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

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.

Figure 1

Panobinostat affects activity and expression of DNA methyltransferases in vitro. (A) Total DNMT activity was evaluated in HepG2 and Hep3B cells treated with 0.1 μM panobinostat for the indicated points in time. Results are the mean remaining DNMT activity ± relative error of three independent experiments and are expressed relative to values of untreated controls with a set value of 1.0 for each point in time. * P < 0.05 vs. untreated controls. (B) and (C) Quantitative RT-PCR analysis of expression of DNMTs in HepG2 (B) and Hep3B (C) cells after treatment with 0.1 μM panobinostat. Results were normalized to the GAPDH level of each sample and represent mean ± relative error of three independent experiments and are expressed relative to mRNA levels of untreated controls at each point in time using the set value 1.0.

Figure 2

Westernblot analysis of DNMT expression. HepG2 and Hep3B cells were incubated with 0.1 μM panobinostat for the indicated points in time. Western blot results show representative examples for expression of DNMT1, DNMT3a and DNMT3b as well as β-actin which served as an internal control. Densitometry values are relative to untreated controls with a set value of 1.0 for each point in time.

Figure 3

Regulation of DNA methylation and expression of target genes after panobinostat treatment. DNA methylation of APC (A) and RASSF1A (B) was detected by quantitative methylation-specific PCR in HepG2 and Hep3B cells treated with 0.1 μM panobinostat. Expression of total mRNA for APC (C) and RASSF1A (D) was analyzed using quantitative real-time RT-PCR and normalization to GAPDH content of each sample. Results are mean ± relative error of three independent experiments and are expressed relative to the untreated controls with a set value of 1.0. * P < 0.05 vs. untreated controls.

Figure 4

Effect of panobinostat on DNMT and target gene expression in vivo. HepG2 xenograft specimens were analyzed for expression of (A) DNMTs after 1, 7 and 28 days of daily i.p. injections of 10 mg/kg panobinostat. Results were normalized to the GAPDH content of each sample and represent mean ± relative error of 5 to 10 independent samples per group and are expressed relative to expression levels of untreated control animals with the set value of 1.0 for each point in time. (B) Methylation status and total expression level of APC and RASSF1A were analyzed at day 7 and day 28 of panobinostat treatment. Results are normalized to levels of untreated controls. Methylation of RASSF1A was not detectable in untreated controls and in treated animals at day 7. * P < 0.05 vs. untreated controls.

Figure 5

Immunohistochemical analysis of DNMT1 and DNMT3a expression. HepG2 xenografts were treated with daily i.p. injections of 10 mg/kg panobinostat for 28 days. Untreated controls show a high nuclear expression of both DNMTs, while a significant reduction in expression is observed in treated samples. Bar diagram shows mean percentage of positive cells ± standard deviation of n = 5 in each group. * P < 0.05 vs. untreated controls. Bar represents 20 μm (400× magnification) for overview and 10 μm (1000 x magnification) for detailed areas (marked by a rectangle).