Effects of a grape-supplemented diet on proliferation and Wnt signaling in the colonic mucosa are greatest for those over age 50 and with high arginine consumption

Holcombe et al. Nutrition Journal (2015) 14:62 DOI 10.1186/s12937-015-0050-z RESEARCH Open Access Effects of a grape-supplemented diet on proliferation and Wnt signaling in the colonic mucosa are greatest for those over age 50 and with high arginine consumption Randall F. Holcombe1*, Micaela Martinez2, Kestutis Planutis1 and Marina Planutiene1 Abstract A diet rich in fruits and vegetables, and a grape-derived compound, resveratrol, have been linked to a reduced incidence of colon cancer. In vitro and in vivo, resveratrol suppresses Wnt signaling, a pathway constitutively activated in over 85 % of colon cancers. Thirty participants were placed on a low resveratrol diet and subsequently allocated to one of three groups ingesting 1/3-to-1 lb (0.15–0.45 kg) of grapes per day for 2 weeks. Dietary information was collected via 24-h recall. Colon biopsies for biomarker analysis were obtained pre- and post-grape and evaluated for the expression of Wnt pathway target genes and for markers of proliferation by RT-PCR and immunohistochemistry. Participants lost an average of 2 · 6 lb (1.2 kg, p = 0 · 0018) during the period of grape ingestion. The expression of CyclinD1 (p < 0 · 01), AXIN2, CD133 (p = 0 · 02) and Ki67 (p = 0 · 002) were all reduced after grape ingestion. Individuals over 50 years of age and those with high dietary arginine consumption had increased basal expression of CyclinD1, AXIN2, cMYC and CD133 (p value range 0 · 04 to <0 · 001) that, following grape ingestion, were reduced to levels seen in younger participants. The reduction in Wnt signaling and mucosal proliferation seen following short-term ingestion of 1/3–1 lb (0.15–0.45 kg) of grapes per day may reduce the risk of mutational events that can facilitate colon carcinogenesis. The potential benefit is most marked for high-risk older individuals and individuals whose diet is high in arginine intake. Dietary grape supplementation may play a role in colon cancer prevention for high-risk individuals. Keywords: Diet, Cancer prevention, Resveratrol, Colon cancer, Aging, Arginine Introduction Studies suggest strongly that a diet rich in fruits and vegetables leads to a lower risk of colorectal cancer (CRC) [1, 2]. Grape seeds and other grape-based products have purported CRC prevention activity [3], are rich in polyphenols and contain resveratrol, anthocyanins, catechins, quercetin and numerous other compounds with chemopreventive potential [4]. The most intensively studied component in grapes is resveratrol which suppresses PI3-kinase, AKT and NF-kB signaling pathways [5] * Correspondence: 1 Division of Hematology & Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustav L. Levy Place, Box 1128, New York, NY 10029, USA Full list of author information is available at the end of the article and may affect tumor growth by a myriad of other mechanisms as well [6, 7]. Systemic administration of resveratrol has been shown to inhibit the growth of intestinal tumors in several different rodent cancer models [8, 9]. For colon cancer prevention, effects are seen over a wide variety of dose ranges depending on individual studies. Tessotore [10] demonstrated activity of very low dose resveratrol of 0.2 mg/kg/day in reducing aberrant crypt foci (ACF) in the colon in an azoymethane-induced tumor model. In another carcinogen-based model, utilizing 1,2-dimethylhydrazine, resveratrol at 8 mg/kg/day reduced both ACF and colonic tumors [11] as did gavage of 60 mg/kg body weight [12]. In genetic models utilizing the APCmin/+ mouse, which harbors a single allele mutation in apc and therefore has intrinsically activated Wnt signaling, © 2015 Holcombe et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Holcombe et al. Nutrition Journal (2015) 14:62 Schneider [13] demonstrated profound activity at dosages as low as 0.3 mg/mouse/day in reducing intestinal tumors. In this study, expression of Wnt target gene cyclinD1 as well as other markers of cell cycling was reduced. Resveratrol, even at low concentrations, blocks Wnt signaling in colon cancer cells in vitro [14]. This pathway is activated in over 85 % of CRC making it an attractive target for a colon cancer prevention agent. Resveratrolrich freeze-dried grape powder has been utilized in a pilot study in normal human volunteers and was found to down-regulate the expression of Wnt pathway target genes CyclinD1 and AXIN2 in colonic mucosa [15]. However, low bioavailability of individual compounds such as resveratrol often results in systemic concentrations too low to be clinically active [16]. Therefore, it is important in consideration of dietary approaches to cancer prevention to consider the aggregate activity of all of the bioactive components in a particular foodstuff [17] and not just single purified compounds. This phase I study was undertaken to evaluate the potential role of a grape-supplemented diet for CRC prevention. The endpoints were biologic biomarkers of proliferation and Wnt signaling in colonic mucosa. During the study, detailed dietary information was collected and analyzed. This study is unique in that the effects of the complete foodstuff, rather than a refined component (ie grape seed extract) or individual substance (ie resveratrol) on biologically relevant cancer prevention endpoints is being investigated. Materials and methods Page 2 of 8 produced by the University of Minnesota. During days 15–30, participants were assigned to one of three grape consumption cohorts: 1/3 lb, 2/3 lb or 1 lb (0.15 kg, 0.30 kg, 0.45 kg) of grapes to be ingested each day. These amounts were selected by the investigators because 1/3 lb of grapes is equivalent to one “serving” as defined by the United States Department of Agriculture (USDA). Each patient was given a voucher redeemable at a local grocery store for red, seedless grapes and a digital kitchen scale. Compliance with grape consumption was ascertained at the time of the 24 h recall evaluations and again at the end of subject participation. Assignment into the cohorts was determined by a random sequential enrollment algorithm. Limited flexible sigmoidoscopies were performed on day 15 (“pre-grape”) and on day 30 (“post-grape”). Two-to-four rectal mucosal biopsies were obtained for RNA and immunohistochemical analysis. See Fig. 1 for a schematic of the clinical trial design. 24 hour dietary recall Nutrition data system for research (NDSR) is a dietary analysis program designed for the collection and analyses of 24-h dietary recalls and the analysis of food records, menu (...truncated)