J Korean Med Sci.  2011 Dec;26(12):1563-1568. 10.3346/jkms.2011.26.12.1563.

Novel EGFR-TK Inhibitor EKB-569 Inhibits Hepatocellular Carcinoma Cell Proliferation by AKT and MAPK Pathways

• Kim H
• Lim HY

• KMID: 1786011
• DOI: http://doi.org/10.3346/jkms.2011.26.12.1563

Abstract

Epidermal growth factor receptor (EGFR)-targeted therapies have been effective in some cancers, but not in hepatocellular carcinoma (HCC). The aim of this study was to investigate the drug potential to overcome multi-drug resistance in HCC cells. Thirteen drug-sensitive HCC cells were assessed using the CCK-8 assay. G0-G1 arrest was measured by FACS. Western blot analysis was used to detect the key enzymes in both the Ras/Raf and PI3K pathways. When establishing the IC50 of HCC to several drugs, including EKB-569, sorafenib, erlotinib, gefitinib, pazopanib, and brivanib, SK-Hep1 cells treated with EKB-569 have shown the highest (72.8%-86.4%) G0-G1 arrest and decreased the phosphorylation of AKT and ERK at the protein level. We found that EKB-569 had higher efficacy in HCC, compared to first generation, reversible EGFR-TK inhibitors. Furthermore, the combination of sorafenib and EKB-569 showed a synergistic effect to inhibit proliferation of SNU-475, previously the most resistant cell to EGFR-TKIs. Therefore, novel EKB-569 in combination with sorafenib may be able to overcome HCC resistance to EGFR-TK inhibitors.

Keyword

Epidermal Growth Factor Receptor (EGFR)-Tyrosine Kinase Inhibitors (TKIs); EKB-569; Multi-drug Resistance; Hepatocellular Carcinoma (HCC) Cells

MeSH Terms

Aminoquinolines/*pharmacology
Aniline Compounds/*pharmacology
Antineoplastic Agents/*pharmacology
Antineoplastic Combined Chemotherapy Protocols/pharmacology
Benzenesulfonates/pharmacology
Carcinoma, Hepatocellular/*drug therapy/pathology
Cell Cycle Checkpoints/drug effects
Cell Line, Tumor
Cell Proliferation/drug effects
Drug Resistance, Neoplasm
Drug Synergism
Humans
Liver Neoplasms/drug therapy/pathology
Mitogen-Activated Protein Kinases/*metabolism
Phosphorylation
Proto-Oncogene Proteins c-akt/*metabolism
Pyridines/pharmacology
Receptor, Epidermal Growth Factor/*antagonists & inhibitors

Figure

• 

  Fig. 1 Detection of EKB-569-induced G0-G1 arrest by FACS analysis. SK-Hep1 cells were treated with the indicated concentrations of EKB-569 for 24 hr. EKB-569 induced G0-G1 arrest in SK-Hep1.

• 

  Fig. 2 EKB-569 decreased the phosphorylation of AKT and ERK at the protein level. SK-Hep1 cells were treated with the indicated concentrations of EKB-569 for 24 hr, and Western blotting of total-, pERK, total-, pAKT, CDK4, p21, and β-actin (loading control) was performed. Immunoblotting for each protein was done at least twice, using independently prepared lysates.

• 

  Fig. 3 Effect of sorafenib/EKB-569 co-treatment on the EGFR-targeted resistant SNU-475 cell line. SNU-475 cells were treated with sorafenib alone, EKB-569 alone, or sorafenib plus EKB-569 at the indicated concentrations, for 72 hr. Cellular viability was measured using the CCK-8 assay.

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