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Open Access Highly Accessed Research article

Inhibition of DNA methyltransferase activity and expression by treatment with the pan-deacetylase inhibitor panobinostat in hepatocellular carcinoma cell lines

Steffen Zopf1*, Matthias Ocker4, Daniel Neureiter2, Beate Alinger2, Susanne Gahr13, Markus F Neurath1 and Pietro Di Fazio4

Author Affiliations

1 Department of Medicine 1, University Hospital Erlangen, Ulmenweg 18, Erlangen, 91054, Germany

2 Institute of Pathology, Paracelsus Medical University, Salzburg, Austria

3 Department of Pneumology, Klinikum Nuremberg Nord, Nuremberg, Germany

4 Institute for Surgical Research, Philipps University Marburg, Marburg, Germany

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BMC Cancer 2012, 12:386  doi:10.1186/1471-2407-12-386

Published: 3 September 2012

Abstract

Background

Hepatocellular 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.

Methods

We 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.

Results

Our 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.

Conclusion

We 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.

Keywords:
DNA methyltransferase; Deacetylase inhibitor; Epigenetics; Transcriptional control; Hepatocellular carcinoma