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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. 2015
Received: 26 March 2015
Accepted: 10 June 2015
Published: 19 June 2015
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.
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 , are rich in polyphenols and contain resveratrol, anthocyanins, catechins, quercetin and numerous other compounds with chemopreventive potential . The most intensively studied component in grapes is resveratrol which suppresses PI3-kinase, AKT and NF-kB signaling pathways  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  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  as did gavage of 60 mg/kg body weight . In genetic models utilizing the APCmin/+ mouse, which harbors a single allele mutation in apc and therefore has intrinsically activated Wnt signaling, Schneider  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 . This pathway is activated in over 85 % of CRC making it an attractive target for a colon cancer prevention agent. Resveratrol-rich 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 . However, low bioavailability of individual compounds such as resveratrol often results in systemic concentrations too low to be clinically active . 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  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
Clinical trial design and conduct
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, menus, and recipes. Nutrient intake from both food and supplemental sources are captured and quantified. Dietary interviews took place over the telephone and/or in person and data was entered directly into NDSR. Food portion estimation visual aids and a kitchen scale were provided to respondents to assist in portion size estimation.
Quantitative Real-time PCR (qRT-PCR)
RNA was extracted from one-to-two of the biopsies obtained from each of the “pre-grape” and “post-grape” sigmoidoscopies using standard methodologies. Rectal mucosal biopsies were analyzed by quantitative real-time PCR for the expression of genes associated with proliferation and Wnt signaling including: ornithine decarboxylase (ODC1), nemo-like kinase (NLK), cJUN, cMYC, cyclinD1 (CCND1), lymphoid enhancing factor-1 (LEF1, TCF7L3), axinII (AXIN2), forkhead box protein 3 (FOXO3) and CD133 (Prominin1, PROM1). Primer pairs were obtained from Qiagen (Valencia, CA) with cycling parameters as defined by the manufacturer. Wnt target gene mRNA levels were all normalized to the housekeeping gene ribosomal RNA (rRNA). The relative RNA expression was calculated by the comparative threshold cycle method. All experiments were repeated in triplicate.
One-to-two of the biopsies obtained from each of the “pre-grape” and “post-grape” sigmoidoscopies were immediately placed into 10 % formalin for processing and subsequently paraffin embedded, sectioned, and placed onto slides for Ki67 immunohistochemistry (IHC). The percentage of cells expressing Ki67 in the lower 1/3 of colonic crypts was determined by fluorescence confocal microscopy. A minimum of 50 crypts were evaluated for each set of biopsies.
Patient characteristics were compared with 1-way ANOVA with a Newman-Keuls post-test. Dietary changes seen with grape supplementation, pre- and post-grape gene expression by qRT-PCR and Ki67 expression scored by IHC were analyzed with a Wilcoxon matched pairs signed-rank test. Linear regression analysis was utilized to generate r2 and p values for the comparison of AXIN2 and CCND1 expression. Comparison of expression levels stratified for age or for dietary arginine intake was undertaken with an unpaired t-test. Statistical significance was defined at a level of p < 0 · 05.
Clinical trial and dietary analysis results
Overall group (mean ± SE)
1 pound cohort (mean ± SE)
2 pound cohort (mean ± SE)
3 pound cohort (mean ± SE)
43 · 28 ± 2 · 28
46 · 00 ± 3 · 02
43 · 45 ± 4 · 79
40 · 10 ± 3 · 98
Initial weight (lb)
172 · 1 ± 6 · 55
175 · 3 ± 16 · 30
171 · 4 ± 8 · 22
169 · 2 ± 7 · 67
28 · 12 ± 1 · 07
27 · 89 ± 2 · 42
27 · 58 ± 1 · 63
29 · 01 ± 1 · 09
Dietary changes observed with grape supplementation
At conclusion of grape intervention
Direction of change
172 · 1 ± 6 · 55
170 · 7 ± 7 · 65
0 · 0018
1960 ± 131 · 2
1984 ± 125 · 1
233 · 4 ± 15 · 8
271 · 3 ± 18 · 2
0 · 0048
Calories from carbohydrate (%)
46 · 99 ± 1 · 58
51 · 87 ± 1 · 53
0 · 0089
Total sugars (gm)
92 · 49 ± 7 · 86
131 · 9 ± 9 · 44
0 · 0001
Calories from fat (%)
35 · 32 ± 1 · 44
30 · 77 ± 1 · 20
0 · 011
1 · 32 ± 0 · 13
1 · 53 ± 0 · 11
0 · 0035
2566 ± 189
3004 ± 203
0 · 0014
Vitamin B6 (mg)
1 · 957 ± 0 · 16
2 · 254 ± 0 · 21
0 · 0193
Biochanin A (mg)
0 · 133 ± 0 · 07
0 · 013 ± 0 · 01
0 · 0067
486 · 2 ± 41 · 8
433 · 0 ± 41 · 1
289 · 3 ± 34 · 93
262 · 0 ± 28 · 06
2 · 53 ± 1 · 11
0 · 61 ± 0 · 20
4798 ± 726
4064 ± 702
4 · 503 ± 0 · 41
4 · 276 ± 0 · 34
Changes in markers of proliferation and Wnt signaling
Results related to changes in proliferation and Wnt signaling markers were not statistically different across the three cohorts. In general the magnitude of the changes was slightly greater in the 1 lb/day (0.45 kg) cohort than the 2/3 lb/day (0.30 kg) and 1/3 lb/day (0.15 kg) cohorts but the direction of the changes were consistent throughout all 3 groups. Each cohort contained only ten participants and therefore data for all participants were combined for presentation in this report. RT-PCR analysis revealed a significant reduction in the expression of CyclinD1, a proliferation marker and target gene for Wnt signaling, following grape ingestion (Fig. 2; p < 0.01). A trend toward reduction in another Wnt target gene, AXIN2 was seen and the pre- and post-grape expression levels of CyclinD1 correlated significantly with the corresponding levels of AXIN2 (Fig. 1). The expression of CD133, a colonic stem cell marker was also significantly reduced (p = 0 · 02). The expression of Ki67 protein, as measured by the percentage of expressing cells in the lower 1/3 of colonic crypts, was significantly reduced following grape ingestion (p = 0 · 002; Fig. 2). While the expression of several of the other genes tested had consistent trends, none, including cMYC, reached statistical significance.
Effect of age and grape diet on markers of proliferation and Wnt signaling
Effect of arginine intake and grape diet on markers of proliferation and Wnt signaling
Participants were able to tolerate up to a pound of grapes per day for 2 weeks with full compliance and without any adverse consequences. No patients with diabetes were enrolled on the trial based on a recommendation from the Institutional Review Board (Ethics Committee) because of the high sugar content of grapes. While on study, participants ingested less fat and lost, on average, 1 · 2 kg of weight. While weight loss with grapes has been reported previously in the popular press , to our knowledge this is the first confirmation of this effect under controlled clinical trial conditions.
The effects of a grape supplemented diet on Wnt signaling and markers of proliferation were consistent with prior in vitro  studies using resveratrol and an in vivo  study using resveratrol supplements and freeze-dried grape powder. Inhibition of Wnt signaling and reduced colonic mucosal proliferation suggests potential cancer preventative effects of a grape-containing diet. Whether these effects are due to resveratrol alone or other constituents of grapes, or a combination of constituents, remains to be defined. This study was of short duration and did not address long-term effects on the appearance of aberrant crypt foci or pre-cancerous colonic adenomas. The significant changes noted after a short period of exposure suggests that intermittent consumption of grapes may be sufficient and this should be considered in the design of future dietary-based cancer prevention trials.
As the human colon ages, the inflammatory process leading to mucosal injury and the regenerative capacity of the epithelium are affected . Telomere shortening, methylation of mucosal healing-associated genes, and alterations of growth factor signaling occur and have been postulated to affect the regenerative capacity of the epithelium. In rats, an increase in the proliferation rate of colonic mucosa is seen in conjunction with senescence . Diet restriction increases intestinal apoptosis in aging rats, perhaps providing protection from an age-related accumulation of DNA alterations . Resveratrol has been shown to improve survival of mice on a high calorie diet , possibly through its effects on suirtuin-1 (SIRT1) activation . Indeed, small molecule SIRT1 activators have been proposed for the treatment of age-related disorders . In our study, colonic proliferation was significantly higher in older participants compared to younger participants. The effects of grape ingestion were most dramatically seen in the older population, with a reduction of both Wnt target genes and markers of mucosal proliferation.
High levels of dietary arginine and the polyamine synthesis pathway have been linked to colon carcinogenesis in several types of animal models [25, 26]. This has also been implicated in human colon carcinogenesis  and a randomized trial has demonstrated that an inhibitor of polyamine synthesis, DFMO, reduces the incidence of colon adenomas with high malignant potential . Ornithine decarboxylase (ODC) catalyzes the rate-limiting step in the biosynthesis of polyamines and is inhibited by resveratrol, suggesting that one of the molecular mechanisms through which resveratrol may be operating is polyamine pathway inhibition . In our study, dietary arginine consumption was ascertained through the diet survey instrument. When stratified for arginine consumption, the effect on colonic mucosal proliferation was most marked in participants who had higher levels of arginine ingestion. Based on the ongoing dietary monitoring and post-grape intervention data, this effect appears to have been due to the grape consumption and not due to a reduction in arginine consumption over the course of the study.
This study has significant implications for colon cancer prevention. The reduction in Wnt signaling and mucosal proliferation seen following relatively short-term ingestion of 1/3–1 lb of grapes should reduce the risk for the development of mutational events that can ultimately result in colon carcinogenesis. The potential benefit is most marked for older individuals and individuals whose diet is high in arginine intake. Both of these groups have an increased incidence of colon cancer [27, 30]. Subsequent studies should focus on other high-risk populations such as patients with a history of colonic adenomas (polyps) and individuals with an inherited genetic predisposition for colon cancer. Finally, patients with inflammatory bowel disease may benefit from the anti-proliferative effects of a grape-supplemented diet.
Supported by a grant from the Gateway for Cancer Foundation to RFH.
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