1. Volume 41, Issue 6, Pages 1008-1016 (December 2004)
Targeting the epidermal growth factor receptor by gefitinib for treatment of hepatocellular carcinoma 
Michael Höpfner, Andreas P. Sutter, Alexander Huether, Detlef Schuppan, Martin Zeitz, Hans Scherübl 
Journal of Hepatology 
Volume 41, Issue 6, Pages (December 2004)
DOI: /j.jhep
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
mRNA and protein expression of EGFR and IGFR in hepatocellular carcinoma cells. (A) mRNA expression of IGFRβ-1 (lanes 2 and 5), EGFR-1 (lanes 3 and 4) and EGFRvIII (lanes 6 and 7) were evaluated in HepG2 (lanes 2, 3 and 6) and Huh-7 cells (lanes 4, 5 and 7). The expression of the housekeeping gene β-actin (amplicon: 822bp) in HepG2 (lane 8) and Huh-7 cells (lane 9) was analyzed for standardization. Lane 1: 100bp DNA ladder. (B, C) Flow cytometric analysis of EGFR and IGFR-β1 protein expression in HepG2 (B) and Huh-7 cells (C). Black lines: cells stained with antibodies against either EGFR or IGFR-β1. Grey lines: isotypic controls.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Gefitinib-induced growth inhibition. Gefitinib caused a time- and dose-dependent growth inhibition in HepG2 and Huh-7 cells which was significant for 10–25μM. The IC50 values amounted to 12.2±0.6μM in HepG2 (A) and 16.6±0.4μM in Huh-7 cells (B). Data are given as percentage of untreated controls (means±SEM of four independent experiments).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Induction of cell cycle arrest by gefitinib. After 24h of incubation with gefitinib HepG2 (A) and Huh-7 cells (B) dose-dependently accumulated in the G0/G1-phase of the cell cycle. Accordingly, the proportion of cells in the S-phase decreased. In HepG2 cells, gefitinib additionally induced an arrest in the G2/M phase at high concentrations (20–25μM). Means of four independent experiments are shown. The difference of the proportion of cells in a particular phase of the cell cycle versus control was significant for 10–25μM in either cell line. *, statistically significant (P<0.05).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Gefitinib-induced apoptosis. In Huh-7 (A) and HepG2 (B) cells, gefitinib (1-20μM) dose-dependently induced caspase-3 activation after 24h of incubation. To monitor changes in the mitochondrial membrane potential (ΔΨM), Huh-7 and HepG2 cells were stained with JC-1 and analyzed by flow cytometry. After 3–12h of incubation with gefitinib (5–20μM), Huh-7 cells (C, top) showed a time- and dose-dependent hyperpolarization of ΔΨM, which returned to control values after 24h. In parallel there was a time- and dose-dependent increase in mitochondrial volume (C, bottom). While HepG2 cells demonstrated gefitinib-induced changes in ΔΨM (D, top), no appreciable changes in mitochondrial volume were observed (D, bottom). Data are given as percentage of untreated controls (means±SEM of three independent experiments for each cell line).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Effects of gefitinib on MAP kinase activity and on the expression of Bcl-2 and Bcl-XL. (A) HepG2 cells were treated with gefitinib (1–25μM) for 4–24h. Phosphorylation of ERK1/2 was analyzed by Western blotting using antibodies against the active form (p-ERK) or against total ERK1/2. Gefitinib induced a time- and dose-dependent decrease of p-ERK in HepG2 cells. (B) The antiapoptotic proteins Bcl-XL and Bcl-2 were dose-dependently downregulated by gefitinib. Representative results out of three independent experiments are shown.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions