The pan-deacetylase inhibitor panobinostat (LBH 589) inhibits DNA methyltransferases in HCC in vitro and in vivo

The novel cinnamic hydroxamic acid pan-deacetylase inhibitor panobinostat (LBH 589) has recently described to posesss cytotoxic effects against HCC by inhibition of protein deacetylases and histone deacetylases. We therefore applied panobinostat to human HCC cell lines (HepG2, Hep3B) in vitro and to a xenograft model in vivo to determine its effect on another epigenetic target, the DNA methyltransferases (DNMT´s).The human hepatoma cell lines HepG2 and Hep3B were treated with panobinostat at 0.1µM and analyzed after 6 to 72h. Furthermore we used a HepG2 xenograft models with NMRI mice. When subcoutaneous tumors reached a diameter of 7mm, daily i.p. treatment with panobinotstat (10mg/kg) or vehicle (saline solution) was started up to 4 weeks.The cell lines responded to panobinostat treatment with a reduction of cell proliferation in vitro and a significant growth delay of tumors in vivo.We analyzed DNMT activity by a specific colorimetric assay, presenting a significant reduction of DNMT activity at 6 to 72h in both cell lines in vitro and in the HepG2 xenograft in vivo.Accordingly we analyzed the expression of DNMT1, DNMT3a and DNMT3b by quantitative PCR and western blotting. We observed a downregulation especially of DNMT1 and DNMT3a in vivo compared to untreated controls. These findings were confirmed by the in vivo data. To evaluate the effect of the DNMT downregulation, we analyzed the expression of known hypermethylated genes in HCC (APC, RASSF1A, CFTR). We were able to verify a reduced expression of the hypermethylated genes by methylation specific quantitative PCR and a consecutive up regulation of unmethylated genes.

In conclusion the pan-deacetylase inhibitor presents a wide epigenetic modulation by inhibiting deacetylases and moreover by downregulating DNMT´s in HCC.

HCC - LBH 589 - epigenetic