Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. It is intrinsically resistant toward standard chemotherapy, making it imperative to develop novel selective chemotherapeutic agents. The Wnt/beta-catenin pathway plays critical roles in development and oncogenesis, and is dysregulated in HCC. This study aims to evaluate the activity of three small molecule antagonists of the Tcf4/beta-catenin complex (PKF118-310, PKF115-584, and CGP049090) on HCC cell lines in vitro and in vivo. All three chemicals displayed dose-dependent cytotoxicity in vitro against all three HCC cell lines (HepG2, Hep40, and Huh7), but were at least 10-times less cytotoxic to normal hepatocytes (from three donors) by using ATP assay. In HepG2 and Huh7 cells, treatment with the antagonists decreased Tcf4/beta-catenin binding capability and transcriptional activity, associated with down-regulation of the endogenous Tcf4/beta-catenin target genes c-Myc, cyclin D1, and survivin. In HepG2 and Huh7 cells, treatment with the antagonists induced apoptosis and cell cycle arrest at the G1/S phase. All antagonists suppressed in vivo tumor growth in a HepG2 xenograft model, associated with apoptosis and reduced c-Myc, cyclin D1, and survivin expressions. Our results suggest that these three antagonists of the Tcf4/beta-catenin complex are potential chemotherapeutic agents which may offer a pathway specific option for the clinical management of HCC. (c) 2009 UICC.