Volume 42, Issue 2, Pages 210-217 (February 2005) Apoptosis on hepatoma cells but not on primary hepatocytes by histone deacetylase inhibitors valproate and ITF2357  Sorin Armeanu, Anita Pathil, Sascha Venturelli, Paolo Mascagni, Thomas S. Weiss, Martin Göttlicher, Michael Gregor, Ulrich M. Lauer, Michael Bitzer  Journal of Hepatology  Volume 42, Issue 2, Pages 210-217 (February 2005) DOI: 10.1016/j.jhep.2004.10.020 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Accumulation of acetylated histones in HCC cells. HepG2 cells were cultured with 1mM VPA or 0.2μM ITF2357 for 24h. Western blot analysis using antibodies against acetylated histone H3 (Ac-H3) were performed after 24h of incubation with VPA (lane 3) or ITF2357 (lane 2) and compared to untreated cells (lane 1). Equal protein loading was verified by the detection of tubulin. Journal of Hepatology 2005 42, 210-217DOI: (10.1016/j.jhep.2004.10.020) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Antiproliferative effect on hepatoma cell lines. HepG2, HuH-7 and PLC/PRF/5 were cultured for five days in the presence of various concentrations of VPA (A) and ITF2357 (B). Cell viability was assayed in a SRB cytotoxicity assay; the data plotted represent the mean of measurements of four individual experiments related to untreated cells, including single SD. Hundred percent viability represents the optical density of untreated cells. Journal of Hepatology 2005 42, 210-217DOI: (10.1016/j.jhep.2004.10.020) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 VPA and ITF2357 were found to be non-toxic for primary human hepatocytes (PHH). PHH from a single donor (A and B) were treated for 48h in the presence of different concentrations of VPA (A) or ITF2357 (B). Toxicity was monitored by measuring ALT, AST, and LDH in the cellular supernatants. PHH from four further donors (C and D) were investigated using VPA (1 and 2mM) or ITF2357 (0.2 and 0.5μM). The relative activity of ALT, AST and LDH release into the supernatants compared to untreated controls are shown as a mean of the four independent experiments including single SD. Albumin secretion as a marker of PHH differentiation was measured, too (white columns). As a control for cellular toxicity LDH release of equally treated HepG2 hepatoma cells was determined (dotted columns in C and D). (E) Caspase-3 activation in PHH from one single donor and HepG2 cells was fluorometrically determined in cellular extracts 48h after incubation without any HDAC-I (control) or either with VPA or ITF2357 at the indicated concentration. Results are representative for three independent experiments. Journal of Hepatology 2005 42, 210-217DOI: (10.1016/j.jhep.2004.10.020) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Induction of apoptotsis in hepatoma cell lines. (A) Apoptosis induction in dependence of HDAC-I concentration and incubation time. HepG2 cells were cultured in the presence of TSA, VPA or ITF2357 at different concentrations for 12, 24, 48 or 72h without renewal of the medium. Subsequently, the fraction of apoptotic cells was determined by FACS analysis (sub-2n DNA). (B) Apoptosis phenomena in different hepatoma cells. HepG2, HuH-7 and PLC/PRF/5 cells were incubated for 48h with different concentrations of TSA, VPA or ITF2357 and the rate of apoptosis was determined by FACS analysis (sub-2n DNA). Journal of Hepatology 2005 42, 210-217DOI: (10.1016/j.jhep.2004.10.020) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Western blot analysis of HDAC-I modulated proteins. PLC/PRF/5 cells were treated with 1μM TSA (lane 2), 1mM (lane 3) or 2mM VPA (lane 4), and 0.2μM (lane 5) or 0.5μM ITF2357 (lane 6) for 48h. Western blot analysis using cellular lysates was performed with antibodies against (A) p21cip/waf, (B) Bcl-XL, (C) Bid, or (D) Bax. Untreated control cells were loaded in lane 1. Equal protein loading was verified by tubulin staining. Journal of Hepatology 2005 42, 210-217DOI: (10.1016/j.jhep.2004.10.020) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Inhibition of apoptotic cell death by transfection of Bcl-XL. HepG2 cells were transfected with a Bcl-XL gene encoding plasmid (psFFV-Bcl-XL) or control plasmid pNeo (pcDNANeo). (A) Western Blot analysis of intracellular Bcl-XL protein in the absence (c) or presence of VPA or ITF (ITF2357). Equal protein loading was verified by tubulin staining. (B) Rate of transfected (GFP positive) apoptotic cells by FACS analysis in the absence (control) or presence of HDAC-I (VPA; ITF2357). Shown are the mean of 3 experiments and single SD. Journal of Hepatology 2005 42, 210-217DOI: (10.1016/j.jhep.2004.10.020) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Schematic summary of VPA and ITF2357 induced reactions in hepatoma cells. Incubation of hepatoma cells leads to an inhibition of cellular proliferation (left side) and induction of apoptotic cell death (right side). Upregulation of p21cip/waf might be the major factor being responsible for inhibition of hepatoma cell proliferation. The modulation of pro- and anti-apoptotic genes leads to apoptosis induction; therefore, it is tempting to speculate that an altered ratio of proapoptotic (e.g. Bax and tBid) and antiapoptotic (e.g. Bcl-XL) cellular proteins is the major component that makes human hepatoma cell lines susceptible to apoptotic cell death. [This figure appears in colour on the web.] Journal of Hepatology 2005 42, 210-217DOI: (10.1016/j.jhep.2004.10.020) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions