7% and 47. 4%, respectively. Trichostatin A mw At later points in time, DNMT activity was stably reduced by approximately 20% in both cell lines, except for the 24 and 72 h time point in HepG2, where an in hibition of more than 40% was observed. Expression of DNMT1, DNMT3a and DNMT3b were then investigated by quantitative real time RT PCR. Panobinostat treatment significantly repressed mRNA for DNMT1 and DNMT3a in both cell lines while no changes were observed in DNMT3b levels. These findings were corroborated by westernblot analysis showing a strong reduction of DNMT1 and DNMT3a protein in both cell lines but not of DNMT3b. Here, only a transient decrease in protein levels was observed after 24 to 48 h in both cell lines.

Although mRNA levels in total were rapidly decreased by panobi nostat, protein expression was significantly reduced after only 24 h and remained suppressed until 72 h for DNMT1 and DNMT3a. Effects of panobinostat on target gene methylation and expression in vitro We next investigated whether the inhibition of DNMT activity and expression is also reflected on the methyla tion pattern of known hypermethylated tumor suppres sor genes. In order to do so, quantitative methylation specific PCR was performed for APC and RASSF1A in cells treated with 0. 1 uM panobinostat for 6 to 72 h and expressed relative to the levels of untreated controls at the given points in time. Overall, Hep3B cells seemed to be more sensitive to the DACi mediated inhibition of DNA methylation as shown by a significant and strong reduction of methylated APC after only 6 h.

While methylation was suppressed by approximately 80% here, APC methylation returned to the level of untreated controls after 24 h. RASSF1A showed a slight reduction in methylation at 12 h but only proved to be significant at 72 h. In HepG2, APC methylation was significantly reduced after only 24 h of treatment while no change Drug_discovery was observed for RASSF1A. In line with the reduction of methylation, an increased expression of APC was observed in both cell lines, reaching the highest level at 48 h for Hep3B and at 72 h for HepG2, respectively. Observation of methylation of RASSF1A showed no significant change in expression induced by panobinostat. Panobinostat influences methylation and gene expression pattern in vivo To address whether panobinostat also influences expres sion of DNMTs and related target genes in vivo, we ana lyzed HepG2 xenograft samples from a previously described nude mouse model. Animals were treated with daily intraperitoneal injections of 10 mg kg panobi nostat. After only 1 day expression of all DNMTs were reduced by approximately 40% compared to untreated controls. The observed reduction in expression was sta tistically significant for DNMT1 and DNMT3a.