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DOI: 10.1055/s-0034-1397178
Inducing active demethylation through 5-Azacytidine open a new aspect for treatment of Hepatocellular Carcinoma
Background:
Hepatocellular carcinoma (HCC) is the fifth deadliest cancer worldwide. Global deregulation of the methylation status is one of the main causes of HCC. The oxidation of 5-methylcytosine (5mC) triggers the formation of 5-hydroxymethylcytosine (5hmC). 5hmC can be depleted from the genome through down-regulation of the TET proteins leading to the demethylation of previously silenced genes. 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. Our aim was to ascertain, if 5-Aza also induces an active demethylation by increasing the expression of 5hmC mediated by the proteins TET2 and TET3, which may lead to new approaches in cancer therapy.
Methods and Results:
The expression of 5mC and 5hmC in primary human hepatocytes (hHeps), HCC cells (Huh-7, HLE, HLF) and tissue sections from healthy and HCC patient cohorts (55 patients) was studied by immunofluorescence (IF) staining and immunohistochemistry: The expression of 5hmC was lower in HCC tissue than in healthy tissue samples. This finding was confirmed by comparing HCC cells to hHeps. The expression of TET2 and TET3 was lower in HCC tissues and cell lines than in non-tumor liver tissues and hHeps. In order to evaluate, if the expressions of TETs and 5hmC are inducible by 5-Aza, HCC cell lines were treated with different concentrations of 5-Aza (0 – 20µM). Even at the highest concentration, no LDH release and toxic effect were registered after 48 hours of incubation. Interestingly, the expressions of TETs and of 5hmC-5mC were increased, while the proliferation marker PCNA indicated an inhibition of cell proliferation.
Conclusion:
Our data exhibit a decrease of 5hmC and an increase of 5mC in HCCs through down-regulation of the TETs. However, the expression levels of 5hmC and the TETs can be re-induced by 5-Aza through active demethylation, which is a novel function of the drug. We suggest that the loss of 5-hmC expression is an additional marker for hepatocellular carcinoma diagnosis. This would open up new avenues in the therapy of HCC.
Corresponding author: Nuessler, Andreas K
E-Mail: andreas.nuessler@med.uni-tuebingen.de