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      Vitamin C enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7

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          Abstract

          Background

          5-Azacytidine (5-AZA), a DNA methyl transferase inhibitor, is a clinically used epigenetic drug for cancer therapy. Recently, we have shown that 5-AZA upregulates ten-eleven translocation (TET) protein expression in hepatocellular carcinoma (HCC) cells, which induce active demethylation. Vitamin C facilitates TET activity and enhances active demethylation. The aim of this study is to investigate whether vitamin C is able to enhance the effect of 5-AZA on active demethylation and to evaluate its consequence in HCC cell lines.

          Methods

          HCC cell lines (Huh7 and HLE) were treated with 5-AZA and vitamin C. After 48 h of treatment, viability (resazurin conversion), toxicity (lactose dehydrogenase (LDH) release), and proliferation ((proliferating cell nuclear antigen (PCNA)) of single- and combined-treated cells were assessed. The effect of the treatment on 5-hydroxymethylcytosine (5hmC) intensity (immunofluorescence (IF) staining), TET, Snail, GADD45B, and P21 mRNA (real-time PCR) and protein expression (Western blot) were investigated.

          Results

          Our results indicated that vitamin C enhances the anti-proliferative and apoptotic effect of 5-AZA in HCC cell lines. By further analyzing the events leading to cell cycle arrest, we have shown for the first time in HCC that the combination of 5-AZA and vitamin C leads to an enhanced downregulation of Snail expression, a key transcription factor governing epithelial-mesenchymal transition (EMT) process, and cell cycle arrest.

          Conclusions

          We conclude that when combined with 5-AZA, vitamin C enhances TET activity in HCC cells, leading to induction of active demethylation. An increase in P21 expression as a consequence of downregulation of Snail accompanied by the induction of GADD45B expression is the main mechanism leading to cell cycle arrest in HCCs.

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          Most cited references 36

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          5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy.

           J K Christman (2002)
          5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of anti-metabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However, because of 5-azacytidine's general toxicity, other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that, when present in DNA, it inhibited DNA methylation, led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here, the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed.
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            5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.

            DNA methylation dynamics influence brain function and are altered in neurological disorders. 5-hydroxymethylcytosine (5-hmC), a DNA base that is derived from 5-methylcytosine, accounts for ∼40% of modified cytosine in the brain and has been implicated in DNA methylation-related plasticity. We mapped 5-hmC genome-wide in mouse hippocampus and cerebellum at three different ages, which allowed us to assess its stability and dynamic regulation during postnatal neurodevelopment through adulthood. We found developmentally programmed acquisition of 5-hmC in neuronal cells. Epigenomic localization of 5-hmC-regulated regions revealed stable and dynamically modified loci during neurodevelopment and aging. By profiling 5-hmC in human cerebellum, we found conserved genomic features of 5-hmC. Finally, we found that 5-hmC levels were inversely correlated with methyl-CpG-binding protein 2 dosage, a protein encoded by a gene in which mutations cause Rett syndrome. These data suggest that 5-hmC-mediated epigenetic modification is critical in neurodevelopment and diseases.
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              Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals.

              DNA hydroxymethylation and its mediated DNA demethylation are critical for multiple cellular processes, for example, nuclear reprogramming, embryonic development, and many diseases. Here, we demonstrate that a vital nutrient ascorbic acid (AA), or vitamin C (Vc), can directly enhance the catalytic activity of Tet dioxygenases for the oxidation of 5-methylcytosine (5mC). As evidenced by changes in intrinsic fluorescence and catalytic activity of Tet2 protein caused by AA and its oxidation-resistant derivatives, we further show that AA can uniquely interact with the C-terminal catalytic domain of Tet enzymes, which probably promotes their folding and/or recycling of the cofactor Fe(2+). Other strong reducing chemicals do not have a similar effect. These results suggest that AA also acts as a cofactor of Tet enzymes. In mouse embryonic stem cells, AA significantly increases the levels of all 5mC oxidation products, particularly 5-formylcytosine and 5-carboxylcytosine (by more than an order of magnitude), leading to a global loss of 5mC (∼40%). In cells deleted of the Tet1 and Tet2 genes, AA alters neither 5mC oxidation nor the overall level of 5mC. The AA effects are however restored when Tet2 is re-expressed in the Tet-deficient cells. The enhancing effects of AA on 5mC oxidation and DNA demethylation are also observed in a mouse model deficient in AA synthesis. Our data establish a direct link among AA, Tet, and DNA methylation, thus revealing a role of AA in the regulation of DNA modifications.
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                Author and article information

                Contributors
                andreas.nuessler@gmail.com
                Journal
                Clin Epigenetics
                Clin Epigenetics
                Clinical Epigenetics
                BioMed Central (London )
                1868-7075
                1868-7083
                30 April 2016
                30 April 2016
                2016
                : 8
                Affiliations
                [ ]Eberhard Karls University Tuebingen, BG Trauma Clinic, SWI, Schnarrenbergstraße 95, 72076 Tuebingen, Germany
                [ ]CSIR - Centre for Cellular and Molecular Biology, Uppal Road, Habsiguda, Hyderabad India
                [ ]Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
                [ ]Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany
                Article
                213
                10.1186/s13148-016-0213-6
                4851801
                27134688
                © Sajadian et al. 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: The Ministerium für Ländlichen Raum und Verbraucherschutz / Baden-Württemberg
                Award ID: BW 05110214
                Funded by: The Ministerium für Ländlichen Raum und Verbraucherschutz / Baden-Württemberg
                Award ID: BW 05110214
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: SFB/TTR77
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Genetics

                5-azacytidine, vitamin c, tets, 5hmc, snail, gadd45b, emt/met

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