DNA Methylation In Colon Research Articles

Effects of β-carotene on Expression of Selected MicroRNAs, Histone Acetylation, and DNA Methylation in Colon Cancer Stem Cells.

BackgroundBeta-carotene (BC) is a carotenoid which exerts anti-cancer effects in several types of cancer, including colorectal cancer. Epigenetic modifications of genes, such as histone deacetylation and DNA hypermethylation, have also been detected in various types of cancer. To understand the molecular mechanism underlying cancer preventive and therapeutic effects of BC, microRNAs (miRNAs), histone acetylation, and global DNA methylation in colon cancer stem cells (CSCs) were investigated.MethodsHCT116 colon cancer cells positive for expression of CD44 and CD133 were sorted by flow cytometry and used in subsequent experiments. Cell proliferation was examined by the MTT assay and self-renewal capacity was analyzed by the sphere formation assay. The miRNA sequencing array was used to detect miRNAs regulated by BC. Histone acetylation levels were measured by the Western blot analysis. mRNA expression of DNA methyltransferases (DNMTs) was examined by qPCR and global DNA methylation levels were determined by enzyme-linked immunosorbent assay.ResultsTreatment of CD44+CD133+ colon CSCs with BC caused a reduction in both cell proliferation and sphere formation. Analysis of the miRNA sequencing array showed that BC regulated expression of miRNAs associated with histone acetylation. Histone H3 and H4 acetylation levels were elevated by BC treatment. In addition, BC treatment down-regulated DNMT3A mRNA expression and global DNA methylation in colon CSCs.ConclusionsThese results suggest that BC regulates epigenetic modifications for its anti-cancer effects in colon CSCs.

A lifelong exposure to a Western-style diet, but not aging, alters global DNA methylation in mouse colon.

BACKGROUND/OBJECTIVESPrevious studies have indicated that when compared to young mice, old mice have lower global DNA methylation and higher p16 promoter methylation in colonic mucosa, which is a common finding in colon cancer. It is also known that a Western-style diet (WSD) high in fat and calories, and low in calcium, vitamin D, fiber, methionine and choline (based on the AIN 76A diet) is tumorigenic in colons of mice. Because DNA methylation is modifiable by diet, we investigate whether a WSD disrupts DNA methylation patterns, creating a tumorigenic environment.SUBJECTVIES/METHODSWe investigated the effects of a WSD and aging on global and p16 promoter DNA methylation in the colon. Two month old male C57BL/6 mice were fed either a WSD or a control diet (AIN76A) for 6, 12 or 17 months. Global DNA methylation, p16 promoter methylation and p16 expression were determined by LC/MS, methyl-specific PCR and real time RT-PCR, respectively.RESULTSThe WSD group demonstrated significantly decreased global DNA methylation compared with the control at 17 months (4.05 vs 4.31%, P = 0.019). While both diets did not change global DNA methylation over time, mice fed the WSD had lower global methylation relative to controls when comparing all animals (4.13 vs 4.30%, P = 0.0005). There was an increase in p16 promoter methylation from 6 to 17 months in both diet groups (P < 0.05) but no differences were observed between diet groups. Expression of p16 increased with age in both control and WSD groups.CONCLUSIONSIn this model a WSD reduces global DNA methylation, whereas aging itself has no affect. Although the epigenetic effect of aging was not strong enough to alter global DNA methylation, changes in promoter-specific methylation and gene expression occurred with aging regardless of diet, demonstrating the complexity of epigenetic patterns.

Supplementation of Seaweeds Extracts Suppresses Azoxymethane-induced Aberrant DNA Methylation in Colon and Liver of ICR Mice

Background:Seamustard and seatangle are commonly consumed seaweeds in Korea and rich sources of non-digestible polysaccharides which possess biological activities. However anti-mutagenic and anti-cancer activities of these seaweeds under physiological condition have not been clarified yet. The objective of this study was to investigate the effect of seaweeds consumption on azoxymethane (AOM) -induced DNA methylation at N7 and O6 position of guanine base, an indicator of DNA damage related to cancer initiation.Methods:Thirty ICR mice were divided into five groups and fed one of the following diets for two weeks: control diet, diet containing 10% water-soluble or water-insoluble fraction of seamustard or seatangle. After two weeks of experimental diet AOM was injected at 6 hours before sacrifice and N7-methylguanine (N7-meG) and O6-methylguanine (O6-meG) from the colon and liver DNA were quantified using a gas chromatography-mass spectroscopy.Results:Water-soluble fractions of both seamustard and seatangle significantly reduced AOM-induced production of N7-meG guanine in colon and liver. Also water-soluble fractions of these seaweeds suppressed the level of methylation at O6-guanine of colon and liver directly responsible for tumorigenesis. While water-insoluble fraction of seamustard suppressed the production of N7-meG in liver this seaweed fraction decreased O6-meG and the ratio of O6/N7-meG in liver. Water insoluble fraction of seatangle decreased both O6- and N7-meG in colon and liver. Supplementation of all seaweeds extracts increased fecal weight of animals and the increase of fecal weight by water-insoluble fraction of seaweeds were higher than that by water-soluble fraction.Conclusion:Seamustard and seatangle intake may effectively prevent colon and liver carcinogenesis by decreasing DNA damage and the mechanism of inhibiting carcinogenesis by seaweeds in a long term study are warranted.

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

Older age and inadequate folate intake are strongly implicated as important risk factors for colon cancer and each is associated with altered DNA methylation. This study was designed to determine the effects of aging and dietary folate on select features of DNA methylation in the colon that are relevant to carcinogenesis. Old (18 mo; n = 34) and young (4 mo; n = 32) male C57BL/6 mice were randomly divided into 3 groups and fed diets containing 0, 4.5, or 18 μmol folate/kg (deplete, replete, and supplemented groups, respectively) for 20 wk. Genomic DNA methylation and p16 promoter methylation in the colonic mucosa were analyzed by liquid chromatography/electrospray ionization/MS and methylation-specific PCR, respectively. p16 gene expression was determined by real-time RT-PCR. Old mice had significantly lower genomic DNA methylation compared with young mice at each level of dietary folate (4.5 ± 0.2, 4.8 ± 0.1, and 4.9 ± 0.1 vs. 6.0 ± 0.1, 5.3 ± 0.2, and 5.9 ± 0.2%, in folate-deplete, -replete, and -supplemented groups, respectively, P < 0.05) and markedly higher p16 promoter methylation (61.0 ± 2.7, 69.7 ± 6.9, and 87.1 ± 13.4 vs. 10.8 ± 3.6, 8.4 ± 1.8, and 4.9 ± 1.7%, respectively, P < 0.05). In old mice, genomic and p16 promoter DNA methylation each increased in a manner that was directly related to dietary folate (Ptrend = 0.009). Age-related enhancement of p16 expression occurred in folate-replete (P = 0.001) and folate-supplemented groups (P = 0.041), but not in the folate-deplete group. In conclusion, aging decreases genomic DNA methylation and increases promoter methylation and expression of p16 in mouse colons. This effect is dependent on the level of dietary folate.

Dietary Folate and Selenium Affect Dimethylhydrazine-Induced Aberrant Crypt Formation, Global DNA Methylation and One-Carbon Metabolism in Rats

Several observations suggest a role for DNA methylation in cancer pathogenesis. Although both selenium and folate deficiency have been shown to cause global DNA hypomethylation and increased cancer susceptibility, the nutrients have different effects on one-carbon metabolism. Thus, the purpose of this study was to investigate the interactive effects of dietary selenium and folate. Weanling, Fischer-344 rats (n = 23/diet) were fed diets containing 0 or 2.0 mg selenium (as selenite)/kg and 0 or 2.0 mg folate/kg in a 2 x 2 factorial design. After 3 and 4 wk of a 12-wk experiment, 19 rats/diet were injected intraperitoneally with dimethylhydrazine (DMH, 25 mg/kg) and 4 rats/diet were administered saline. Selenium deficiency decreased (P < 0.05) colonic DNA methylation and the activities of liver DNA methyltransferase and betaine homocysteine methyltransferase and increased plasma glutathione concentrations. Folate deficiency increased (P < 0.05) the number of aberrant crypts per aberrant crypt foci, the concentration of colonic S-adenosylhomocysteine and the activity of liver cystathionine synthase. Selenium and folate interacted (P < 0.0001) to influence one-carbon metabolism and cancer susceptibility such that the number of aberrant crypts and the concentrations of plasma homocysteine and liver S-adenosylhomocysteine were the highest and the concentrations of plasma folate and liver S-adenosylmethionine and the activity of liver methionine synthase were the lowest in rats fed folate-deficient diets and supplemental selenium. These results suggest that selenium deprivation ameliorates some of the effects of folate deficiency, probably by shunting the buildup of homocysteine (as a result of folate deficiency) to glutathione.

The effect of dietary folate on genomic and p53-specific DNA methylation in rat colon.

Folate is an important mediator in the transfer of methyl groups for DNA methylation, abnormalities of which are considered to play an important mechanistic role in colorectal carcinogenesis. This study investigated the time-dependent effects of dietary folate on genomic and p53 (in the promoter region and exons 6-7) DNA methylation in rat colon, and how these changes are related to steady-state levels of p53 transcript. Despite a marked reduction in plasma and colonic folate concentrations, a large increase in plasma homocysteine (an accurate inverse indicator of folate status), and a progressive decrease in colonic S-adenosylmethionine (SAM; the primary methyl donor for methylations) to S-adenosylhomocysteine (SAH; a potent inhibitor of methylations) ratio, isolated folate deficiency did not induce significant genomic DNA hypomethylation in the colon. Paradoxically, isolated folate deficiency increased the extent of genomic DNA methylation in the colon at an intermediate time point (P = 0.022). Folate supplementation did not modulate colonic SAM, SAH and SAM to SAH ratios, and genomic DNA methylation at any time point. The extent of p53 methylation in the promoter and exons 6-7 was variable over time at each of the CpG sites examined, and no associations with time or dietary folate were observed at any CpG site except for site 1 in exons 6-7 at week 5. Dietary folate deprivation progressively decreased, whereas supplementation increased, steady-state levels of p53 transcript over 5 weeks (P < 0.05). Steady-state levels of p53 mRNA correlated directly with plasma and colonic folate concentrations (P = 0.41-0.49, P < 0.002) and inversely with plasma homocysteine and colonic SAH levels (r = -0.37-0.49, P < 0.006), but did not significantly correlates with either genomic or p53 methylation within the promoter region and exons 6-7. The data indicate that isolated folate deficiency, which significantly reduces steady-state levels of colonic p53 mRNA, is not associated with a significant degree of genomic or p53 DNA hypomethylation in rat colon. This implies that neither genomic or p53 hypomethylation within exons 6-7 nor aberrant p53 methylation within the promoter region is likely a mechanism by which folate deficiency enhances colorectal carcinogenesis in the rat.