ABSTRACT
BACKGROUND: Oxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1) gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS), we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2) and impaired fasting glucose (IFG). METHODS: In this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA) and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student's two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount. RESULTS: The intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate. CONCLUSION: This vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a result of the ROS scavenging antioxidant rich diet, leading to lower activity of DNA demethylating enzymes. Our results suggest the hypothesis of CpG demethylation via DNA repair enzymes under these circumstances. NIDDM2 and IFG patients benefit from this simple dietary intervention involving epigenetic and DNA repair mechanisms.
ABSTRACT
BACKGROUND: The tumor suppressor genes p15(INK)4(b) and p16(INK)4(a) as well as the estrogen receptor-α (ESR1) gene are abnormally methylated and expressed in colon cancer. The cancer-preventative abilities of several bioactive food components have been linked to their estrogenic and epigenetic activities. METHODS: The effect of folic acid, zebularine, resveratrol, genistein and epigallocatechin-3-gallate (EGCG) on tumor cell growth, promoter methylation of ESR1, p15(INK)4(b) and p16(INK)4(a) and gene expression of ESR1 and ESR2 was analyzed in Caco-2 cells. Gene expression was measured using real-time PCR, and promoter CpG methylation was assessed using bisulfite conversion and methylation-specific PCR. RESULTS: After exposure to a high concentration of folic acid (20 µmol/l), enhanced cancer cell growth and concomitant increased methylation of the ESR1 (3.6-fold), p16(INK)4(a) and p15(INK)4(b) promoters was observed. A lower concentration of folic acid (2 µmol/l) decreased cell growth. The phytoestrogens genistein and resveratrol enhanced expression of ESR1 (genistein 200 µmol/l: 2.1-fold; resveratrol 50 µmol/l: 6.3-fold) and ESR2 (2.6- and 3.6-fold, respectively). Genistein and resveratrol treatment increased promoter methylation of ESR1 (genistein 200 µmol/l: 2.9-fold; resveratrol 50 µmol/l: 1.4-fold). For p16(INK)4(a), increased methylation was found after exposure to 10 µmol/l resveratrol, but for p15(INK)4(b), decreased methylation was found. Both components showed growth-inhibitory activities. For EGCG, growth inhibition at 100 µmol/l and suppressed promoter methylation of tumor suppressor genes (p16(INK)4(a): 0.9-fold; p15(INK)4(b): 0.6-fold) was seen. CONCLUSIONS: Our results show that these food compounds regulate ESR and tumor suppressor gene expression by multiple mechanisms including epigenetic processes. An improved understanding of these epigenetic effects could therefore support specific dietary concepts of epigenetic cancer prevention and intervention.
