Cepharanthine hydrochloride inhibits the Wnt/β-catenin/Hedgehog signaling axis in liver cancer

Cepharanthine, a biscoclaurine alkaloid isolated from the roots of Stephania cephalantha Hayata, has been reported to demonstrate antitumor activity across multiple cancer types; however, the mechanisms are still under investigation. High transcriptional responses by both the Hedgehog and Wnt pathways are frequently associated with specific human cancers, including liver cancer. To investigate whether these signaling pathways are involved in the pharmaceutical action of cepharanthine, we investigated Hedgehog and Wnt signaling in models of liver cancer treated with a semi-synthetic cepharanthine derivative, cepharanthine hydrochloride (CH), in vitro and in vivo. By using MTT cytotoxic, scratch, Transwell, colony formation and flow cytometry assays, the pharmaceutical effect of CH was assessed. The compound was found to inhibit cellular proliferation and invasion, and promote apoptosis. Subsequent mechanistic investigations revealed that CH suppressed the Hedgehog/Gli1 signaling pathway by inhibiting Gli1 transcription and its transcriptional activity. CH also inhibited Wnt/beta-catenin signaling, and the pathway was found to be an upstream regulator of Hedgehog signaling in CH-treated liver cancer cells. Finally, the antitumor effects of CH were demonstrated in an in vivo xenograft tumor model. Immunohistochemical analysis indicated that Gli1 protein levels were diminished in CH-treated xenografts, compared with that noted in the controls. In summary, our results highlight a novel pharmaceutical antitumor mechanism of cepharanthine and provide support for CH as a clinical therapy for refractory liver cancer and other Wnt/Hedgehog-driven cancers.