Abstract
BackgroundHepatocellular carcinoma (HCC) still represents an unmet medical need. Epigenetic inactivation of tumor suppressor genes like RASSF1A or APC by overexpression of DNA methyltransferases (DNMTs) has been shown to be common in HCC and to be linked to the overall prognosis of patients. Inhibitors of protein and histone deacetylases (DACi) have been demonstrated to possess strong anti-tumor effects in HCC models.MethodsWe therefore investigated whether DACi also has any influence on the expression and activity of DNMTs and methylated target genes in HepG2 and Hep3B cell culture systems and in a xenograft model by immunohistochemistry, westernblotting, RT-qPCR and methylation-specific PCR.ResultsOur findings demonstrate a rapid inhibition of DNMT activity 6 h after treatment with 0.1 μM of the pan-DACi panobinostat. A downregulation of DNMT mRNAs and protein were also observed at later points in time. This loss of DNMT activity and expression was paralleled by a diminished methylation of the target genes RASSF1A and APC and a concomitant re-expression of APC mRNA and protein. Analysis of HepG2 xenograft specimens confirmed these results in vivo.ConclusionWe suggest a dual mode of action of DACi on DNA methylation status: a rapid inhibition of enzyme activity due to interference with posttranslational acetylation and a delayed effect on transcriptional control of DNMT genes by HDAC or miRNA mechanisms.
Highlights
Hepatocellular carcinoma (HCC) still represents an unmet medical need
At later points in time, DNA methyltransferases (DNMTs) activity was stably reduced by approximately 20% in both cell lines, except for the 24 and 72 h time point in HepG2, where an inhibition of more than 40% was observed (Figure 1A)
Expression of DNMT1, DNMT3a and DNMT3b were investigated by quantitative real-time RT-PCR
Summary
Epigenetic inactivation of tumor suppressor genes like RASSF1A or APC by overexpression of DNA methyltransferases (DNMTs) has been shown to be common in HCC and to be linked to the overall prognosis of patients. Changes in chromatin have recently been identified to contribute to tumorigenesis [5] These reversible modifications are considered to contribute to tumor suppressor gene inactivation by means of DNA methylation, histone modifications or miRNA expression. Genes that are commonly affected by DNA methylation include both the tumor suppressors RASSF1A (Ras association domain family 1 isoform A) and APC (adenomatous polyposis coli) Both genes have been shown to be commonly inactivated in human hepatocellular carcinoma and to influence the overall prognosis of patients and represent interesting targets for reversing DNA methylation status [13,14,15,16,17]
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