Volume 41, Issue 5, Pages 799-807 (November 2004) Peripheral benzodiazepine receptor ligands induce apoptosis and cell cycle arrest in human hepatocellular carcinoma cells and enhance chemosensitivity to paclitaxel, docetaxel, doxorubicin and the Bcl-2 inhibitor HA14-1  Andreas P. Sutter, Kerstin Maaser, Patricia Grabowski, Gesine Bradacs, Kirsten Vormbrock, Michael Höpfner, Antje Krahn, Bernhard Heine, Harald Stein, Rajan Somasundaram, Detlef Schuppan, Martin Zeitz, Hans Scherübl  Journal of Hepatology  Volume 41, Issue 5, Pages 799-807 (November 2004) DOI: 10.1016/j.jhep.2004.07.015 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Expression of PBR in hepatocellular carcinoma (HCC) cells. (A) PBR mRNA expression in Huh-7 (lane 1) and HepG2 cells (lane 2) detected by RT-PCR. The expression of the housekeeping gene β-actin (amplicon: 822bp) in Huh-7 (lane 3) and HepG2 cells (lane 4) was analyzed for standardization. (B) PBR protein expression in HCC cells was shown by flow cytometry. Huh-7 (left panel) and HepG2 cells (right panel) were stained with a polyclonal anti-PBR antibody with (gray area) or without (gray line) previous membrane permeabilization. Black line: isotypic control. Journal of Hepatology 2004 41, 799-807DOI: (10.1016/j.jhep.2004.07.015) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Localization of PBR in HCC tissues and cells. (A, B) Immunohistochemical detection of PBR (red) in HCC tissue and in adjacent non-neoplastic tissue (NO). (A) Bar=200μm; (B) Bar=100μM. (C–E) For immunocytochemistry, PBR was immunostained with a polyclonal antibody (C), and mitochondria were marked with CMTMRos (D). Superposition of both fluorescence images resulted in a bright yellow color (E), indicating a co-localization of PBR and mitochondria in Huh-7 cells. Bar=10μM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). Journal of Hepatology 2004 41, 799-807DOI: (10.1016/j.jhep.2004.07.015) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Anti-proliferative effects of PBR ligands. The PBR ligands FGIN-1-27 and PK 11195 induced a time- and dose-dependent growth inhibition in Huh-7 (A) and HepG2 (B) cells. Growth inhibition was significant for 10–100μM FGIN-1-27, and for 25–100μM PK 11195 (P<0.05). Means of four independent experiments are shown. (C) PBR- and tumor cell specificity of the growth inhibitory effects. In contrast to PBR ligands, FGIN-1-52 did not inhibit the growth of Huh-7 (white columns) or HepG2 cells (hatched columns; Fig. 2C, left panel). Non-malignant human primary keratinocytes remained nearly unaffected by incubation with 50μM FGIN-1-27 (white columns) or 75μM PK 11195 (black columns), in contrast to their inhibitory actions in Huh-7 and HepG2 cells (Fig. 2C, right panel). Journal of Hepatology 2004 41, 799-807DOI: (10.1016/j.jhep.2004.07.015) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Mitochondrial alterations and apoptosis induction by PBR ligands. Huh-7 (black symbols) and HepG2 cells (open symbols) cells were incubated with 10–100μM FGIN-1-27 (squares) or PK 11195 (circles). FGIN-1-27 and PK 11195 dose-dependently decreased the ΔΨM after 24h of incubation (A), induced caspase-3 activation after 48h of incubation (B), and increased the proportion of apoptotic cells measured as subdiploidy after 96h of incubation (C). Data are shown as means±SEM of 4 independent experiments. *Statistical significance (P<0.05) compared to untreated controls. Journal of Hepatology 2004 41, 799-807DOI: (10.1016/j.jhep.2004.07.015) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 PBR ligands modulate the expression of pro- and anti-apoptotic proteins. Modulation of protein expression in HepG2 cells was assessed by Western blot analysis after 96h of treatment with PBR ligands. The anti-apoptotic proteins Bcl-XL and Bcl-2 were dose-dependently downregulated by PBR ligands, whereas the pro-apoptotic protein Bax was upregulated. A representative result out of three independent experiments is shown. Journal of Hepatology 2004 41, 799-807DOI: (10.1016/j.jhep.2004.07.015) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Induction of cell cycle arrest in the G0/G1 and G2/M phases by PBR ligands. After 24h of incubation of Huh-7 (A) and HepG2 cells (B) with PBR ligands, cells accumulated in the G0/G1 phase (white columns) of the cell cycle, while the proportion of cells in the S phase (hatched columns) decreased. Means of four independent experiments are shown. When compared to control, significant differences of the proportion of cells in the G0/G1 phase of the cell cycle were observed in response to FGIN-1-27 and PK 11195 from 50–100μM (P<0.05). Additionally, a significant (P<0.05) arrest in the G2/M phase (black columns) was observed in HepG2 cells after treatment with FGIN-1-27 or PK 11195 (100μM). Journal of Hepatology 2004 41, 799-807DOI: (10.1016/j.jhep.2004.07.015) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Synergistic growth inhibition of HCC cells by cytostatic drugs or the Bcl-2 inhibitor HA14-1 when combined with the PBR ligand FGIN-1-27. Combination treatment with sub-IC50 concentrations of cytostatic agents (paclitaxel, docetaxel, doxorubicin) or the Bcl-2 inhibitor HA14-1 and the PBR-ligand FGIN-1-27 for 96h led to synergistic growth inhibitory effects in Huh-7 (A) and HepG2 cells (B). Black bars indicate the values of the calculated additive growth inhibition. Data are given as percentage of untreated controls (means±SEM of at least 3 independent experiments). Journal of Hepatology 2004 41, 799-807DOI: (10.1016/j.jhep.2004.07.015) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 8 Synergistic apoptotic effects of a combination of PBR ligands and paclitaxel in Huh-7 cells. The combination of either the PBR ligand FGIN-1-27 or PK 11195 with paclitaxel led to a synergistic increase of hypodiploid Huh-7 cells after 96h of treatment. The percentage of sub-G1 apoptotic cells is noted on each histogram. Representative data from three independent experiments are shown. Journal of Hepatology 2004 41, 799-807DOI: (10.1016/j.jhep.2004.07.015) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions