title: Induction of active demethylation and 5hmC formation by 5-azacytidine is TET2 dependent and suggests new treatment strategies against hepatocellular carcinoma
creator: Sajadian, Sahar Olsadat
creator: Ehnert, Sabrina
creator: Vakilian, Haghighat
creator: Koutsouraki, Eirini
creator: Damm, Georg
creator: Seehofer, Daniel
creator: Thasler, Wolfgang
creator: Dooley, Steven
creator: Baharvand, Hossein
creator: Sipos, Bence
creator: Nussler, Andreas K.
subject: ddc-610
subject: 610 Medical sciences Medicine
description: Background: Global deregulation of DNA methylation is one of the crucial causes of hepato cellular carcinoma (HCC). It has been reported that the anti-cancer drug 5-azacytidine (5-AZA) mediates the activation of tumor suppressor genes through passive demethylation by inhibiting DNMT1. Recent evidence suggests that active demethylation which is mediated by ten-eleven translocation (TET) proteins may also be an important step to control global methylation. However, there exists a controversial discussion in which TET proteins are involved in the demethylation process in HCC. Therefore, we firstly wanted to identify which of the TETs are involved in demethylation and later to study whether or not 5-AZA could trigger the TET-dependent active demethylation process in HCC. HCC cell lines (Huh-7, HLE, HLF), primary human hepatocytes (hHeps), and tissues from both healthy (55 patients) and HCC patients (55 patients) were included in this study; mRNA levels of isocitrate dehydrogenase (IDH1, 2) and TETs (TET1–3) were studied via qPCR and confirmed by Western blot. The expression of 5hmC/5mC was determined by immunohistochemistry in human HCC tissues and the corresponding adjacent healthy liver. HCC cell lines were stimulated with 5-AZA (0–20 μM) and viability (Resazurin conversion), toxicity (LDH release), proliferation (PCNA), and 5hmC/5mC distribution were assessed. In addition, knockdown experiments on TET proteins in HCC cell lines using short interference RNAs (siRNAs), in the presence and absence of 5-AZA, were performed.   Results: Our data applying qPCR, immunofluorescence, and Western blotting clearly show that TET2 and TET3 but not TET1 were significantly decreased in HCC tissue and different HCC cell lines compared to non-tumor liver tissues and hHeps. In addition, we show here for the first time applying knockdown experiments that 5-AZA is able to trigger an active TET2-dependent demethylation process with concomitant significant changes in 5hmC/5mC in HCC cell lines and hHeps.   Conclusions: Our data clearly show that the expression and activity of TET2 and TET3 proteins but not TET1 are impaired in hepatocellular carcinoma leading to the reduction of 5hmC in HCCs. Furthermore, this study identified a novel function of 5-azacytidine in promoting a TET-mediated generation of 5hmC suggesting that the availability of 5-AZA in cancer cells will have various effects on different epigenetic targets. These findings may open new therapeutic strategies for epigenetic drugs to treat HCC.  
publisher: BioMed Central
date: 2015
type: Article
type: info:eu-repo/semantics/article
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/19415/1/13148_2015_Article_133.pdf
identifier: DOI:http://dx.doi.org/10.1186/s13148-015-0133-x
identifier: urn:nbn:de:bsz:16-heidok-194152
identifier:   Sajadian, Sahar Olsadat ; Ehnert, Sabrina ; Vakilian, Haghighat ; Koutsouraki, Eirini ; Damm, Georg ; Seehofer, Daniel ; Thasler, Wolfgang ; Dooley, Steven ; Baharvand, Hossein ; Sipos, Bence ; Nussler, Andreas K.  (2015) Induction of active demethylation and 5hmC formation by 5-azacytidine is TET2 dependent and suggests new treatment strategies against hepatocellular carcinoma.  Clinical Epigenetics, 7 (98).  pp. 1-14.  ISSN 1868-7083     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/19415/
rights: info:eu-repo/semantics/openAccess
rights: Please see front page of the work (Sorry, Dublin Core plugin does not recognise license id)
language: eng