J Korean Med Sci.  2011 Feb;26(2):214-221. 10.3346/jkms.2011.26.2.214.

Could HBx Protein Expression Affect Signal Pathway Inhibition by Gefitinib or Selumetinib, a MEK Inhibitor, in Hepatocellular Carcinoma Cell Lines?

• Park YK
• Kim KM
• Lee YJ
• Kim KH
• Lee SG
• Lee D
• Shim JH
• Lim YS
• Lee HC
• Chung YH
• Lee YS
• Suh DJ

• KMID: 1782109
• DOI: http://doi.org/10.3346/jkms.2011.26.2.214

Abstract

Hepatitis B virus X (HBx) protein has been known to play an important role in development of hepatocellular carcinoma (HCC). The aim of this study is to find out whether HBx protein expression affects antiproliferative effect of an epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor and a MEK inhibitor in HepG2 and Huh-7 cell lines. We established HepG2 and Huh-7 cells transfected stably with HBx gene. HBx protein expression increased pERK and pAkt expression as well as beta-catenin activity in both cells. Gefitinib (EGFR-TK inhibitor) inhibited pERK and pAkt expression and beta-catenin activity in both cells. Selumetinib (MEK inhibitor) reduced pERK level and beta-catenin activity but pAkt expression was rather elevated by selumetinib in these cells. Reduction of pERK levels was much stronger with selumetinib than gefitinib in both cells. The antiproliferative efficacy of selumetinib was more potent than that of gefitinib. However, the antiproliferative effect of gefitinib, as well as selumetinib, was not different between cell lines with or without HBx expression. Signal pathway activation by HBx might not be strong enough to attenuate the antiproliferative effect of EGFR-TK inhibitor. Future experiments are needed to understand the role of HBx protein expression in HCC treatment using molecular targeting agent.

Keyword

Hepatocellular Carcinoma; HBx protein; Gefitinib; Selumetinib; EGFR; MEK

MeSH Terms

Animals
Antineoplastic Agents/*pharmacology
Benzimidazoles/*pharmacology
Carcinoma, Hepatocellular/*metabolism
Cell Line, Tumor/drug effects
Cell Proliferation
Extracellular Signal-Regulated MAP Kinases/metabolism
Humans
Liver Neoplasms/*metabolism
Mitogen-Activated Protein Kinase Kinases/*antagonists & inhibitors
Protein Kinase Inhibitors/*pharmacology
Proto-Oncogene Proteins c-akt
Quinazolines/*pharmacology
Receptor, Epidermal Growth Factor/antagonists & inhibitors
Signal Transduction/*drug effects
Trans-Activators/*metabolism
beta Catenin/metabolism

Figure

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  Fig. 1 Establishment of HBx protein expressing HCC cell lines. RT-PCR with HBx primer (A) and immunofluorescence (IF) staining (× 400) of HepG2 (B) and Huh-7 cells (C) after pEGFP-N1 or pEG-HBx transfection in HepG2 and Huh-7 cells.

• 

  Fig. 2 pERK and pAkt expression and β-catenin activity changes by gefitinib or selumetinib in HepG2 and Huh-7 cells. Western blotting was performed for pERK, EGFR and pMEK in pEGFP-N1 or pEG-HBx transfected HepG2 and Huh-7 cells with or without TGF-α treatment (A). pERK, pAkt, β-catenin expression was also measured after the treatment of gefitinib (B) or selumetinib (C) in both cells. β-catenin activity was measured in Huh-7 cells with or without the treatment of gefitinib or selumetinib by TCF reporter gene assay (D). *P < 0.001.

• 

  Fig. 3 Antiproliferative effect of gefitinib in HepG2 and Huh-7 cells. MTS assay was performed in pEGFP-N1 or pEG-HBx transfected HepG2 (A) or Huh-7 cells (B) with or without gefitinib.

• 

  Fig. 4 Antiproliferative effect of selumetinib in HepG2 and Huh-7 cells. MTS assay was performed in pEGFP-N1 or pEG-HBx transfected HepG2 (A) or Huh-7 cells (B) with or without selumetinib.

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  Fig. 5 Antiproliferative effect of gefitinib or selumetinib in primary cultured HCC cell line. MTS assay was performed in primary cultured HCC cell line with gefitinib or selumetinib.

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  Fig. 6 Suggested role of HBx protein in signal pathways of hepatocarcinogenesis.

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