Antioxidant and Anti-Inflammatory Properties of Walnut Constituents: Focus on Personalized Cancer Prevention and the Microbiome.

Walnuts have been lauded as a 'superfood', containing a remarkable array of natural constituents that may have additive and/or synergistic properties that contribute to reduced cancer risk. Walnuts are a rich source of polyunsaturated fatty acids (PUFAs: alpha-linolenic acid, ALA), tocopherols, antioxidant polyphenols (including ellagitannins), and prebiotics, including fiber (2 g/oz). There is a growing body of evidence that walnuts may contribute in a positive way to the gut microbiome, having a prebiotic potential that promotes the growth of beneficial bacteria. Studies supporting this microbiome-modifying potential include both preclinical cancer models as well as several promising human clinical trials. Mediated both directly and indirectly via its actions on the microbiome, many of the beneficial properties of walnuts are related to a range of anti-inflammatory properties, including powerful effects on the immune system. Among the most potent constituents of walnuts are the ellagitannins, primarily pedunculagin. After ingestion, the ellagitannins are hydrolyzed at low pH to release ellagic acid (EA), a non-flavonoid polyphenolic that is subsequently metabolized by the microbiota to the bioactive urolithins (hydroxydibenzo[b,d]pyran-6-ones). Several urolithins, including urolithin A, reportedly have potent anti-inflammatory properties. These properties of walnuts provide the rationale for including this tree nut as part of a healthy diet for reducing overall disease risk, including colorectal cancer. This review considers the latest information regarding the potential anti-cancer and antioxidant properties of walnuts and how they may be incorporated into the diet to provide additional health benefits.

Effect of Nuts on Gastrointestinal Health.

Nuts are high nutrient-dense foods containing healthy lipids, dietary fiber, and bioactive phytochemicals, including vitamins and minerals. Although the beneficial effect of nut consumption on different chronic diseases has been well documented, especially in relation to their cardiometabolic benefits, less scientific evidence is available on their possible beneficial effects on gastrointestinal health. In this narrative review, we summarize the most important findings and new research perspectives in relation to the importance of nut consumption on gastrointestinal health. The integrity of the cell wall structure, cell size and particle size after mastication are known to play a crucial role in energy, nutrient and bioactive release from nuts during digestion, therefore affecting bioaccessibility. Other mechanisms, such as cell wall composition, thickness and porosity, as well as stability of the membranes surrounding the oil bodies within the cell, are also important for energy extraction. As the undigested nutrients and phytochemicals are delivered to the colon, effects on gut microbiota composition are predicted. Although the overall effect of nut consumption on microbial alpha- and beta-diversity has been inconsistent, some scientific evidence suggests an increase in fecal butyrate after almond consumption, and a beneficial role of walnuts on the prevention of ulcerative colitis and protection against the development of gastric mucosal lesions.

Combination of naproxen and a chemically-stable eicosapentaenoic acid analog provide additive tumor protection in Pirc rats.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. Patients with the genetic disorder Familial Adenomatous Polyposis (FAP) develop hundreds to thousands of polyps that unless removed by prophylactic colectomy will progress to CRC at an early age. Nonsteroidal anti-inflammatory drugs (NSAIDs) and the ω-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA), have been evaluated for their chemopreventive potential in delaying CRC onset in high-risk patients. In our study, we determined whether the NSAID, naproxen, alone or in combination with a chemically-stable EPA analog (TP-252), affects tumor formation in the ApcPirc rat model. When compared to control diet, animals fed naproxen or HD TP-252 had 66% and 82% fewer tumors, respectively. However, animals fed a combination of naproxen and HD TP-252, exhibited a 95% reduction in tumor formation and a 98% reduction in tumor volume, respectively. To elucidate potential mechanisms of tumor protection, a comprehensive, targeted lipidomic analysis was performed on colonic mucosa to determine changes in eicosanoid metabolism. Animals receiving TP-252 alone or in combination with naproxen had significantly reduced mucosal levels of proinflammatory ω-6 eicosanoids (PGE2 , 5-HETE and 14,15-DiHETrE), along with a simultaneous increase in anti-inflammatory EPA-derived ω-3 eicosanoids. A comprehensive lipidomic analysis also uncovered several potential pharmacodynamic (PD) lipid biomarkers, including resolvin E2, 9-HEPE, 12-HEPE and 18-HEPE, that were significantly correlated with tumor protection. Further studies with this drug combination should be focused on dose optimization and the role of EPA-derived lipid mediators in CRC initiation and progression.

The Epithelial-Stromal Microenvironment in Early Colonic Neoplasia.

Stromal cells play a central role in promoting the progression of colorectal cancer. Here, we analyze molecular changes within the epithelial and stromal compartments of dysplastic aberrant crypt foci (ACF) formed in the ascending colon, where rapidly developing interval cancers occur. We found strong activation of numerous neutrophil/monocyte chemokines, consistent with localized inflammation. The data also indicated a decrease in interferon signaling and cell-based immunity. The immune checkpoint and T-cell exhaustion gene PDCD1 was one of the most significantly upregulated genes, which was accompanied by a decrease in cytotoxic T-cell effector gene expression. In addition, CDKN2A expression was strongly upregulated in the stroma and downregulated in the epithelium, consistent with diverse changes in senescence-associated signaling on the two tissue compartments. IMPLICATIONS: Decreased CD8 T-cell infiltration within proximal colon ACF occurs within the context of a robust inflammatory response and potential stromal cell senescence, thus providing new insight into potential promotional drivers for tumors in the proximal colon.

Fatty acid metabolism and colon cancer protection by dietary methyl donor restriction.

INTRODUCTION: A methyl donor depleted (MDD) diet dramatically suppresses intestinal tumor development in Apc-mutant mice, but the mechanism of this prevention is not entirely clear. OBJECTIVES: We sought to gain insight into the mechanisms of cancer suppression by the MDD diet and to identify biomarkers of cancer risk reduction. METHODS: A plasma metabolomic analysis was performed on ApcΔ14/+ mice maintained on either a methyl donor sufficient (MDS) diet or the protective MDD diet. A group of MDS animals was also pair-fed with the MDD mice to normalize caloric intake, and another group was shifted from an MDD to MDS diet to determine the durability of the metabolic changes. RESULTS: In addition to the anticipated changes in folate one-carbon metabolites, plasma metabolites related to fatty acid metabolism were generally decreased by the MDD diet, including carnitine, acylcarnitines, and fatty acids. Some fatty acid selectivity was observed; the levels of cancer-promoting arachidonic acid and 2-hydroxyglutarate were decreased by the MDD diet, whereas eicosapentaenoic acid (EPA) levels were increased. Machine-learning elastic net analysis revealed a positive association between the fatty acid-related compounds azelate and 7-hydroxycholesterol and tumor development, and a negative correlation with succinate and ß-sitosterol. CONCLUSION: Methyl donor restriction causes dramatic changes in systemic fatty acid metabolism. Regulating fatty acid metabolism through methyl donor restriction favorably effects fatty acid profiles to achieve cancer protection.

Epithelial Cell-specific Deletion of Microsomal Prostaglandin E Synthase-1 Does Not Influence Colon Tumor Development in Mice.

Activation of the COX-2/microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E2 (PGE2) signaling axis is a hallmark of many cancers, including colorectal cancer, prompting the implementation of prevention strategies targeting COX-2 activity. We have previously shown that targeting the downstream terminal PGE2 synthase, mPGES-1 (Ptges), specifically reduces inducible PGE2 formation without disrupting synthesis of other essential prostanoids, thereby conferring dramatic cancer protection against colon carcinogenesis in multiple mouse models. In order to accelerate its development as a viable drug target, and to better understand the mechanisms by which PGE2 influences colon carcinogenesis, we recently developed a conditional Ptges knockout mouse model (cKO). To evaluate the functional role of Ptges directly within the colonic epithelia, cKO mice were crossed with carbonic anhydrase 1 (Car1)-Cre mice (cKO.Car1), and colon tumors were induced using the azoxymethane/dextran sodium sulfate protocol. Unexpectedly, epithelial-specific blockade of Ptges failed to protect mice against colon tumor development. Further studies using the cKO mouse model will be necessary to pinpoint the cell type-specific location of mPGES-1 and its control of inducible PGE2 formation that drives tumor formation in the colon.

Single-Cell-Derived Primary Rectal Carcinoma Cell Lines Reflect Intratumor Heterogeneity Associated with Treatment Response.

PURPOSE: The standard treatment of patients with locally advanced rectal cancer consists of preoperative chemoradiotherapy (CRT) followed by surgery. However, the response of individual tumors to CRT is extremely diverse, presenting a clinical dilemma. This broad variability in treatment response is likely attributable to intratumor heterogeneity (ITH). EXPERIMENTAL DESIGN: We addressed the impact of ITH on response to CRT by establishing single-cell-derived cell lines (SCDCL) from a treatment-naïve rectal cancer biopsy after xenografting. RESULTS: Individual SCDCLs derived from the same tumor responded profoundly different to CRT in vitro. Clonal reconstruction of the tumor and derived cell lines based on whole-exome sequencing revealed nine separate clusters with distinct proportions in the SCDCLs. Missense mutations in SV2A and ZWINT were clonal in the resistant SCDCL, but not detected in the sensitive SCDCL. Single-cell genetic analysis by multiplex FISH revealed the expansion of a clone with a loss of PIK3CA in the resistant SCDCL. Gene expression profiling by tRNA-sequencing identified the activation of the Wnt, Akt, and Hedgehog signaling pathways in the resistant SCDCLs. Wnt pathway activation in the resistant SCDCLs was confirmed using a reporter assay. CONCLUSIONS: Our model system of patient-derived SCDCLs provides evidence for the critical role of ITH for treatment response in patients with rectal cancer and shows that distinct genetic aberration profiles are associated with treatment response. We identified specific pathways as the molecular basis of treatment response of individual clones, which could be targeted in resistant subclones of a heterogenous tumor.

A role for ceramide glycosylation in resistance to oxaliplatin in colorectal cancer.

There is growing evidence to support a role for the ceramide-metabolizing enzyme, glucosylceramide synthase (GCS), in resistance to a variety of chemotherapeutic agents. Whether GCS contributes to oxaliplatin resistance in colorectal cancer (CRC) has not yet been determined. We have addressed this potentially important clinical issue by examining GCS function in two panels of oxaliplatin-resistant, isogenic CRC cell lines. Compared to parental cell lines, oxaliplatin-resistant cells have increased expression of GCS protein associated with increased levels of the pro-survival ceramide metabolite, glucosylceramide (GlcCer). Inhibition of GCS expression by RNAi-mediated gene knockdown resulted in a reduction in cellular GlcCer levels, with restored sensitivity to oxaliplatin. Furthermore, oxaliplatin-resistant CRC cells displayed lower ceramide levels both basally and after treatment with oxaliplatin, compared to parental cells. GlcCer, formed by GCS-mediated ceramide glycosylation, is the precursor to a complex array of glycosphingolipids. Differences in cellular levels and species of gangliosides, a family of glycosphingolipids, were also seen between parental and oxaliplatin-resistant CRC cells. Increased Akt activation was also observed in oxaliplatin-resistant CRC cell lines, together with increased expression of the anti-apoptotic protein survivin. Finally, this study shows that GCS protein levels are greatly increased in human CRC specimens, compared to matched, normal colonic mucosa, and that high levels of UGCG gene expression are significantly associated with decreased disease-free survival in colorectal cancer patients. These findings uncover an important cellular role for GCS in oxaliplatin chemosensitivity and may provide a novel cellular target for augmenting chemotherapeutic drug effectiveness in CRC.

Colon Cancer Prevention with Walnuts: A Longitudinal Study in Mice from the Perspective of a Gut Enterotype-like Cluster.

There is limited understanding of how walnut consumption inhibits the development of colorectal cancer. A possible mechanism may involve alterations to the gut microbiota. In this study, the effects of walnut on gut microbiota were tested in a mouse tumor bioassay using the colonotropic carcinogen, azoxymethane (AOM) added to the total Western diet (TWD). 16S rRNA pyrosequencing identified three enterotype-like clusters (E1, E2, and E3) in this murine model. E1, E2, and E3 are associated with AOM exposure, walnut consumption, and TWD diet, respectively. E2 and E3 showed distinct taxonomic and functional characteristics, while E1 represented an intermediate state. At the family level, E1 and E3 were both enriched with Bacteroidaceae, but driven by two different operational taxonomic units (OTU; OTU-2 for E1, OTU-4 for E3). E2 was overrepresented with Porphyromonadaceae and Lachnospiraceae, with OTU-3 (family Porphyromonadaceae) as the "driver" OTU for this cluster. Functionally, E3 is overrepresented with genes of glycan biosynthesis and metabolism, xenobiotic metabolism, and lipid metabolism. E2 is enriched with genes associated with cell motility, replication and repair, and amino acid metabolism. Longitudinally, E2 represents the gut microbial status of early life in these mice. In comparison with E1 and E3, E2 is associated with a moderate lower tumor burden (P = 0.12). Our results suggest that walnuts may reduce the risk of colorectal cancer within a Western diet by altering the gut microbiota. Our findings provide further evidence that colorectal cancer risk is potentially modifiable by diet via alterations to the microbiota.

Methyl Donor Deficiency Blocks Colorectal Cancer Development by Affecting Key Metabolic Pathways.

Our understanding of the role of folate one-carbon metabolism in colon carcinogenesis remains incomplete. Previous studies indicate that a methyl donor-deficient (MDD) diet lacking folic acid, choline, methionine, and vitamin B12 is associated with long-lasting changes to the intestinal epithelium and sustained tumor protection in Apc-mutant mice. However, the metabolic pathways by which the MDD diet affects these changes are unknown. Colon samples harvested from ApcΔ14/+ mice fed the MDD diet for 18 weeks were profiled using a GC-MS and LC-MS/MS metabolomics platform. Random forest and pathway analyses were used to identify altered metabolic pathways, and associated gene expression changes were analyzed by RT-PCR. Approximately 100 metabolites affected by the MDD diet were identified. As expected, metabolites within the methionine cycle, including methionine (-2.9-fold, P < 0.001) and betaine (-3.3-fold, P < 0.001), were reduced. Elevated homocysteine (110-fold, P < 0.001) was associated with increased flux through the transsulfuration pathway. Unexpectedly, levels of deoxycholic acid (-4.5-fold, P < 0.05) and several other secondary bile acids were reduced. There were also unexpected reductions in the levels of carnitine (-2.0-fold, P < 0.01) and a panel of acylcarnitines involved in fatty acid ß-oxidation. Finally, metabolites involved in redox balance, including ascorbate and hypotaurine, were found to be persistently elevated. These findings provide clues to the molecular changes underlying MDD-mediated tumor protection and identify regulatable metabolic pathways that may provide new targets for colon cancer prevention and treatment. IMPLICATIONS: Metabolomic profiling reveals molecular changes underlying MDD-induced tumor protection and may provide new targets for colorectal cancer prevention and treatment.