title: High-throughput screening for identification of inhibitors of EpCAM-dependent growth of hepatocellular carcinoma cells.
abstract: The cancer stem cell marker, EpCAM, is an important indicator of Wnt/β-catenin signaling activation and a functional component of hepatocellular tumor-initiating cells. A high-throughput screening assay was developed to identify inhibitors of EpCAM-dependent growth of hepatocellular carcinoma (HCC) cells. EpCAM(+) and EpCAM(-) HCC cell lines were assessed for differential sensitivity to a Wnt/β-catenin pathway inhibitor. Libraries comprising 22 668 pure compounds and 107 741 crude or partially purified natural product extracts were tested, and 12 pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized in terms of effects on EpCAM expression. Treatment of EpCAM(+) Hep3B cells resulted in loss of EpCAM expression as assessed by flow cytometry. This reduction was incomplete (most cells continued to express EpCAM), but resulted in generation of cell populations expressing lower levels of EpCAM. Sublethal concentrations (~IC50 ) reduced median EpCAM expression to 28% of control after 1 day and 19% of control after 2 days. Reduction in EpCAM expression preceded growth inhibition suggesting that a threshold of EpCAM expression may be required for growth of EpCAM-dependent cells. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of EpCAM-dependent cell growth.
authors.affiliation: Molecular Targets Laboratory, National Cancer Institute, Frederick, MD, USA. henrichcj@mail.nih.gov
authors.forename: Curtis J
authors.initials: CJ
authors.lastname: Henrich
authors.forename: Anuradha
authors.initials: A
authors.lastname: Budhu
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Yu
authors.forename: Jason R
authors.initials: JR
authors.lastname: Evans
authors.forename: Ekaterina I
authors.initials: EI
authors.lastname: Goncharova
authors.forename: Tanya T
authors.initials: TT
authors.lastname: Ransom
authors.forename: Xin W
authors.initials: XW
authors.lastname: Wang
authors.forename: James B
authors.initials: JB
authors.lastname: McMahon
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001688:Biological Products / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors* / Q000378:metabolism; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D000071858:Epithelial Cell Adhesion Molecule; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D054852:Small Molecule Libraries / Q000494:pharmacology; D051153:Wnt Proteins / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D001688:Biological Products; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D054852:Small Molecule Libraries; D051153:Wnt Proteins; D051176:beta Catenin
keywords: Wnt/β-catenin; epithelial cell adhesion molecule; hepatocellular carcinoma; high-throughput screening