known to modulate gastrointestinal function. As larger data sets become available, this methodology can be extended to validation and, ultimately, identification of the main nutritional components that modulate intestinal maturation and function.

The Smart Choices front-of-package nutrition labeling program: rationale and development of the nutrition criteria.

The goal of the Smart Choices Program (SCP) is to provide a simple front-of-the-package icon system to direct consumers to smarter food choices in the supermarket, which will eventually lead to more balanced diets and to more beneficial foods as food manufacturers renovate products to meet the nutrition criteria for carrying the icon. The SCP was developed by a coalition of scientists and nutrition educators, experts with experience with dietary guidelines, public health organizations, and food manufacturers in response to consumer confusion over multiple front-of-the-package systems based on different criteria. Representatives from different government organizations acted as observers. The process of developing the program was facilitated by the nonprofit Keystone Center, an organization that develops consensus solutions to complex health and social policy changes. The nutrition criteria for receiving the SCP icon are specific for product category by indicating "smarter" products within that category. A calorie indicator noting calories per serving and servings per package accompanies the SCP icon to remind consumers that calories do count, even for smarter food choices. For a product to qualify, it first has to be below the threshold for "nutrients to limit" and then (in most cases) it must be above the threshold for one or more nutrients or food groups to encourage. The criteria are based on the 2005 Dietary Guidelines and other consensus science and are transparent and available on the SCP website. This article describes the nutrition criteria and rationales for their selection.

Evaluation of fecal mRNA reproducibility via a marginal transformed mixture modeling approach.

BACKGROUND: Developing and evaluating new technology that enables researchers to recover gene-expression levels of colonic cells from fecal samples could be key to a non-invasive screening tool for early detection of colon cancer. The current study, to the best of our knowledge, is the first to investigate and report the reproducibility of fecal microarray data. Using the intraclass correlation coefficient (ICC) as a measure of reproducibility and the preliminary analysis of fecal and mucosal data, we assessed the reliability of mixture density estimation and the reproducibility of fecal microarray data. Using Monte Carlo-based methods, we explored whether ICC values should be modeled as a beta-mixture or transformed first and fitted with a normal-mixture. We used outcomes from bootstrapped goodness-of-fit tests to determine which approach is less sensitive toward potential violation of distributional assumptions. RESULTS: The graphical examination of both the distributions of ICC and probit-transformed ICC (PT-ICC) clearly shows that there are two components in the distributions. For ICC measurements, which are between 0 and 1, the practice in literature has been to assume that the data points are from a beta-mixture distribution. Nevertheless, in our study we show that the use of a normal-mixture modeling approach on PT-ICC could provide superior performance. CONCLUSIONS: When modeling ICC values of gene expression levels, using mixture of normals in the probit-transformed (PT) scale is less sensitive toward model mis-specification than using mixture of betas. We show that a biased conclusion could be made if we follow the traditional approach and model the two sets of ICC values using the mixture of betas directly. The problematic estimation arises from the sensitivity of beta-mixtures toward model mis-specification, particularly when there are observations in the neighborhood of the the boundary points, 0 or 1. Since beta-mixture modeling is commonly used in approximating the distribution of measurements between 0 and 1, our findings have important implications beyond the findings of the current study. By using the normal-mixture approach on PT-ICC, we observed the quality of reproducible genes in fecal array data to be comparable to those in mucosal arrays.