Resistant starch supplementation increases crypt cell proliferative state in the rectal mucosa of older healthy participants.

There is strong evidence that foods containing dietary fibre protect against colorectal cancer, resulting at least in part from its anti-proliferative properties. This study aimed to investigate the effects of supplementation with two non-digestible carbohydrates, resistant starch (RS) and polydextrose (PD), on crypt cell proliferative state (CCPS) in the macroscopically normal rectal mucosa of healthy individuals. We also investigated relationships between expression of regulators of apoptosis and of the cell cycle on markers of CCPS. Seventy-five healthy participants were supplemented with RS and/or PD or placebo for 50 d in a 2 × 2 factorial design in a randomised, double-blind, placebo-controlled trial (the Dietary Intervention, Stem cells and Colorectal Cancer (DISC) Study). CCPS was assessed, and the expression of regulators of the cell cycle and of apoptosis was measured by quantitative PCR in rectal mucosal biopsies. SCFA concentrations were quantified in faecal samples collected pre- and post-intervention. Supplementation with RS increased the total number of mitotic cells within the crypt by 60 % (P = 0·001) compared with placebo. This effect was limited to older participants (aged ≥50 years). No other differences were observed for the treatments with PD or RS as compared with their respective controls. PD did not influence any of the measured variables. RS, however, increased cell proliferation in the crypts of the macroscopically-normal rectum of older adults. Our findings suggest that the effects of RS on CCPS are not only dose, type of RS and health status-specific but are also influenced by age.

Adherence to the World Cancer Research Fund/American Institute for Cancer Research cancer prevention recommendations and WNT-pathway-related markers of bowel cancer risk.

Bowel cancer risk is strongly influenced by lifestyle factors including diet and physical activity. Several studies have investigated the effects of adherence to the World Cancer Research Fund (WCRF)/American Institute for Cancer Research (AICR) cancer prevention recommendations on outcomes such as all-cause and cancer-specific mortality, but the relationships with molecular mechanisms that underlie the effects on bowel cancer risk are unknown. This study aimed to investigate the relationships between adherence to the WCRF/AICR cancer prevention recommendations and wingless/integrated (WNT)-pathway-related markers of bowel cancer risk, including the expression of WNT pathway genes and regulatory microRNA (miRNA), secreted frizzled-related protein 1 (SFRP1) methylation and colonic crypt proliferative state in colorectal mucosal biopsies. Dietary and lifestyle data from seventy-five healthy participants recruited as part of the DISC Study were used. A scoring system was devised including seven of the cancer prevention recommendations and smoking status. The effects of total adherence score and scores for individual recommendations on the measured outcomes were assessed using Spearman's rank correlation analysis and unpaired t tests, respectively. Total adherence score correlated negatively with expression of Myc proto-oncogene (c-MYC) (P=0·039) and WNT11 (P=0·025), and high adherers had significantly reduced expression of cyclin D1 (CCND1) (P=0·042), WNT11 (P=0·012) and c-MYC (P=0·048). Expression of axis inhibition protein 2 (AXIN2), glycogen synthase kinase (GSK3ß), catenin ß1 (CTNNB1) and WNT11 and of the oncogenic miRNA miR-17 and colonic crypt kinetics correlated significantly with scores for individual recommendations, including body fatness, red meat intake, plant food intake and smoking status. The findings from this study provide evidence for positive effects of adherence to the WCRF/AICR cancer prevention recommendations on WNT-pathway-related markers of bowel cancer risk.

Non-digestible carbohydrates supplementation increases miR-32 expression in the healthy human colorectal epithelium: A randomized controlled trial.

Colorectal cancer (CRC) risk is modulated by diet and there is convincing evidence of reduced risk with higher non-digestible carbohydrates (NDCs) consumption. Resistant starch (RS), a NDC, positively modulates the expression of oncogenic microRNAs, suggesting that this could be a mechanism through which NDCs protect against CRC. The present study aimed to investigate the effects of supplementation with two NDCs, RS, and polydextrose (PD), on microRNA expression in the macroscopically-normal human rectal epithelium using samples from the DISC Study, a randomized, double-blind, placebo-controlled dietary intervention. We screened 1008 miRNAs in pooled post-intervention rectal mucosal samples from participants allocated to the double placebo group and those supplemented with both RS and PD. A total of 111 miRNAs were up- or down-regulated by at least twofold in the RS + PD group compared with the control group. From these, eight were selected for quantification in individual participant samples by qPCR, and fold-change direction was consistent with the array for seven miRNAs. The inconsistency for miR-133b and the lower fold-change values observed for the seven miRNAs is probably because qPCR of individual participant samples is a more robust and sensitive method of quantification than the array. miR-32 expression was increased by approximately threefold (P = 0.033) in the rectal mucosa of participants supplemented with RS + PD compared with placebo. miR-32 is involved in the regulation of processes such as cell proliferation that are dysregulated in CRC. Furthermore, miR-32 may affect non-canonical NF-κB signaling via regulation of TRAF3 expression and consequently NIK stabilization.

The cancer risk related to meat and meat products.

Introduction: Meat has been classified by International Agency for Research on Cancer (IARC) as carcinogenic to humans. The evidence and the implications for health are reviewed. Sources of data: Evidence was obtained from published reports and systematic reviews published before and since the IARC decision. Areas of agreement: Epidemiology indicates that processed meat products are associated with increased risk of colorectal cancer. Evidence for red meat and for other cancers remains tentative. Areas of controversy: Several mechanisms for mutagenic effects of meat consumption have been identified but it is not clear which cause cancer in humans. The extent to which complete abstention from meat protects against cancer is also uncertain. Growing points: Prospective studies on meat consumption in western populations will continue to illuminate the details of carcinogenesis, and effective strategies for reducing risk. Areas timely for developing research: Further studies on the precise mechanisms of carcinogenesis in human populations would assist both food manufacturers and the general public to minimize risk.

Transcriptomics and proteomics show that selenium affects inflammation, cytoskeleton, and cancer pathways in human rectal biopsies.

Epidemiologic studies highlight the potential role of dietary selenium (Se) in colorectal cancer prevention. Our goal was to elucidate whether expression of factors crucial for colorectal homoeostasis is affected by physiologic differences in Se status. Using transcriptomics and proteomics followed by pathway analysis, we identified pathways affected by Se status in rectal biopsies from 22 healthy adults, including 11 controls with optimal status (mean plasma Se = 1.43 µM) and 11 subjects with suboptimal status (mean plasma Se = 0.86 µM). We observed that 254 genes and 26 proteins implicated in cancer (80%), immune function and inflammatory response (40%), cell growth and proliferation (70%), cellular movement, and cell death (50%) were differentially expressed between the 2 groups. Expression of 69 genes, including selenoproteins W1 and K, which are genes involved in cytoskeleton remodelling and transcription factor NFκB signaling, correlated significantly with Se status. Integrating proteomics and transcriptomics datasets revealed reduced inflammatory and immune responses and cytoskeleton remodelling in the suboptimal Se status group. This is the first study combining omics technologies to describe the impact of differences in Se status on colorectal expression patterns, revealing that suboptimal Se status could alter inflammatory signaling and cytoskeleton in human rectal mucosa and so influence cancer risk.-Méplan, C., Johnson, I. T., Polley, A. C. J., Cockell, S., Bradburn, D. M., Commane, D. M., Arasaradnam, R. P., Mulholland, F., Zupanic, A., Mathers, J. C., Hesketh, J. Transcriptomics and proteomics show that selenium affects inflammation, cytoskeleton, and cancer pathways in human rectal biopsies.

Procyanidin induces apoptosis of esophageal adenocarcinoma cells via JNK activation of c-Jun.

Procyanidins are polymeric flavanols found in fruits and vegetables and have shown anticarcinogenic/chemopreventive properties. We previously showed that oligomeric procyanidin extracted from apples induced cell cycle arrest and apoptosis in esophageal adenocarcinoma (OA) cells. To understand the mechanism of action, we determined transcriptomic changes induced by procyanidin in OA cells. Pathway analysis implicated mitogen-activated protein kinase signaling pathways in eliciting these responses. Procyanidin induced the activation of JNK and p38 and the phosphorylation and expression of c-Jun. Inhibition of JNK but not p38 kinase activity prevented the procyanidin-induced phosphorylation and expression of c-Jun. Knockdown of the expression of JNK1, JNK2, or JUN diminished procyanidin-induced effects on cell proliferation and apoptosis. c-Jun is a component of the transcription factor AP-1 and AP-1 binding sites are overrepresented in the promoters of procyanidin-induced genes. This indicates that JNK activation of c-Jun by procyanidin leads to the induction of apoptosis of OA cells and suggests a role for a c-Jun-mediated transcriptional program. These data provide a mechanistic understanding of how procyanidin specifically targets a distinct pathway involved in the induction of apoptosis in OA cells and will inform future studies investigating its use as a chemopreventive/therapeutic agent.

TrxR1 and GPx2 are potently induced by isothiocyanates and selenium, and mutually cooperate to protect Caco-2 cells against free radical-mediated cell death.

Currently, there is significant interest in the field of diet-gene interactions and the mechanisms by which food compounds regulate gene expression to modify cancer susceptibility. From a nutrition perspective, two key components potentially exert cancer chemopreventive effects: isothiocyanates (ITCs), present in cruciferous vegetables, and selenium (Se) which, as selenocysteine, is an integral part of selenoproteins. However, the role of these compounds in the expression of key selenoenzymes once the cancer process has been initiated still needs elucidation. Therefore, this investigation examined the effect of two forms of selenium, selenium-methylselenocysteine and sodium selenite, both individually and in combination with two ITCs, sulforaphane or iberin, on the expression of the two selenoenzymes, thioredoxin reductase 1 (TrxR1) and gastrointestinal glutathione peroxidase (GPx2), which are targets of ITCs, in Caco-2 cells. Co-treatment with both ITCs and Se induced expression of TrxR1 and GPx2 more than either compound alone. Moreover, pre-treatment of cells with ITC+Se enhanced cytoprotection against H(2)O(2)-induced cell death through a ROS-dependent mechanism. Furthermore, a single and double knockdown of TrxR1 and/or GPx2 suggested that both selenoproteins were responsible for protecting against H(2)O(2)-induced cell death. Together, these data shed new light on the mechanism of interactions between ITC and Se in which translational expression of the enhanced transcripts by the former is dependent on an adequate Se supply, resulting in a cooperative antioxidant protective effect against cell death.

Quantitative profiling of CpG island methylation in human stool for colorectal cancer detection.

PURPOSE: The aims of this study were to investigate the use of quantitative CGI methylation data from stool DNA to classify colon cancer patients and to relate stool CGI methylation levels to those found in corresponding tissue samples. METHODS: We applied a quantitative methylation-specific PCR assay to determine CGI methylation levels of six genes, previously shown to be aberrantly methylated during colorectal carcinogenesis. Assays were performed on DNA from biopsies of "normal" mucosa and stool samples from 57 patients classified as disease-free, adenoma, or cancer by endoscopy, and in tumour tissue from cancer patients. Additionally, CGI methylation was analysed in stool DNA from an asymptomatic population of individuals covering a broad age range (mean = 47 ± 24 years) RESULTS: CGI methylation levels in stool DNA were significantly higher than in DNA from macroscopically normal mucosa, and a significant correlation between stool and mucosa was observed for ESR1 only. Multivariate statistical analyses using the methylation levels of each CGI in stool DNA as a continuous variable revealed a highly significant (p = 0.003) classification of cancer vs. non-cancer (adenoma + disease-free) patients (sensitivity = 65 %, specificity = 81 %). CONCLUSION: CGI methylation profiling of stool DNA successfully identified patients with cancer despite the methylation status of CGIs in stool DNA not generally reflecting those in DNA from the colonic mucosa.

Colorectal cancer cells Caco-2 and HCT116 resist epigenetic effects of isothiocyanates and selenium in vitro.

PURPOSE: It is relatively unknown how different dietary components, in partnership, regulate gene expression linked to colon pathology. It has been suggested that the combination of various bioactive components present in a plant-based diet is crucial for their potential anticancer activities. This study employed a combinatorial chemopreventive strategy to investigate the impact of selenium and/or isothiocyanates on DNA methylation processes in colorectal carcinoma cell lines. METHODS: To gain insights into the epigenetic-mediated changes in gene expression in response to these dietary constituents cultured Caco-2 and HCT116 cells were exposed for up to 12 days to different concentrations of selenium methylselenocysteine and selenite (ranging from 0.2 to 5 µM) either alone or in combination with sulforaphane and iberin (ranging from 6 to 8 µM), and changes to gene-specific (p16(INK4A) and ESR1), global (LINE-1) methylation and DNMT expression were quantified using real-time PCR-based assays. RESULTS: No effects on the methylation of CpG islands in ESR1, p16(INK4A) or of LINE-1, a marker of global genomic methylation, were observed after exposure of Caco-2 and HCT116 cells to selenium or isothiocyanates. Only transient changes in DNMT mRNA expression, which occurred mostly in the treatment groups containing isothiocyanates, were observed, and these occurred only for specific DNMT transcripts and did not lead to the modification of the aberrant methylation status present in these cells. CONCLUSION: These data suggest that treatment for colon cancer cells with selenium and/or isothiocyanates, either individually or in combination does not impact abnormal methylation patterns of key genes involved in the complex multistep process of colon carcinogenesis in vitro.

Metabolomics of fecal extracts detects altered metabolic activity of gut microbiota in ulcerative colitis and irritable bowel syndrome.

(1)H NMR spectroscopy of aqueous fecal extracts has been used to investigate differences in metabolic activity of gut microbiota in patients with ulcerative colitis (UC) (n = 13), irritable bowel syndrome (IBS) (n = 10), and healthy controls (C) (n = 22). Up to four samples per individual were collected over 2 years giving a total of 124 samples. Multivariate discriminant analysis, based on NMR data from all three groups, was able to predict UC and C group membership with good sensitivity and specificity; classification of IBS samples was less successful and could not be used for diagnosis. Trends were detected toward increased taurine and cadaverine levels in UC with increased bile acid and decreased branched chain fatty acids in IBS relative to controls; changes in short chain fatty acids and amino acids were not significant. Previous PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the same fecal material had shown alterations of the gut microbiota when comparing UC and IBS groups with controls. Hierarchical cluster analysis showed that DGGE profiles from the same individual were stable over time, but NMR spectra were more variable; canonical correlation analysis of NMR and DGGE data partly separated the three groups and revealed a correlation between the gut microbiota profile and metabolite composition.

Diet-Associated Inflammation Modulates Inflammation and WNT Signaling in the Rectal Mucosa, and the Response to Supplementation with Dietary Fiber.

Inflammation drives colorectal cancer development, and colorectal cancer risk is influenced by dietary factors, including dietary fiber. Hyperactive WNT signaling occurs in colorectal cancer and may regulate inflammation. This study investigated (i) relationships between the inflammatory potential of diet, assessed using the Energy-adjusted Dietary Inflammatory Index (E-DII), and markers of WNT signaling, and (ii) whether DII status modulated the response to supplementation with two types of dietary fiber. Seventy-five healthy participants were supplemented with resistant starch and/or polydextrose (PD) or placebo for 50 days. Rectal biopsies were collected before and after intervention and used to assess WNT pathway gene expression and crypt cell proliferation. E-DII scores were calculated from food frequency questionnaire data. High-sensitivity C-reactive protein (hsCRP) and fecal calprotectin concentrations were quantified. hsCRP concentration was significantly greater in participants with higher E-DII scores [least square means (LSM) 4.7 vs. 2.4 mg/L, P = 0.03]. Baseline E-DII score correlated with FOSL1 (ß = 0.503, P = 0.003) and WNT11 (ß = 0.472, P = 0.006) expression, after adjusting for age, gender, body mass index, endoscopy procedure, and smoking status. WNT11 expression was more than 2-fold greater in individuals with higher E-DII scores (LSM 0.131 vs. 0.059, P = 0.002). Baseline E-DII modulated the effects of PD supplementation on FOSL1 expression (P = 0.04). More proinflammatory diets were associated with altered WNT signaling and appeared to modulate the effects of PD supplementation on expression of FOSL1 This is the first study to investigate relationships between the E-DII and molecular markers of WNT signaling in rectal tissue of healthy individuals.Prevention Relevance: Our finding that more inflammatory dietary components may impact large bowel health through effects on a well-recognized pathway involved in cancer development will strengthen the evidence base for dietary advice to help prevent bowel cancer.

Patterns of DNA methylation in individual colonic crypts reveal aging and cancer-related field defects in the morphologically normal mucosa.

Methylation of CpG islands (CGIs) in the promoter regions of tumour suppressor genes is common in colorectal cancer and occurs also in an age-dependent manner in the morphologically normal colorectal mucosa. In this study, we quantified the level of methylation of six genes associated with the Wnt signalling pathway (adenomatous polyposis coli, DKK1, WIF1, SFRP1, SFRP2 and SFRP5) together with long-interspersed nuclear element-1 as a surrogate for global methylation. DNA methylation was analysed in 260 individual colorectal crypts obtained from eight female patients with no evidence of colorectal disease and five with colorectal cancer. Significant variation in methylation levels for each of the six genes existed between crypts from the same biopsy. The variation in both global and gene-specific CGI methylation between crypts from the same individual was significantly less than that between individuals. Bisulphite sequencing provided insight into the mechanism of aberrant methylation showing that CGI methylation occurs in an 'all-or-none' manner by the directional spreading of methylation from further upstream. Univariate statistical analyses revealed that there were significant differences in crypt-specific methylation associated with both aging and disease status. A multivariate statistical modelling approach was able to distinguish both subject age and health status based on crypt-specific methylation profiles. Our results indicate that the differential methylation of genes associated with the Wnt signalling pathway affecting individual morphologically normal crypts may contribute to the age-dependent generation of the colonic field defect and, in combination with mutations, to the stepwise development of colorectal neoplasia.

Ulcerative colitis and irritable bowel patients exhibit distinct abnormalities of the gut microbiota.

BACKGROUND: Previous studies suggest a link between gut microbiota and the development of ulcerative colitis (UC) and irritable bowel syndrome (IBS). Our aim was to investigate any quantitative differences in faecal bacterial compositions in UC and IBS patients compared to healthy controls, and to identify individual bacterial species that contribute to these differences. METHODS: Faecal microbiota of 13 UC patients, 11 IBS patients and 22 healthy volunteers were analysed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE) using universal and Bacteroides specific primers. The data obtained were normalized using in-house developed statistical method and interrogated by multivariate approaches. The differentiated bands were excised and identified by sequencing the V3 region of the 16S rRNA genes. RESULTS: Band profiles revealed that number of predominant faecal bacteria were significantly different between UC, IBS and control group (p < 10-4). By assessing the mean band numbers in UC (37 ± 5) and IBS (39 ± 6), compared to the controls (45 ± 3), a significant decrease in bacterial species is suggested (p = 0.01). There were no significant differences between IBS and UC. Biodiversity of the bacterial species was significantly lower in UC (µ = 2.94, σ = 0.29) and IBS patients (µ = 2.90, σ = 0.38) than controls (µ = 3.25, σ = 0.16; p = 0.01). Moreover, similarity indices revealed greater biological variability of predominant bacteria in UC and IBS compared to the controls (median Dice coefficients 76.1% (IQR 70.9 - 83.1), 73.8% (IQR 67.0 - 77.5) and 82.9% (IQR 79.1 - 86.7) respectively). DNA sequencing of discriminating bands suggest that the presence of Bacteroides vulgatus, B. ovatus, B. uniformis, and Parabacteroides sp. in healthy volunteers distinguishes them from IBS and UC patients. DGGE profiles of Bacteroides species revealed a decrease of Bacteroides community in UC relative to IBS and controls. CONCLUSION: Molecular profiling of faecal bacteria revealed abnormalities of intestinal microbiota in UC and IBS patients, while different patterns of Bacteroides species loss in particular, were associated with UC and IBS.

Involvement of KLF4 in sulforaphane- and iberin-mediated induction of p21(waf1/cip1).

Sulforaphane (SF; 4-methylsulfinylbutyl isothiocyanate), a dietary compound derived from broccoli, may exhibit chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21(waf1/cip1)), but the exact molecular mechanism has not been determined. Here we evaluate the role of the transcription factor Kruppel-like factor 4 (KLF4) in mediating the induction of p21(waf1/cip1) and cellular differentiation by SF and iberin (IB; 3-methylsulphinyl propyl isothiocyanate), also derived from broccoli. Exposure of Caco-2 and Caco-2/TC7 cells to SF and IB increased expression of both KLF4 and p21(waf1/cip1), whereas exposure of HT29 cells resulted only in induction of p21(waf1/cip1). In Caco-2 cells, small interfering RNA knock down of KLF4 expression attenuated induction of p21(waf1/cip1) in response to either SF or IB treatment. Contrary to expectation, prolonged exposure to SF reduced sucrase isomaltase activity, a marker of small intestinal differentiation in Caco-2 cells. Additional support for the SF-mediated induction of p21(waf1/cip1) by KLF4 was obtained from analyses of gastric tissue of Apc(Min/+) mice following acute intervention with SF but not from the analyses of other tissue of the intestinal tract. These results suggest that induction of p21(waf1/cip1) by SF or IB may be partly mediated by KLF4 in some colon cancer cells and tissues.

A comparison of the effects of soya isoflavonoids and fish oil on cell proliferation, apoptosis and the expression of oestrogen receptors alpha and beta in the mammary gland and colon of the rat.

Isoflavonoids and fish oil may be protective against colorectal cancer, but the evidence in relation to breast cancer risk is ambiguous. In the present study, we have investigated the impact of soya-derived isoflavonoids and n-3 fatty acids from fish oil, both individually and in combination, on apoptosis, cell proliferation and oestrogen receptor (ER) expression in the colon and mammary gland of the rat. Female rats were fed diets high in n-3 fatty acids (80 g/kg diet) or soya protein (765 mg/kg diet isoflavones) for 2 weeks, and then killed before the removal of the colon and mammary glands. Cell proliferation and apoptosis were quantified morphologically in whole crypts and terminal end buds. The expressions of ERalpha and ERbeta were measured in colon tissue scrapes and the mammary gland. Fish oil significantly increased apoptosis and decreased mitosis in both tissues, an effect associated with a decrease in the expressions of ERalpha and ERbeta. Soya had no effect on apoptosis in either tissue, but reduced mitosis in the colon (P < 0.001) while increasing it in the mammary gland (P = 0.001). The changes in proliferation were associated with contrasting changes in the ER expression such that fish oil significantly decreased both ERbeta and ERalpha, while soya increased ERalpha and decreased ERbeta. The results may provide a novel mechanism by which n-3 fatty acids could reduce cancer risk, but the interpretation of the results in relation to soya consumption and breast cancer risk requires further investigation.

Flavonoids and intestinal cancers.

Cancers of the gastrointestinal tract are amongst the most common causes of death from cancer, but there is substantial variation in incidence across populations. This is consistent with a major causative role for diet. There is convincing evidence that fruits and vegetables protect against cancers of the upper alimentary tract and the large bowel, and this has focused attention on biologically active phytochemicals, and on flavonoids in particular. Many flavonoids exert anticarcinogenic effects in vitro and in animals, and many of these effects occur via signalling pathways known to be important in the pathogenesis of colorectal, gastric and oesophageal cancers. However dietary flavonoid intakes are generally low and their metabolism in humans is extremely complex. The advent of new post-genomic technologies will do much to address these problems by making it possible to monitor patterns of gene expression in humans to provide essential molecular biomarkers of early disease. By combining such data with knowledge of the dietary exposure and bioavailability of the most effective compounds it will be possible to predict the most effective dietary sources and to properly evaluate the potential role of flavonoids in clinical nutrition.

Environment, diet and CpG island methylation: epigenetic signals in gastrointestinal neoplasia.

The epithelial surfaces of the mammalian alimentary tract are characterised by very high rates of cell proliferation and DNA synthesis, and in humans they are highly susceptible to cancer. The role of somatic mutations as drivers of carcinogenesis in the alimentary tract is well established, but the importance of gene silencing by epigenetic mechanisms is increasingly recognised. Methylation of CpG islands is an important component of the epigenetic code that regulates gene expression during development and normal cellular differentiation, and a number of genes are well known to become abnormally methylated during the development of tumours of the oesophagus, stomach and colorectum. Aberrant patterns of DNA methylation develop as a result of pathological processes such as chronic inflammation, and in response to various dietary factors, including imbalances in the supply of methyl donors, particularly folates, and exposure to DNA methyltransferase inhibitors, which include polyphenols and possibly isothiocyanates from plant foods. However the importance of these environmental interactions in human health and disease remains to be established. Recent moves to modify the exposure of human populations to folate, by mandatory supplementation of cereal foods, emphasise the importance of understanding the susceptibility of the human epigenome to dietary and other environmental effects.

Proteomic analysis reveals field-wide changes in protein expression in the morphologically normal mucosa of patients with colorectal neoplasia.

Models for the pathogenesis of colorectal cancer tend to focus on the localized lesion, with less attention paid to changes in normal-appearing mucosa. Here we used two-dimensional gel electrophoresis and mass spectrometry to define patterns of protein expression in morphologically normal colonic mucosa from 13 healthy subjects, 9 patients with adenomatous polyps, and 9 with cancer. Tumor samples were also compared with the normal mucosa. Systematic gel comparisons identified a total of 839 spots that differed significantly between one or more groups (P < 0.05). Principle component analysis indicated that the first three components accounted for approximately 37% of the total variation and provided clear evidence that flat mucosa from healthy subjects differed significantly from that of patients with polyps or cancer. Sixty-one proteins differed significantly between mucosa from healthy subjects and all other tissue types, and 206 differed significantly between healthy mucosa and polyp mucosa. Several of the proteins showing significant underexpression in tumor tissue were cytokeratins and other cytoskeletal components. In contrast, cytokeratins, including several isoforms of cytokeratin 8, were overexpressed in apparently normal mucosa from polyp and cancer patients compared with mucosa from healthy subjects. These findings indicate that protein expression in the apparently normal colonic mucosal field is modified in individuals with neoplastic lesions at sites distant from the lesion. Recognition and further characterization of this field effect at the molecular level may provide protein biomarkers of susceptibility to colorectal cancer and facilitate development of hypotheses for the role of diet and other environmental factors in its causation.

Cruciferous Vegetables and Risk of Cancers of the Gastrointestinal Tract.

Cancers of the oropharyngeal tissues, oesophagus, stomach, and colorectum are amongst the most common causes of death from cancer throughout the world. Higher consumption of fruits and vegetables is thought to be protective, and cruciferous vegetables are of particular interest because of their unique role as a source of biologically active glucosinolate breakdown products. A literature review of primary studies and meta-analyses indicates that higher consumption of cruciferous vegetables probably reduces the risk of colorectal and gastric cancers by approximately 8% and 19%, respectively. Some studies support the hypothesis that the protective effect against colorectal cancer is modified by genetic polymorphisms of genes regulating the expression of enzymes of the glutathione S-transferase family, but due to contradictory findings the evidence is currently inconclusive. Despite these promising findings, future epidemiological research on the protective effects of cruciferous plants will depend critically upon accurate measurement of dietary exposure, both to the vegetables themselves, and to their active constituents. The development of sensitive chemical assays has facilitated the measurement of urinary excretion of isothiocyanate metabolites as an objective biomarker of intake, but sampling strategies need to be optimized in order to assess long-term exposures at the population level.

Effects of supplementation with nondigestible carbohydrates on fecal calprotectin and on epigenetic regulation of SFRP1 expression in the large-bowel mucosa of healthy individuals.

BACKGROUND: Hyperactive Wnt signaling is frequently observed in colorectal cancer. Higher intakes of dietary fiber [nondigestible carbohydrates (NDCs)] and the fermentation product butyrate are protective against colorectal cancer and may exert their preventative effects via modulation of the Wnt pathway. OBJECTIVES: We investigated the effects of supplementing healthy individuals with 2 NDCs [resistant starch (RS) and polydextrose] on fecal calprotectin concentrations and Wnt pathway-related gene expression. In addition, we determined whether effects on secreted frizzled-related protein 1 (SFRP1) expression are mediated via the epigenetic mechanisms DNA methylation and microRNA expression. DESIGN: In a randomized, double-blind, placebo-controlled trial (the Dietary Intervention, Stem cells and Colorectal Cancer (DISC) Study), 75 healthy participants were supplemented with RS and/or polydextrose or placebo for 50 d in a 2 × 2 factorial design. Pre- and postintervention stool samples and rectal mucosal biopsies were collected and used to quantify calprotectin and expression of 12 Wnt-related genes, respectively. The expression of 10 microRNAs predicted to target SFRP1 was also quantified by quantitative reverse transcriptase-polymerase chain reaction, and DNA methylation was quantified at 7 CpG sites within the SFRP1 promoter region by pyrosequencing. RESULTS: NDC supplementation did not affect fecal calprotectin concentration. SFRP1 mRNA expression was reduced by both RS (P = 0.005) and polydextrose (P = 0.053). RS and polydextrose did not affect SFRP1 methylation or alter the expression of 10 microRNAs predicted to target SFRP1. There were no significant interactions between RS and polydextrose. CONCLUSIONS: RS and polydextrose supplementation did not affect fecal calprotectin concentrations. Downregulation of SFRP1 with RS and polydextrose could result in increased Wnt pathway activity. However, effects on Wnt pathway activity and downstream functional effects in the healthy large-bowel mucosa remain to be investigated. The DISC Study was registered at clinicaltrials.gov as NCT01214681.