Walnuts are composed of a complex array of biologically active constituents with individual cancer-protective properties. Here, we assessed the potential benefit of whole walnut consumption in a mouse tumor bioassay using azoxymethane. In study 1, a modest reduction (1.3-fold) in tumor numbers was observed in mice fed a standard diet (AIN-76A) containing 9.4% walnuts (15% of total fat). In study 2, the effects of walnut supplementation was tested in the Total Western Diet (TWD). There was a significant reduction (2.3-fold; P < 0.02) in tumor numbers in male mice fed TWD containing 7% walnuts (10.5% of total fat). Higher concentrations of walnuts lacked inhibitory effects, particularly in female mice, indicating there may be optimal levels of dietary walnut intake for cancer prevention. Since components of the Mediterranean diet have been shown to affect the gut microbiome, the effects of walnuts were therefore tested in fecal samples using 16S rRNA gene sequencing. Carcinogen treatment reduced the diversity and richness of the gut microbiome, especially in male mice, which exhibited lower variability and greater sensitivity to environmental changes. Analysis of individual operational taxonomic units (OTU) identified specific groups of bacteria associated with carcinogen exposure, walnut consumption, and/or both variables. Correlation analysis also identified specific OTU clades that were strongly associated with the presence and number of tumors. Taken together, our results indicate that walnuts afford partial protection to the colon against a potent carcinogenic insult, and this may be due, in part, to walnut-induced changes to the gut microbiome. Cancer Prev Res; 9(8); 692–703. ©2016 AACR.

Colorectal cancer is the third leading cause of cancer-related deaths in the United States. Approximately 5% (1 in 18) of men and women will be diagnosed with this disease during the course of their lifetime. Several clinical and preclinical studies have identified risk factors that increase the likelihood for developing colorectal cancer. For example, obesity and diets high in red meat and fat are associated with increased risk (1–4). However, the consumption of nutritive foods, such as those comprising the Mediterranean diet, has become a popular approach to mitigating cancer risk (5). The Mediterranean diet is composed mainly of vegetables, fruits, grains, legumes, olive oil, unsaturated fats, and a moderate amount of red wine (6). These whole foods contain a variety of polyphenols and plant bioactive compounds that have modifying activities against inflammation, tumorigenesis, and atherogenesis (6). While dietary patterns undoubtedly exert a powerful effect on the gut microbiome (7), there is accumulating evidence that among the beneficial effects of the Mediterranean diet is the ability to positively impact the composition of the gut microbiota (7, 8).

An important feature of the Mediterranean diet is the consumption of nuts. Nuts contain a large variety of beneficial bioactive components, including monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) and contain only low amounts of saturated fatty acids (SFA), as well as a number of phytochemicals (i.e., phenolic antioxidants), dietary fiber, and minerals (9, 10). PUFAs, including omega-3 fatty acids, reduce inflammation and have also been shown to lower polyp burden in animal models of colon cancer (11). Among all the nuts from the tree nut family, walnuts (Juglans regia) are the most enriched in PUFAs, with the highest ratio of omega-3:omega-6 (1:4.2). Walnuts also contain high levels of γ-tocopherol, a form of vitamin E with proven anticancer benefit (9, 12). A number of studies have identified beneficial effects of walnut consumption in a variety of diseases, including heart disease, diabetes, neurologic disorders, and cancer (9).

Given the healthful composition of walnuts and the proven benefit of many of its individual components on cancer signaling pathways, here we sought to assess the potential benefit of whole walnut consumption in a well-established primary colon cancer model. In the present study, we performed 2 in vivo studies using the organotropic colon carcinogen, azoxymethane (AOM; ref. 13). The effects of walnuts on colon carcinogenesis were tested in 2 basal diets, AIN-76A and a recently developed experimental diet that represents the typical Western dietary pattern, referred to as the Total Western Diet (TWD; ref. 14). To gain further insight into potential mechanisms by which walnuts may affect colon cancer development, fecal samples were collected to examine diet-induced changes to the community structure of the microbiome. Our results demonstrate for the first time that walnut consumption reduces colon tumor development, an effect that is more pronounced in male mice. Importantly, cancer protection is associated with significant alterations to the microbial community structure, which appeared to be associated with tumor suppression.

Animal treatment

Male and female A/J mice were purchased from The Jackson Laboratory. Whole walnut halves were provided by the California Walnut Commission. The amount of walnuts added to the diets was determined on the basis of previous in vivo studies (15, 16). In Study 1, mice (4 week-old) were fed AIN-76A diet (Harlan Laboratories, Inc.) supplemented with 0%, 9.4%, 14.1%, or 18.8% of walnuts by weight, which are equivalent to 0%, 15%, 22.5%, or 30.2% of energy from walnuts, respectively. In Study 2, mice (4 week-old) were fed the TWD (Harlan Laboratories) supplemented with 0%, 3.5%, 7%, or 14% of walnuts by weight, which are equivalent to 0%, 5.2%, 10.5%, or 21.4% of energy from walnuts, respectively. Macronutrient sources and fatty acid composition of AIN-76A and TWD diets are summarized in Supplementary Tables S1 and S2, respectively. Contents of the fat sources were proportionally lowered in each diet to compensate for the addition of walnuts. Walnuts were finely ground and added to each diet, which were prepared freshly each week. The diets and drinking water were given ad libitum.

Treatment with AOM and tissue processing

Five-week-old mice received 6 weekly intraperitoneal injections of AOM (Sigma-Aldrich); the first 3 doses were given at 5 mg/kg of body weight and the last 3 doses at 10 mg/kg of body weight. This protocol reduces morbidity and allows animals to adapt to the treatment. Control mice were injected with vehicle alone (0.9% NaCl). Ten weeks after the last injection, mice were sacrificed, and colons were harvested for further analyses. Colons were flushed immediately with ice-cold PBS and excised longitudinally. Specimens were fixed-flat in 10% neutral-buffered formalin for 4 hours and stored in 70% ethanol. Tissues were Swiss-rolled, paraffin-embedded, and sectioned at 5-μm thickness. Animal experiments were conducted after approval by the Animal Care Committee (ACC/IACUS) and Center for Comparative Medicine (CCM) at UCHC.

Quantification of lesions

Whole-mount colons were stained with 0.2% methylene blue, and the number and size of aberrant crypt foci (ACF) and tumors were scored under a dissecting microscope. Colon tumor load per mouse was determined using tumor diameter to calculate the spherical tumor volume, V = (4/3) × π × r3.

Immunohistochemistry and immunofluorescence.

Tissue sections were deparaffinized, antigen-retrieved, blocked, and incubated with anti-β-catenin (1:100; Sigma-Aldrich), anti-Ki67 (1:600; Cell Signaling), anti-pStat3 (1:400; Cell Signaling), or anti-p21 (1:100; Santa Cruz Biotechnology). Sections were incubated with secondary antibody conjugated with the peroxidase micropolymers (Vector Laboratories Inc.), and signal was detected using DAB solution (Vector Laboratories). Tissues were counterstained with hematoxylin. Images were captured using QCapture PRO software (QImaging).

Mouse colonoscopy

Mouse colonoscopy was performed using a modified Olympus human choledochoscope, consisting of an Olympus Exera CV-160 camera system with an Olympus CHF B160 camera unit with an insertion diameter of 3 mm as described previously (17). Mice were anesthetized by intraperitoneal injection of ketamine/xylazine solution consisting of 0.6-mL ketamine (100 mg/mL), 0.4-mL xylazine (20 mg/mL), and 4-mL saline, and the mixture was injected in a volume of about 8 μL/g body weight, as described earlier (18). To clear intestinal contents, colons were flushed with sterile Hanks' balanced salt solution using an 18-G gavage needle inserted to a depth of 4 cm. The tip of the endoscope was inserted slowly into the colon to a maximum depth of 4 cm. Number of colon tumors was scored during the procedure.

Microbiome analyses

Fecal samples were collected from 20-week-old mice and stored at −80°C immediately after collection for subsequent microbiome analysis. Total bacterial DNA was extracted from fecal samples by using the Power Soil DNA Extraction Kit (MoBio Laboratories) according to manufacturer's instructions. Bacterial 16S rDNA was amplified using the 27F/534R primer set (27F 5′-AGAGTTTGATCCTGGCTCAG-3′, 534R 5′-ATTACCGCGGCTGCTGG-3′). PCR reactions were performed using the Phusion High-Fidelity PCR Mastermix (Invitrogen) with the following condition: 95°C for 2 minutes (1 cycle), 95°C for 20 seconds, 56°C for 30 seconds, and 72°C for 1 minute (30 cycles). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter) according to manufacturer's protocol. DNA sequencing was conducted on an Illumina Miseq using paired end 300 base reads according to manufacturer's protocols.

For the analyses, raw reads were filtered according to length and quality criteria. Filter-pass reads were assembled using Flash Assembly, for which the minimum overlap requirement is 30-bp and the maximum mismatch ratio is 10% (19). After assembly, chimeric sequences were removed using the USEARCH software on the basis of the UCHIME algorithm (20). Operational taxonomic units (OTU) were selected using the de novo OTU selection protocol with a 97% similarity threshold. Taxonomy assignments of OTUs were performed by comparing sequences to RDP classifier (cutoff = 0.5). A total of 548,254 assembled reads were generated for the 54 samples. On average, 10,153 reads per sample with range from 4,883 to 22,182 were obtained. To normalize the sequence depth of each sample, 4,883 reads (the minimum sample number of reads) were randomly picked from each sample for further analysis. The R-package “Phyloseq” was used for alpha and beta diversity analysis (21). The ‘reads’ abundance was log-transformed for comparison between groups.

Statistical analyses

For tumor studies, statistical analyses were performed using GraphPad Prism V software (GraphPad Software, Inc.). Data are shown as mean ± SEM. P values were calculated by the Student t test or one-way ANOVA with Bonferroni multiple comparison tests where appropriate as indicated in the figure legends. P < 0.05 was considered statistically significant. For microbiome analyses, a 2-sided Student t-test was used for significance testing for normally distributed variables. The Mann–Whitney U test was used for significance testing of variables that did not show a normal distribution. A Spearman correlation test was used for correlation analysis between gut bacteria and tumor number. The statistical tests and plotting were done in R with packages “plyr”, “ggplot2.”

Effects of walnuts on colon carcinogenesis using a purified diet (Study 1)

In Study 1, we tested the effects of walnuts added to a purified diet, AIN-76A, at increasing concentrations of 0%, 9.4%, 14.1%, and 18.8% by weight on colon carcinogenesis in A/J mice (Supplementary Table S1; Fig. 1A). As shown in Fig. 1B, control mice showed significant weight gain at higher concentrations of walnuts. Mice that received AOM exhibited only a minimal increase in body weight gain that was similar between groups (Fig. 1B). Colonoscopy was performed 8 weeks after the last injection of AOM. As shown in Fig. 1C, tumor development was confirmed in the AOM-treated mice in the distal (∼3 cm) portion of the colon. Tumor numbers were estimated by the colonoscopic images and showed a modest decline (33%, P = 0.35) in mice fed 9.4% walnuts (Fig. 1C).

Figure 1.

Effect of walnut supplementation on AOM-induced colon tumor formation assessed by colonoscopy (Study 1). A, experimental design for Study 1 is depicted. Fecal samples were collected at the time point indicated (circled number). B, body weight change with walnut supplementation (0%, 9.4%, 14.4%, and 18.8%) incorporated into AIN-76A diet, with or without AOM treatment. C, mouse colonoscopy identifying the presence and location of colon tumors at 8 weeks after AOM exposure is completed. The location of distal colon tumors are indicated by the arrows. Bar graph showing a quantification of tumor numbers visualized by colonoscopy (n = 5, 5, 3, 3, 5 for 0% without AOM, 0%, 9.4%, 14.4%, 18.8% with AOM, respectively). D, colon tumor formation (number, volume, and size) was evaluated 10 weeks after the last injection of AOM. E, AOM treatment resulted in the development of small colonic lesions, including ACF (i) and microadenomas (ii). AOM-induced colon tumors exhibited polypoid (iii) or flat (iv) morphologies. n = 12, 12, 11, 14 in 0%, 9.4%, 14.4%, and 18.8%, respectively. Bars indicate means ± SEM. N. normal; T, tumor.

Figure 1.

Effect of walnut supplementation on AOM-induced colon tumor formation assessed by colonoscopy (Study 1). A, experimental design for Study 1 is depicted. Fecal samples were collected at the time point indicated (circled number). B, body weight change with walnut supplementation (0%, 9.4%, 14.4%, and 18.8%) incorporated into AIN-76A diet, with or without AOM treatment. C, mouse colonoscopy identifying the presence and location of colon tumors at 8 weeks after AOM exposure is completed. The location of distal colon tumors are indicated by the arrows. Bar graph showing a quantification of tumor numbers visualized by colonoscopy (n = 5, 5, 3, 3, 5 for 0% without AOM, 0%, 9.4%, 14.4%, 18.8% with AOM, respectively). D, colon tumor formation (number, volume, and size) was evaluated 10 weeks after the last injection of AOM. E, AOM treatment resulted in the development of small colonic lesions, including ACF (i) and microadenomas (ii). AOM-induced colon tumors exhibited polypoid (iii) or flat (iv) morphologies. n = 12, 12, 11, 14 in 0%, 9.4%, 14.4%, and 18.8%, respectively. Bars indicate means ± SEM. N. normal; T, tumor.

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At 10 weeks after the last dose of AOM, all mice were sacrificed and examined for the presence of ACF and tumors. The number and size of ACF in the colons was not significantly affected by dietary walnut consumption in both males and females (Supplementary Fig. S1A). As shown in Fig. 1D, the number, volume, and size of colon tumors did not show significant differences between the walnut diet groups. However, there was a modest reduction in both the number (1.3-fold, P = 0.15) and volume (1.3-fold, P = 0.37) of tumors in mice that consumed the diet with 9.4% walnuts compared with mice on the control diet. Tumor protection was most pronounced in male mice, suggesting a gender-specific benefit of walnut consumption (Supplementary Fig. S1B). Furthermore, there was a modest but nonsignificant trend toward increased tumor number and size at higher concentrations of dietary walnuts (14.1% and 18.8%; Fig. 1D and Supplementary Fig. S1B). Overall, these results indicate that walnut supplementation may suppress both colon tumor initiation and promotion when consumed at lower concentrations, but these effects may be somewhat impaired by increased amounts of dietary walnuts. While AOM treatment induced the development of several distinct morphological subtypes of tumors (ACF, microadenomas and tumors), there was no obvious change in tumor morphology at 10 weeks after AOM in male mice fed 9.4% walnuts (Fig. 1E).

Effects of walnuts on colon carcinogenesis using the TWD (Study 2)

In Study 2, we used a TWD as a base diet and tested for the efficacy of walnuts during AOM-induced colon carcinogenesis (Supplementary Table S2 and Fig. 2A). The TWD is a modified diet that contains fat from multiple sources, which can reflect consumption patterns of a typical American (14). All fat sources in the basal diet for each experimental group have been reduced proportionately to contain approximately 35% total fat by calories in the complete diet. The concentration of walnuts was adjusted to 0%, 3.5%, 7%, and 14% (by weight), which included the recommended daily serving of walnuts, 56.6 g (2 ounce) per day, on the basis of a 2,000 total calorie diet (∼18% by calories; ref. 22).

Figure 2.

Effect of dietary walnut consumption on AOM-induced colon tumor formation (Study 2). A, experimental design for Study 2 is depicted. Fecal samples were collected at the time point indicated (circled number). B, body weight change with walnut supplementation (0%, 3.5%, 7.0%, and 14) incorporated into TWD, with or without AOM treatment. C, colon tumor formation (number, volume, and size) was evaluated 10 weeks after the last injection of AOM. n = 10 mice per group. Bars indicate means ± SEM.

Figure 2.

Effect of dietary walnut consumption on AOM-induced colon tumor formation (Study 2). A, experimental design for Study 2 is depicted. Fecal samples were collected at the time point indicated (circled number). B, body weight change with walnut supplementation (0%, 3.5%, 7.0%, and 14) incorporated into TWD, with or without AOM treatment. C, colon tumor formation (number, volume, and size) was evaluated 10 weeks after the last injection of AOM. n = 10 mice per group. Bars indicate means ± SEM.

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As shown in Fig. 2B, body weight gain of vehicle-treated control mice was not affected by the addition of 7% walnuts (10.5% of total energy from walnuts) compared with controls. Similar to our findings in Study 1, the addition of walnuts to the TWD did not affect the number of colonic ACF when measured 10 weeks after AOM treatment (Supplementary Fig. S2). There was a modest but nonsignificant reduction in the number of tumors in mice fed 7% walnuts compared with the 0% walnut group (1.4-fold, P = 0.13; Fig. 2C). Furthermore, when tumors were stratified by size, mice fed the 3.5% and 7% walnuts had a moderate shift toward smaller tumors (Fig. 2C).

Consistent with Study 1, the tumor-suppressive effects of walnuts were significantly more pronounced in male mice (Fig. 3). As shown in Fig. 3A, males fed 7% walnuts showed a significant reduction in the number of tumors (2.3-fold, P = 0.05) and the size of tumors (1.6-fold, P = 0.03, Fig. 3B) compared with mice fed 0% walnuts. Moreover, there was a significant reduction in the number of smaller tumors. As shown in Fig. 3C, 1- and 2-mm tumors were reduced by 73% and 50%, respectively, compared with the 0% walnut group. Surprisingly, these trends were not observed in female mice in each of the groups.

Figure 3.

Effect of walnut supplementation on AOM-induced colon tumor formation by gender (Study 2). Colon tumor formation in A/J mice fed TWD diet was enumerated by gender. Tumor number (A), volume (B), and size distribution (C) in males and females are shown. Bars represent the means ± SEM. n = 5 mice per group. *, Student t test, P < 0.05.

Figure 3.

Effect of walnut supplementation on AOM-induced colon tumor formation by gender (Study 2). Colon tumor formation in A/J mice fed TWD diet was enumerated by gender. Tumor number (A), volume (B), and size distribution (C) in males and females are shown. Bars represent the means ± SEM. n = 5 mice per group. *, Student t test, P < 0.05.

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Histologic examination of the tumor tissues confirmed that there were smaller tumors in male mice fed 7% walnuts; however, there were no significant differences in key markers involved in tumor promotion including Ki67, β-catenin, pStat3, and p21 (Supplementary Fig. S3). This observation suggested that other factors were involved in suppression of tumorigenesis.

Walnuts consumption increases the richness and diversity of the gut bacterial community

On the basis of accumulating evidence that the Mediterranean diet has beneficial effects on the gut microbiota, we next tested the possibility that walnut consumption might affect gut biodiversity and perhaps modify risks to the colon from carcinogen exposure. In the following analyses, we examined the bacterial DNA extracted from fecal samples collected from TWD-fed mice. As shown in Fig. 4A and 4B, the number of observed OTUs and the Shannon index were higher with a diet supplemented with walnuts, suggesting that walnut consumption increased bacterial richness and diversity. This effect was independent of AOM treatment. In fact, carcinogen treatment was associated with a decline of microbial richness in male mice with 0% walnuts included in the diet (Fig. 4A).

Figure 4.

A comparison of alpha diversity dependent upon carcinogen exposure and walnut consumption (Study 2). Fecal samples collected from mice maintained on TWD diet were analyzed. Observed OTUs (A) and Shannon index (B). The alpha diversity was calculated after normalizing the sequence depth to the same level (4,883 reads per sample). C, NMDS plot of the 54 samples. For easier visualization of the samples from the different treatment groups, the NMDS plot was divided into 4 panels. Left, includes samples treated with AOM; right, control samples treated with NaCl; top, female; bottom, male. Other group information is indicated by shape (walnut level: circle, walnut 0%; square, walnut 3.5%; diamond, walnut 7%; triangle, walnut 14%). D, comparison of within-group distance between the different treatment and diet groups. Data are shown as means ± SEM. Letters (L/M/H) denote significant differences as determined by one-way ANOVA between groups at different walnut level with carcinogen treatment. #, significant difference when comparing the vehicle control group with the carcinogen-treated group at the same 0% walnut level and of the same gender. #, P < 0.05; ##, P < 0.01. *, a significant difference between the 0% walnut group and the 7% walnut group, with the same gender and vehicle control treatment. *, P < 0.05; **, P < 0.01. &, a significant difference between male and female mice when comparing carcinogen treatment at the same walnut concentration. &, P < 0.05; &&, P < 0.01.

Figure 4.

A comparison of alpha diversity dependent upon carcinogen exposure and walnut consumption (Study 2). Fecal samples collected from mice maintained on TWD diet were analyzed. Observed OTUs (A) and Shannon index (B). The alpha diversity was calculated after normalizing the sequence depth to the same level (4,883 reads per sample). C, NMDS plot of the 54 samples. For easier visualization of the samples from the different treatment groups, the NMDS plot was divided into 4 panels. Left, includes samples treated with AOM; right, control samples treated with NaCl; top, female; bottom, male. Other group information is indicated by shape (walnut level: circle, walnut 0%; square, walnut 3.5%; diamond, walnut 7%; triangle, walnut 14%). D, comparison of within-group distance between the different treatment and diet groups. Data are shown as means ± SEM. Letters (L/M/H) denote significant differences as determined by one-way ANOVA between groups at different walnut level with carcinogen treatment. #, significant difference when comparing the vehicle control group with the carcinogen-treated group at the same 0% walnut level and of the same gender. #, P < 0.05; ##, P < 0.01. *, a significant difference between the 0% walnut group and the 7% walnut group, with the same gender and vehicle control treatment. *, P < 0.05; **, P < 0.01. &, a significant difference between male and female mice when comparing carcinogen treatment at the same walnut concentration. &, P < 0.05; &&, P < 0.01.

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To further examine changes to the microbial community structure, we performed diversity analysis using Nonmetric Multidimensional Scaling (NMDS). The NMDS plot showed a clear separation between male and female mice in the presence of AOM exposure and the absence of walnuts (Fig. 4C: males vs. females, AOM). It was apparent that AOM treatment specifically modified microbial diversity in male mice (Fig. 4C: AOM vs. control). However, the addition of walnuts to the diet eliminated this gender difference. Moreover, the microbial communities always clustered together following the inclusion of any concentration of walnuts added to the diet. These initial observations indicate that AOM treatment alone or dietary supplementation with walnuts causes significant alterations to the microbial community structure and that walnuts may exert a greater physiological impact to the male gut microbiome.

To examine the diversity of the bacterial community, we plotted the within-group distance for different concentrations of walnuts. The results showed that female mice had significantly higher values for both the AOM and saline control treatment groups than males, with the exception of mice in the AOM treatment group at a concentration of 7% walnuts (Fig. 4D: circle vs. triangle). The value was lower for both male and female mice at 7% walnuts. Similar to the results obtained for beta diversity analysis, male mice showed a significant difference by AOM treatment (Fig. 4D: triangle in AOM vs. control), confirming that male mice were more sensitive to carcinogen treatment with respect to the overall phenotype of the microbiome. Finally, the addition of walnuts in the control group resulted in an increase in diversity in males but not females (Fig. 4D: control), again showing the greater responsiveness of males to this diet.

Specific phylotypes were associated with walnut consumption and carcinogen exposure

To identify how the key phylotypes of gut bacteria respond to dietary and carcinogen exposures, a cluster analysis of the top 100 OTUs (95% of the total reads) was performed and the data are presented as a heatmap (Fig. 5A). The analyses showed that the OTUs clustered into 9 major phylogenetic clades (Fig. 5A: A–I). By comparing the abundance of each OTU clade between different groups, we identified clades that are specifically associated with carcinogen treatment (Clade A), walnut consumption (Clade B, C), and both variables (Clade E, F, and I). Representing 23.1% of the total reads that were generated, clade A is the most abundant group. Clade A consists entirely of one OTU (OTU 1) from the genus of Akkermansia. The abundance of this clade shows a significant increase in AOM-treated male mice compared with the NaCl-treated mice (Supplementary Fig. S4A).

Figure 5.

Association of specific phylotypes with walnut consumption and carcinogen exposure (Study 2). A, the OTU abundance was log-transformed to plot the cluster heatmap after normalization to the same sequence depth. Cluster analysis was performed using Euclidean distance and Ward method. Rows indicate OTUs. Columns indicate samples. Samples from different group were indicated by the color bar above the heat map (olive, NaCl-walnut 0%; silver, NaCl-walnut 7%; maroon, AOM-walnut 0%; red, AOM-walnut 3.5%; orange, AOM-walnut 7%; yellow, AOM-walnut 14%, navy, female; aqua, male). The heatmap shows a gradient color scale from red, indicating value = 0, to faint yellow, indicating value = 8 (color key on the top right). The samples from different group and gender were clearly separated. A total of 9 OTU clades were observed and indicated by characters in the tree on the left. The abundance of major OTU clusters responding to carcinogen exposure and walnut consumption (the abundance of clades was log-transformed). B, abundance of OTU cluster E in different groups. C, abundance of OTU cluster F in different groups. D, abundance of OTU cluster I in different groups. Differing letters (L/M/H) denote significant differences as determined by a one-way ANOVA among groups of AOM-treated mice fed different walnut concentrations. #, significant differences when comparing the vehicle-treated group with the carcinogen-treated group at the same 0% walnut level and gender-matched. #, P < 0.05; ##, P < 0.01. *, a significant difference between the 0% walnut group and 7% walnut group, gender-matched, and vehicle-treated. *, P < 0.05; **, P < 0.01.

Figure 5.

Association of specific phylotypes with walnut consumption and carcinogen exposure (Study 2). A, the OTU abundance was log-transformed to plot the cluster heatmap after normalization to the same sequence depth. Cluster analysis was performed using Euclidean distance and Ward method. Rows indicate OTUs. Columns indicate samples. Samples from different group were indicated by the color bar above the heat map (olive, NaCl-walnut 0%; silver, NaCl-walnut 7%; maroon, AOM-walnut 0%; red, AOM-walnut 3.5%; orange, AOM-walnut 7%; yellow, AOM-walnut 14%, navy, female; aqua, male). The heatmap shows a gradient color scale from red, indicating value = 0, to faint yellow, indicating value = 8 (color key on the top right). The samples from different group and gender were clearly separated. A total of 9 OTU clades were observed and indicated by characters in the tree on the left. The abundance of major OTU clusters responding to carcinogen exposure and walnut consumption (the abundance of clades was log-transformed). B, abundance of OTU cluster E in different groups. C, abundance of OTU cluster F in different groups. D, abundance of OTU cluster I in different groups. Differing letters (L/M/H) denote significant differences as determined by a one-way ANOVA among groups of AOM-treated mice fed different walnut concentrations. #, significant differences when comparing the vehicle-treated group with the carcinogen-treated group at the same 0% walnut level and gender-matched. #, P < 0.05; ##, P < 0.01. *, a significant difference between the 0% walnut group and 7% walnut group, gender-matched, and vehicle-treated. *, P < 0.05; **, P < 0.01.

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Walnut consumption was associated with enrichment of Clade B, regardless of carcinogen exposure or the concentration of walnuts in the diet. For mice treated with AOM, the lowest abundance of Clade B was found in the 0% walnut group in both males and females (Supplemental Fig. S4B). The relative abundance of Clade C was significantly lower in male mice treated with AOM compared with the vehicle control group at the 0% walnut level (Supplemental Fig. S4C). However, Clade C was observed to repopulate to comparable levels found in the other groups by the addition of walnuts to the TWD. The increase in Clade C observed in the walnut groups was also observed in both male and female mice treated with AOM, suggesting that Clade C is directly associated with walnut consumption (Supplemental Fig. S4C). Clade E appeared to be strongly associated with AOM treatment, as there were almost no reads identified in the vehicle-treated control mice (Fig. 5A and B). Moreover, the enrichment of Clade E in both male and female mice in the AOM-treatment groups was attenuated by the addition of walnuts to the diet. These observations indicate that Clade E is influenced by both carcinogen exposure and consumption of walnuts (Fig. 5B).

There was a phylotype that was significantly dependent upon the concentration of walnuts consumed. Clade F showed the lowest abundance in the 7% walnut group in both male and female mice treated with AOM (Fig. 5C). In the vehicle controls, Clade F was further reduced to near zero in both male and female mice at the 7% walnut concentration. Similarly, a significant modification to the abundance of the phylotype at a walnut concentration of 7% was found in Clade I (Fig. 5D). Clade I was highly enriched at 7% walnuts in both male and female mice regardless of carcinogen treatment. These observations clearly indicate that dietary consumption of 7% walnuts is capable of significantly affecting the levels of specific bacteria groups; for example, reducing (Clade F) and increasing (Clade I) specific bacterial populations. Furthermore, these changes to bacterial populations are modified by either carcinogen exposure and/or by the presence of tumors within the colon.

OTU Clade F is associated with reduced colon tumor development

To further examine the potential influence of the gut microbiota on colon tumor formation, we performed a correlation analysis between OTU clades and the number of tumors. As shown in Fig. 6, 2 OTU clades were correlated with tumor numbers. The abundance of OTU clade F was positively correlated with the number of tumors (r = 0.70; P = 0.05), especially with tumors of 1-mm diameter (r = 0.78; P = 0.02; Fig. 6A and B). A negative correlation was observed between the number of tumors and the abundance of OTU clade I (r = 0.66, P = 0.08; Fig. 6C). Furthermore, a stronger negative correlation was found between the abundance of OTU clade I and tumors of 2-mm diameter in size (r = 0.81, P = 0.01; Fig. 6D). Taken together, these analyses suggest that a reduction in clade F may be an important event that contributes to the suppression of tumor initiation. In addition, clade I may be necessary for suppression of tumor promotion.

Figure 6.

Identification of a specific bacterial signature that are strongly associated with tumor suppression by walnut consumption (Study 2). Correlation analysis OTU clade F and tumor numbers. A, abundance of OTU clade F and the total number of tumors. B, abundance of OTU clade F and number of tumors with a diameter of 1 mm. C, abundance of OTU clade I and total number of tumors. D, abundance of OTU clade I and number of tumors with a diameter of 2 mm.

Figure 6.

Identification of a specific bacterial signature that are strongly associated with tumor suppression by walnut consumption (Study 2). Correlation analysis OTU clade F and tumor numbers. A, abundance of OTU clade F and the total number of tumors. B, abundance of OTU clade F and number of tumors with a diameter of 1 mm. C, abundance of OTU clade I and total number of tumors. D, abundance of OTU clade I and number of tumors with a diameter of 2 mm.

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The concept of exploiting whole foods for their chemopreventive benefit has gained significant traction in recent years (23). Whole foods are composed of a wide array of beneficial nutrients that together may have additive or synergistic properties that contribute to reduced cancer risk. Walnuts may be a particularly attractive whole food approach for cancer prevention. They are readily available, widely consumed, and contain an excellent profile of bioactive components that may exert complex and synergistic effects on tumorigenesis (16, 22, 24–30).

The present study has evaluated the impact of dietary walnut consumption on colon carcinogenesis using a well-established mouse cancer model. Importantly, we used 2 fundamentally distinct basal diets to evaluate the potential contribution of dietary patterns to the nutritive effects of walnuts. AIN-76A diet is a purified diet containing micronutrient profiles optimized for growth and fertility and is widely used in preclinical research to provide “standardized” results among different animal experiments (31). On the other hand, as reported by Hintze and colleagues (14), TWD is formulated to recapitulate the typical American intake of macro- and micronutrients and can be used for dietary chemopreventive studies in rodent models of human disease. Compared with the AIN-76A diet, TWD contains less fat but uses a diverse set of sources to match patterns of fat consumption as reported by the National Health and Nutrition Examination Survey (NHANES), resulting a wide range of dietary fatty acids (14). Interestingly, the TWD diet alone did not cause a significant increase in tumor numbers compared with the AIN-76A diet, which is likely due to the potency of AOM treatment in strain A mice. As we have reported in previous studies (13), strain A mice are highly sensitive to AOM-induced colon carcinogenesis, with up to 40 adenomas developing in the distal colon. On this potent cancer background, the promotional effects of TWD are likely obscured.

On the basis of the 2 studies reported herein, walnut consumption at dietary concentrations of 7% and 9.4% by weight, equivalent to 10% to 15% of total caloric intake, afforded protection against colon cancer. These concentrations are equivalent to the recommended daily serving of walnuts, 56.6 g (2 ounce) per day, on the basis of a 2,000 total calorie diet (∼18% by calories; ref. 22). A higher walnut concentration added to the AIN-76A diet (18.8% by weight; 20% to 30% of total caloric intake) caused significant weight gain in control mice but did not protect against AOM-induced colon tumor development (Figs. 1B and 3A). This result, although not statistically significant, suggests an optimal intake of walnuts for colon cancer protection. A higher walnut concentration added to the AIN-76A diet (18.8% by weight; 20% to 30% of total caloric intake) caused significant weight gain in control mice but did not protect against AOM-induced colon tumor development (Figs. 1B and 3A). This result, although not statistically significant, suggests an optimal intake of walnuts for colon cancer protection.

The concentration-dependent effect of walnuts in Study 1 may be related to a pronounced alteration in the levels of oleic acid that was reduced by about 50% at the highest concentration of walnuts (18.8%; Supplementary Table S1). Oleic acid, also referred to as omega-9 fatty acid, is a major constituent of olive oil that has been shown to promote clearance of excess fatty acids to promote “weight loss” (32) and to possess antitumor activity (33). In a recent study, oleic acid was shown to inhibit TNFα-induced COX-2 expression and PGE2 production in a human glioblastoma cell line (34). It was also reported that the levels of palmitoleic acid and oleic acid were significantly lower in colorectal cancer tissues (35). These results suggest that similar to omega-3 fatty acids, the levels of oleic acid may also be important for both weight control and cancer risk.

Importantly, our results indicate both concentration- and gender-specific effects of walnuts. As shown in Fig. 3, at optimal dietary concentrations of walnuts, male mice exhibited greater protection from colon cancer compared with females. This gender-related effect was observed when walnuts were incorporated into either the AIN-76A or TWD. Although 7% walnut consumption resulted in a 2.3-fold reduction in the number of tumors in male mice fed TWD, there was only a moderate effect on tumor volume, suggesting a more pronounced effect on tumor initiation in this carcinogenesis model.

The protection afforded by walnuts may have its basis in a number of nutritive components contained within the nut. For example, the omega-3 PUFAs are a major component of walnuts, and the long-chain omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to reduce adenomatous polyp number and size in patients with familial adenomatous polyposis (FAP; ref. 36). Moreover, γ-tocopherol is a potent antioxidant with anti-inflammatory activities and is found in significant concentrations in walnuts (12). Ju and colleagues (37) showed that dietary supplementation of 0.3% γ-tocopherol–rich mixture (γ-TmT) on AOM/DSS-treated mice reduced inflammation and inhibited colon tumorigenesis by reducing the levels of PGE2, leukotriene B4, and nitrotyrosine in the colon (37). Polyphenols in walnuts, mainly ellagitannins (ET), also elicit anticancer properties. Dietary ETs are metabolized by gut flora to urolithin A & B (38). In a prostate cancer cell line, urolithin A was reported to induce apoptosis, associated with a decrease in the antiapoptotic protein BCL-2 with increased antiproliferative p21 levels (39). Further molecular analysis on tumor-suppressive mechanisms within the walnut-treated tissues is warranted to better understand the roles of these components.

Emerging evidence indicates profound effects of diet on the gut microbiota (reviewed in ref. 40). Dietary changes can cause dramatic and rapid alterations to bacterial community structure, leading to important alterations in the luminal formation of a wide range of microbial metabolites (41). Early stages of tumor development, particularly at the initiation phase, can be influenced by microbial community structure, mediated in part via the formation of key metabolic products (42). For example, bacteria utilize dietary fiber to produce short-chain fatty acids (SCFA), such as acetate, propionate, and butyrate that have anti-inflammatory properties (reviewed in ref. 43). In fact, walnuts are rich in fiber (6.4%), providing a favorable luminal environment for producing the SCFAs. In the present study, we observed significant alterations to the structure of the gut microbial community by the addition of walnuts to the diet. At each concentration of walnuts tested, there was a marked increase in the diversity and richness of the gut microbiota. This finding underscores the beneficial effects of dietary walnut intake because low bacterial diversity in the gut has often been linked to human diseases, including obesity and inflammatory bowel disease (44, 45).

The walnut-supplemented diets influenced multiple phylotypes. For example, Akkermansia muciniphila is a mucin-degrading commensal bacterium that has been shown to exacerbate intestinal inflammation by disturbing mucosal barrier function (46). In fact, members of this genus have also been found at higher concentrations in patients with colorectal cancer (47) and in mice harboring AOM/DSS-induced colon tumors (48). As shown in Fig. 5A, the Akkermansia genus was exclusively associated with mice exposed to AOM and harboring colon tumors (clade A). Thus, it is possible that A. muciniphila may play an as yet undefined role in colon tumor development. In contrast, we report that walnut consumption is associated with an increased abundance of bacteria belonging to the phylum Furmicutes, including Lactobacillus, Clostridiales, Clostridium, Lachnospiraceae, and Ruminococcaceae. Furmicutes are the most prevalent bacteria within both the human and rodent gut, and many members of this phylum tend to be reduced in patients with colorectal cancer, as well as in the colons of tumor-bearing mice (49). In a recent study by Baxter and colleagues (49), fecal microbiota from patients with colorectal cancer were transplanted into germ-free mice and tumors were then induced by treatment with AOM followed by DSS. Interestingly, the number of colon tumors that developed after 10 weeks was negatively correlated with the abundance of Clostridiales, including several members of the Clostridium Group XIVa (49). Moreover, Lactobacillus and Ruminococcaceae are probiotic species that are generally found at lower levels in both rodent tumor models and patients with colorectal cancer (50, 51). Consistent with these results, similar bacterial signatures were found to be strongly associated with carcinogen treatment (Fig. 5).

While it is clear that walnut consumption alone can reshape the gut community structure into one that has potential antitumor properties, phylotypes associated with both carcinogen exposure and walnut consumption formed a distinct cluster. In addition to phylotypes associated with walnut consumption, many bacteria that belong to the phylum Bacteroidetes have also emerged within this group, including Bacteroides, Bacteroidales, and Porphyromonadaceae. Bacteroidetes are anaerobic bacteria and are present as the second largest bacterial population in the gut after Furmicutes. In fact, many members of the genus Bacteroidales are increased in both rodent models and human subjects (52). Furthermore, a higher colonization rate of Bacteroides has been correlated with advanced colorectal cancer status, suggesting that some bacterial species in this genus may play a direct role in promoting colon carcinogenesis (53).

Our correlation analysis depicted in Fig. 6 identified the presence of a potential microbial signature that could be directly associated with walnut consumption and colon cancer suppression in male mice. Mice harboring the lowest number of tumors tended to have a reduced abundance of the Bactroidetes and Lachnospiraceaes family. These changes were associated with an overpopulation of Ruminococcaceae and Clostridium XIVa genus. This bacterial signature is similar to that previously reported during inflammation-associated colon tumorigenesis in mice (49). This recent study also suggested that distinct bacterial communities would result in distinct luminal metabolite profiles and that butyrate production by members of Clostridium XIVa could contribute to cancer protection (49). Butyrate is a major energy source for colonocytes and a critical mediator of the inflammatory response in the gut (54). It is also potent inhibitor of histone deacetylase (HDAC), which enhances apoptosis and suppresses intestinal inflammation (55, 56). During colon carcinogenesis, butyrate has been shown to inhibit cell proliferation (57). Interestingly, it was recently reported that walnuts were among the most highly effective nuts in generating butyrate during the fermentation process in vitro (58). Thus, it is possible that dietary walnut intake can increase populations of butyrate-producing bacteria, inducing a microbial community structure that is antitumorigenic within the gut.

The present study has further established the existence of marked differences in microbiome signatures between male and female mice. For example, male mice have a significantly lower overall bacterial diversity in the absence of walnut supplementation compared with female mice (Fig. 3). Upon walnut consumption, however, bacterial diversity increases in the male mice, ultimately achieving comparable levels that are present in females. These results suggest that male mice may be more sensitive to diet-induced changes, enabling rapid modifications to their microbial composition. In fact, gender-specific differences in disease susceptibility have been reported in relation to gut microbial community structure. For example, colonization of male commensal bacteria into female mice protected against Type 1 diabetes by changing the levels of testosterone and metabolic profiles generated by the microbiota (59). Alterations to the composition of the gut microbiome and bacterial metabolite profiles can further influence disease risk (60, 61). Therefore, it is possible that the male mice in our study underwent beneficial diet-induced changes to microbial composition, a change that contributed to the establishment of a protective luminal environment.

In summary, our results demonstrate that dietary walnut consumption can suppress colon carcinogenesis when provided at optimal concentrations in the diet, protection that is associated with both AIN-76A and a modified Western diet formulation. In addition, walnut intake significantly modifies the microbial community structure in the large intestine, potentially establishing a protective luminal microenvironment. Most importantly, we have identified a unique bacterial signature that is associated with tumor suppression by walnut consumption. Additional studies are required to confirm these results and to elucidate the possible mechanisms by which walnut dietary supplementation may contribute to protection against colon cancer development.

No potential conflicts of interest were disclosed.

Conception and design: M. Nakanishi, G.M. Weinstock, D.W. Rosenberg

Development of methodology: M. Nakanishi, Y. Chen, G.M. Weinstock, D.W. Rosenberg

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Nakanishi, Y. Chen, E. Weinstock, G.M. Weinstock, D.W. Rosenberg

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Nakanishi, Y. Chen, V. Qendro, G.M. Weinstock, D.W. Rosenberg

Writing, review, and/or revision of the manuscript: M. Nakanishi, Y. Chen, G.M. Weinstock, D.W. Rosenberg

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Chen, S. Miyamoto, G.M. Weinstock, D.W. Rosenberg

Study supervision: M. Nakanishi, D.W. Rosenberg

Other [Covered part of the wet lab work (generation of library and sequencing) and the analysis of the data generated]: V. Qendro

Other (contributed to discussions regarding data analysis and data generation of themicrobiomedata): E. Weinstock

The authors thank Nicole Horelik and Yuichi Igarashi for their technical assistance.

This study was financially supported by American Institute for Cancer Research (AICR; D.W. Rosenberg) and the California Walnut Commission. The content is solely the responsibility of the authors and does not necessarily represent the official views of the AICR and the California Walnut Commission; sponsors had no input on interpretation or reporting of findings.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Hawk
ET
,
Levin
B
. 
Colorectal cancer prevention
.
J Clin Oncol
2005
;
23
:
378
91
.
2.
Rudolph
RE
,
Dominitz
JA
,
Lampe
JW
,
Levy
L
,
Qu
P
,
Li
SS
, et al
Risk factors for colorectal cancer in relation to number and size of aberrant crypt foci in humans
.
Cancer Epidemiol Biomarkers Prev
2005
;
14
:
605
8
.
3.
Stevens
RG
,
Swede
H
,
Rosenberg
DW
. 
Epidemiology of colonic aberrant crypt foci: review and analysis of existing studies
.
Cancer Lett
2007
;
252
:
171
83
.
4.
Turini
ME
,
DuBois
RN
. 
Primary prevention: phytoprevention and chemoprevention of colorectal cancer
.
Hematol Oncol Clin North Am
2002
;
16
:
811
40
.
5.
Castro-Quezada
I
,
Roman-Vinas
B
,
Serra-Majem
L
. 
The Mediterranean diet and nutritional adequacy: a review
.
Nutrients
2014
;
6
:
231
48
.
6.
Ostan
R
,
Lanzarini
C
,
Pini
E
,
Scurti
M
,
Vianello
D
,
Bertarelli
C
, et al
Inflammaging and cancer: a challenge for the Mediterranean diet
.
Nutrients
2015
;
7
:
2589
621
.
7.
Del Chierico
F
,
Vernocchi
P
,
Dallapiccola
B
,
Putignani
L
. 
Mediterranean diet and health: food effects on gut microbiota and disease control
.
Int J Mol Sci
2014
;
15
:
11678
99
.
8.
De Filippis
F
,
Pellegrini
N
,
Vannini
L
,
Jeffery
IB
,
La Storia
A
,
Laghi
L
, et al
High-level adherence to a Mediterranean diet beneficially impacts the gut microbiota and associated metabolome
.
Gut.
2015
Sep 28.
[Epub ahead of print]
.
9.
Hayes
D
,
Angove
MJ
,
Tucci
J
,
Dennis
C
. 
Walnuts (Juglansregia) chemical composition and research in human health
.
Crit Rev Food Sci Nutr
2016
;56
:1231–41
.
10.
Falasca
M
,
Casari
I
,
Maffucci
T
. 
Cancer chemoprevention with nuts
.
J Natl Cancer Inst
2014
;
106
.
pii: dju238
.
11.
Cockbain
AJ
,
Toogood
GJ
,
Hull
MA
. 
Omega-3 polyunsaturated fatty acids for the treatment and prevention of colorectal cancer
.
Gut
2012
;
61
:
135
49
.
12.
Yang
CS
,
Li
G
,
Yang
Z
,
Guan
F
,
Chen
A
,
Ju
J
. 
Cancer prevention by tocopherols and tea polyphenols
.
Cancer Lett
2013
;
334
:
79
85
.
13.
Rosenberg
DW
,
Giardina
C
,
Tanaka
T
. 
Mouse models for the study of colon carcinogenesis
.
Carcinogenesis
2009
;
30
:
183
96
.
14.
Hintze
KJ
,
Benninghoff
AD
,
Ward
RE
. 
Formulation of the Total Western Diet (TWD) as a basal diet for rodent cancer studies
.
J Agric Food Chem
2012
;
60
:
6736
42
.
15.
Hardman
WE
,
Ion
G
. 
Suppression of implanted MDA-MB 231 human breast cancer growth in nude mice by dietary walnut
.
Nutr Cancer
2008
;
60
:
666
74
.
16.
Nagel
JM
,
Brinkoetter
M
,
Magkos
F
,
Liu
X
,
Chamberland
JP
,
Shah
S
, et al
Dietary walnuts inhibit colorectal cancer growth in mice by suppressing angiogenesis
.
Nutrition
2012
;
28
:
67
75
.
17.
Nakanishi
M
,
Menoret
A
,
Belinsky
GS
,
Giardina
C
,
Godman
CA
,
Vella
AT
, et al
Utilizing endoscopic technology to reveal real-time proteomic alterations in response to chemoprevention
.
Proteomics Clin Appl
2007
;
1
:
1660
6
.
18.
Becker
C
,
Fantini
MC
,
Neurath
MF
. 
High resolution colonoscopy in live mice
.
Nat Protoc
2006
;
1
:
2900
4
.
19.
Magoc
T
,
Salzberg
SL
. 
FLASH: fast length adjustment of short reads to improve genome assemblies
.
Bioinformatics
2011
;
27
:
2957
63
.
20.
Edgar
RC
,
Haas
BJ
,
Clemente
JC
,
Quince
C
,
Knight
R
. 
UCHIME improves sensitivity and speed of chimera detection
.
Bioinformatics
2011
;
27
:
2194
200
.
21.
McMurdie
PJ
,
Holmes
S
. 
phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data
.
PLoS ONE
2013
;
8
:
e61217
.
22.
Hardman
WE
. 
Walnuts have potential for cancer prevention and treatment in mice
.
J Nutr
2014
;
144
:
555S
60S
.
23.
Mehta
RG
,
Murillo
G
,
Naithani
R
,
Peng
X
. 
Cancer chemoprevention by natural products: how far have we come?
Pharm Res
2010
;
27
:
950
61
.
24.
Soriano-Hernandez
AD
,
Madrigal-Perez
DG
,
Galvan-Salazar
HR
,
Arreola-Cruz
A
,
Briseno-Gomez
L
,
Guzman-Esquivel
J
, et al
The protective effect of peanut, walnut, and almond consumption on the development of breast cancer
.
Gynecol Obstet Invest
2015
;
80
:
89
92
.
25.
Kim
H
,
Yokoyama
W
,
Davis
PA
. 
TRAMP prostate tumor growth is slowed by walnut diets through altered IGF-1 levels, energy pathways, and cholesterol metabolism
.
J Med Food
2014
;
17
:
1281
6
.
26.
Le
V
,
Esposito
D
,
Grace
MH
,
Ha
D
,
Pham
A
,
Bortolazzo
A
, et al
Cytotoxic effects of ellagitannins isolated from walnuts in human cancer cells
.
Nutr Cancer
2014
;
66
:
1304
14
.
27.
Reiter
RJ
,
Tan
DX
,
Manchester
LC
,
Korkmaz
A
,
Fuentes-Broto
L
,
Hardman
WE
, et al
A walnut-enriched diet reduces the growth of LNCaP human prostate cancer xenografts in nude mice
.
Cancer Invest
2013
;
31
:
365
73
.
28.
Vanden Heuvel
JP
,
Belda
BJ
,
Hannon
DB
,
Kris-Etherton
PM
,
Grieger
JA
,
Zhang
J
, et al
Mechanistic examination of walnuts in prevention of breast cancer
.
Nutr Cancer
2012
;
64
:
1078
86
.
29.
Davis
PA
,
Vasu
VT
,
Gohil
K
,
Kim
H
,
Khan
IH
,
Cross
CE
, et al
A high-fat diet containing whole walnuts (Juglans regia) reduces tumour size and growth along with plasma insulin-like growth factor 1 in the transgenic adenocarcinoma of the mouse prostate model
.
The Br J Nutr
2012
;
108
:
1764
72
.
30.
Hardman
WE
,
Ion
G
,
Akinsete
JA
,
Witte
TR
. 
Dietary walnut suppressed mammary gland tumorigenesis in the C(3)1 TAg mouse
.
Nutr Cancer
2011
;
63
:
960
70
.
31.
Report of the American Institute of Nurtition ad hoc Committee on Standards for Nutritional Studies
.
J Nutr
1977
;
107
:
1340
8
.
32.
Lim
JH
,
Gerhart-Hines
Z
,
Dominy
JE
,
Lee
Y
,
Kim
S
,
Tabata
M
, et al
Oleic acid stimulates complete oxidation of fatty acids through protein kinase A-dependent activation of SIRT1-PGC1alpha complex
.
J Biol Chem
2013
;
288
:
7117
26
.
33.
Owen
RW
,
Haubner
R
,
Wurtele
G
,
Hull
E
,
Spiegelhalder
B
,
Bartsch
H
. 
Olives and olive oil in cancer prevention
.
Eur J Cancer Prev
2004
;
13
:
319
26
.
34.
Lamy
S
,
Ben Saad
A
,
Zgheib
A
,
Annabi
B
. 
Olive oil compounds inhibit the paracrine regulation of TNF-alpha-induced endothelial cell migration through reduced glioblastoma cell cyclooxygenase-2 expression
.
J Nutr Biochem
2016
;
27
:
136
45
.
35.
Zhang
J
,
Zhang
L
,
Ye
X
,
Chen
L
,
Gao
Y
,
Kang
JX
, et al
Characteristics of fatty acid distribution is associated with colorectal cancer prognosis
.
Prostaglandins Leukot Essent Fatty Acids
2013
;
88
:
355
60
.
36.
Hull
MA
. 
Nutritional agents with anti-inflammatory properties in chemoprevention of colorectal neoplasia
.
Recent Results Cancer Res
2013
;
191
:
143
56
.
37.
Ju
J
,
Hao
X
,
Lee
MJ
,
Lambert
JD
,
Lu
G
,
Xiao
H
, et al
A gamma-tocopherol-rich mixture of tocopherols inhibits colon inflammation and carcinogenesis in azoxymethane and dextran sulfate sodium-treated mice
.
Cancer Prev Res (Phila)
2009
;
2
:
143
52
.
38.
Garcia-Munoz
C
,
Vaillant
F
. 
Metabolic fate of ellagitannins: implications for health, and research perspectives for innovative functional foods
.
Crit Rev Food Sci Nutr
2014
;
54
:
1584
98
.
39.
Sanchez-Gonzalez
C
,
Ciudad
C
,
Noe
V
,
Izquierdo-Pulido
M
. 
Health benefits of walnut polyphenols: An exploration beyond their lipid profile
.
Crit Rev Food Sci Nutr.
2015
Sep 29.
[Epub ahead of print]
.
40.
Albenberg
LG
,
Wu
GD
. 
Diet and the intestinal microbiome: associations, functions, and implications for health and disease
.
Gastroenterology
2014
;
146
:
1564
72
.
41.
Turnbaugh
PJ
,
Ridaura
VK
,
Faith
JJ
,
Rey
FE
,
Knight
R
,
Gordon
JI
. 
The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice
.
Sci Transl Med
2009
;
1
:
6ra14
.
42.
Manzat-Saplacan
RM
,
Mircea
PA
,
Balacescu
L
,
Chira
RI
,
Berindan-Neagoe
I
,
Balacescu
O
. 
Can we change our microbiome to prevent colorectal cancer development?
Acta Oncologica
2015
;
54
:
1085
95
.
43.
Louis
P
,
Hold
GL
,
Flint
HJ
. 
The gut microbiota, bacterial metabolites and colorectal cancer
.
Nat Rev Microbiol
2014
;
12
:
661
72
.
44.
Qin
J
,
Li
R
,
Raes
J
,
Arumugam
M
,
Burgdorf
KS
,
Manichanh
C
, et al
A human gut microbial gene catalogue established by metagenomic sequencing
.
Nature
2010
;
464
:
59
65
.
45.
Turnbaugh
PJ
,
Hamady
M
,
Yatsunenko
T
,
Cantarel
BL
,
Duncan
A
,
Ley
RE
, et al
A core gut microbiome in obese and lean twins
.
Nature
2009
;
457
:
480
4
.
46.
Ganesh
BP
,
Klopfleisch
R
,
Loh
G
,
Blaut
M
. 
Commensal Akkermansia muciniphila exacerbates gut inflammation in Salmonella Typhimurium-infected gnotobiotic mice
.
PLoS ONE
2013
;
8
:
e74963
.
47.
Weir
TL
,
Manter
DK
,
Sheflin
AM
,
Barnett
BA
,
Heuberger
AL
,
Ryan
EP
. 
Stool microbiome and metabolome differences between colorectal cancer patients and healthy adults
.
PLoS ONE
2013
;
8
:
e70803
.
48.
Zackular
JP
,
Baxter
NT
,
Iverson
KD
,
Sadler
WD
,
Petrosino
JF
,
Chen
GY
, et al
The gut microbiome modulates colon tumorigenesis
.
MBio
2013
;
4
:
e00692
13
.
49.
Baxter
NT
,
Zackular
JP
,
Chen
GY
,
Schloss
PD
. 
Structure of the gut microbiome following colonization with human feces determines colonic tumor burden
.
Microbiome
2014
;
2
:
20
.
50.
Mira-Pascual
L
,
Cabrera-Rubio
R
,
Ocon
S
,
Costales
P
,
Parra
A
,
Suarez
A
, et al
Microbial mucosal colonic shifts associated with the development of colorectal cancer reveal the presence of different bacterial and archaeal biomarkers
.
J Gastroenterol
2015
;
50
:
167
79
.
51.
Zhu
Q
,
Jin
Z
,
Wu
W
,
Gao
R
,
Guo
B
,
Gao
Z
, et al
Analysis of the intestinal lumen microbiota in an animal model of colorectal cancer
.
PLoS ONE
2014
;
9
:
e90849
.
52.
Marchesi
JR
,
Dutilh
BE
,
Hall
N
,
Peters
WH
,
Roelofs
R
,
Boleij
A
, et al
Towards the human colorectal cancer microbiome
.
PLoS ONE
2011
;
6
:
e20447
.
53.
Wu
N
,
Yang
X
,
Zhang
R
,
Li
J
,
Xiao
X
,
Hu
Y
, et al
Dysbiosis signature of fecal microbiota in colorectal cancer patients
.
Microb Ecol
2013
;
66
:
462
70
.
54.
Velazquez
OC
,
Lederer
HM
,
Rombeau
JL
. 
Butyrate and the colonocyte. Implications for neoplasia
.
Dig Dis Sci
1996
;
41
:
727
39
.
55.
Mariadason
JM
. 
HDACs and HDAC inhibitors in colon cancer
.
Epigenetics
2008
;
3
:
28
37
.
56.
Davie
JR
. 
Inhibition of histone deacetylase activity by butyrate
.
J Nutr
2003
;
133
:
2485S
93S
.
57.
Hamer
HM
,
Jonkers
D
,
Venema
K
,
Vanhoutvin
S
,
Troost
FJ
,
Brummer
RJ
. 
Review article: the role of butyrate on colonic function
.
Aliment Pharmacol Ther
2008
;
27
:
104
19
.
58.
Schlormann
W
,
Birringer
M
,
Lochner
A
,
Lorkowski
S
,
Richter
I
,
Rohrer
C
, et al
In vitro fermentation of nuts results in the formation of butyrate and c9,t11 conjugated linoleic acid as chemopreventive metabolites
.
Eur J Nutr.
2015
Aug 19.
[Epub ahead of print]
.
59.
Markle
JG
,
Frank
DN
,
Mortin-Toth
S
,
Robertson
CE
,
Feazel
LM
,
Rolle-Kampczyk
U
, et al
Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity
.
Science
2013
;
339
:
1084
8
.
60.
Nicholson
JK
,
Holmes
E
,
Kinross
J
,
Burcelin
R
,
Gibson
G
,
Jia
W
, et al
Host-gut microbiota metabolic interactions
.
Science
2012
;
336
:
1262
7
.
61.
Clemente
JC
,
Ursell
LK
,
Parfrey
LW
,
Knight
R
. 
The impact of the gut microbiota on human health: an integrative view
.
Cell
2012
;
148
:
1258
70
.