Older Age and Dietary Folate Are Determinants of Genomic and p16-Specific DNA Methylation in Mouse Colon

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Multiple B-Vitamin Inadequacy Amplifies Alterations Induced by Folate Depletion in P53 Expression and its Downstream Effector MDM2

Folate is required for biological methylation and nucleotide synthesis, aberrations of which are thought to be the mechanisms that enhance colorectal carcinogenesis produced by folate inadequacy. These functions of folate also depend on the availability of other B-vitamins that participate in “onecarbon metabolism,” including B2, B6 and B12. Our study therefore investigated whether combined dietary restriction of these vitamins amplifies aberrations in the epigenetic and genetic integrity of the p53 gene that is induced by folate depletion alone. Ninety-six mice were group pair-fed diets with different combinations of B-vitamin depletion over 10 weeks. DNA and RNA were extracted from epithelial cells isolated from the colon. Within the hypermutable region of p53 (exons 5–8), DNA strand breaks were induced within exons 6 and 8 by folate combined with B2, B6 and B12 restriction (p \u3c 0.05); such effects were not significantly induced by mild folate depletion alone. Similarly, a minor degree of hypomethylation of exon 6 produced by isolated folate depletion was significantly amplified (p ≤ 0.05) by simultaneous depletion of all 4 B-vitamins. Furthermore, the expression of p53 and MDM2 were significantly decreased (p ≤ 0.05) by the combined depletion state but not by folate depletion alone. These data indicate that inadequacies of other 1-carbon vitamins may amplify aberrations of the p53 gene induced by folate depletion alone, implying that concurrent inadequacies in several of these vitamins may have added tumorigenic potential beyond that observed with isolated folate depletion

Moderate Folate Depletion Modulates the Expression of Selected Genes Involved in Cell Cycle, Intracellular Signaling and Folate Uptake in Human Colonic Epithelial Cell Lines

Folate deficiency may affect gene expression by disrupting DNA methylation patterns or by inducing base substitution, DNA breaks, gene deletions and gene amplification. Changes in expression may explain the inverse relationship observed between folate status and risk of colorectal cancer. Three cell lines derived from the normal human colon, HCEC, NCM356 and NCM460, were grown for 32–34 days in media containing 25, 50, 75 or 150 nM folic acid, and the expression of genes involved in cell-cycle checkpoints, intracellular signaling, folate uptake and cell adhesion and migration was determined. Expression of Folate Receptor 1 was increased with decreasing media folate in all cell lines, as was p53, p21, p16 and β-catenin. With decreasing folate, the expression of both E-cadherin and SMAD-4 was decreased in NCM356. APC was elevated in NCM356 but unchanged in the other lines. No changes in global methylation were detected. A significant increase in p53 exon 7–8 strand breaks was observed with decreasing folate in NCM460 cells. The changes observed are consistent with DNA damage-induced activation of cell-cycle checkpoints and cellular adaptation to folate depletion. Folate-depletion-induced changes in the Wnt/APC pathway as well as in genes involved in cell adhesion, migration and invasion may underlie observed relationships between folate status and cancer risk

Fecal microbiota transplanted from old mice promotes more colonic inflammation, proliferation, and tumor formation in azoxymethane-treated A/J mice than microbiota originating from young mice

ABSTRACTAging is a strong risk factor for colorectal cancer (CRC). It is well established that gut microbial dysbiosis can play a role in the etiology of CRC. Although the composition of the gut microbial community changes with age and is reported to become more pro-inflammatory, it is unclear whether such changes are also pro-tumorigenic for the colon. To address this gap, we conducted fecal microbiota transplants (FMT) from young (DY, ~6 wk) and old (DO, ~72 wk) donor mice into young (8 wk) recipient mice that were pre-treated with antibiotics. After initiating tumorigenesis with azoxymethane, recipients were maintained for 19 wk during which time they received monthly FMT boosters. Compared to recipients of young donors (RY), recipients of old donors (RO) had an approximately 3-fold higher prevalence of histologically confirmed colon tumors (15.8 vs 50%, Chi2 P = .03), approximately 2-fold higher proliferating colonocytes as well as significantly elevated colonic IL-6, IL-1β and Tnf-α. Transcriptomics analysis of the colonic mucosa revealed a striking upregulation of mitochondria-related genes in the RO mice, a finding corroborated by increased mitochondrial abundance. Amongst the differences in fecal microbiome observed between DY and DO mice, the genera Ruminoclostridium, Lachnoclostridium and Marvinbryantia were more abundant in DY mice while the genera Bacteroides and Akkermansia were more abundant in DO mice. Amongst recipients, Ruminoclostridium and Lachnoclostridium were higher in RY mice while Bacteroides was higher in RO mice. Differences in fecal microbiota were observed between young and old mice, some of which persisted upon transplant into recipient mice. Recipients of old donors displayed significantly higher colonic proliferation, inflammation and tumor abundance compared to recipients of young donors. These findings support an etiological role for altered gut microbial communities in the increased risk for CRC with increasing age and establishes that such risk can be transmitted between individuals

Associations Between Single Nucleotide Polymorphisms in Folate Uptake and Metabolizing Genes with Blood Folate, Homocysteine, and DNA Uracil Concentrations

Background—Folate is an essential nutrient that supports nucleotide synthesis and biological methylation reactions. Diminished folate status results in chromosome breakage and is associated with several diseases, including colorectal cancer. Folate status is also inversely related to plasma homocysteine concentrations—a risk factor for cardiovascular disease. Objective—We sought to gain further understanding of the genetic determinants of plasma folate and homocysteine concentrations. Because folate is required for the synthesis of thymidine from uracil, the latter accumulating and being misincorporated into DNA during folate depletion, the DNA uracil content was also measured. Design—Thirteen single nucleotide polymorphisms (SNPs) in genes involved in folate uptake and metabolism, including folate hydrolase (FOLH1), folate polyglutamate synthase (FPGS), γ-glutamyl hydrolase (GGH), methylene tetrahydrofolate reductase (MTHFR), methionine synthase (MTR), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC1), were studied in a cohort of 991 individuals. Results—The MTHFR 677TT genotype was associated with increased plasma homocysteine and decreased plasma folate. MTHFR 1298A\u3eC and RFC1 intron 5A\u3eG polymorphisms were associated with significantly altered plasma homocysteine concentrations. The FOLH1 1561C\u3eT SNP was associated with altered plasma folate concentrations. The MTHFR 677TT genotype was associated with a ≈34% lower DNA uracil content (P = 0.045), whereas the G allele of the GGH – 124T\u3eG SNP was associated with a stepwise increase in DNA uracil content (P = 0.022). Conclusion—Because the accumulation of uracil in DNA induces chromosome breaks, mutagenic lesions, we suggest that, as for MTHFR C677T, the GGH – 124 T\u3eG SNP may modulate the risk of carcinogenesis and therefore warrants further attention

Correction: Paternal B Vitamin Intake Is a Determinant of Growth, Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring.

[This corrects the article DOI: 10.1371/journal.pone.0151579.]

Curcumin and Salsalate Suppresses Colonic Inflammation and Procarcinogenic Signaling in High-Fat-Fed, Azoxymethane-Treated Mice

High-fat diets (HFDs) and excess adiposity increase proinflammatory cytokines in the colon, altering gene expression in a manner that promotes the development of colorectal cancer (CRC). Thus, compounds that reduce this biochemical inflammation are potential chemopreventive agents. Curcumin (CUR), a dietary polyphenol, and salsalate (SAL), a non-steroidal anti-inflammatory drug, are both anti-inflammatories. We investigated the inhibitory effects of CUR with or without SAL on inflammatory cytokines and procarcinogenic signaling in azoxymethane (AOM)-treated A/J mice. A sub-tumorigenic AOM dose was chosen to produce a biochemical and molecular procarcinogenic colonic environment without tumors. Mice were fed either a HFD (60% of kilocalories) or low-fat diet (LFD) (10% of kilocalories). One HFD treatment group received 0.2% CUR in the diet; one received 0.2% CUR + 0.15% SAL; and one received 0.4% CUR + 0.3% SAL. The HFD mice developed 30% greater fat mass than the LFD mice (<i>p</i> < 0.05). The colonic concentrations of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the HFD mice were decreased by 50–69% by the high-dose combination regimen (<i>p</i> < 0.015). Only the combination regimens significantly suppressed phosphorylation of protein kinase B (Akt) and nuclear factor-κB (NF-κB) p65 (<i>p</i> < 0.044). The combination of CUR and SAL reduces the concentration of proinflammatory cytokines and diminishes activation of Akt and NF-κB more effectively than CUR alone, providing a scientific basis for examining whether this combination mitigates the risk of CRC in obese individuals

Polymorphisms in uracil-processing genes, but not one-carbon nutrients, are associated with altered DNA uracil concentrations in an urban Puerto Rican population123

Background: Five genes—UNG, SMUG1, MBD4, TDG, and DUT—are involved in the repair or prevention of uracil misincorporation into DNA, an anomaly that can cause mutagenic events that lead to cancer. Little is known about the determinants of uracil misincorporation, including the effects of single nucleotide polymorphisms (SNPs) in the abovementioned genes. Because of their metabolic function, folate and other one-carbon micronutrients may be important factors in the control of uracil misincorporation

Offspring gene expression FPKM values

File includes raw gene expression values as Fragments Per Kilobase of transcript per Million mapped reads (FPKM) from Cufflinks v2.2.