Anti-proliferative Effects of Pinocembrin Isolated From Anomianthus dulcis on Hepatocellular Carcinoma Cells

Abstract

Background: Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer. Anomianthus dulcis (Dunal) J.Sinclair (syn. Uvaria dulcis) has been used in Thai traditional medicine in various therapeutic indications. Phytochemical constituents of A. dulcis have been isolated and identified. However, their effects on liver cancer and the associated mechanisms have not been elucidated. Methods: Dry flowers of A. dulcis were extracted using organic solvents, and chromatographic methods were used to purify the secondary metabolites. The chemical structures of the pure compounds were elucidated by analysis of spectroscopic data. Cytotoxicity against HCC cells was examined using SRB assay, and the effects on cell proliferation were determined using flow cytometry. The mechanisms underlying HCC inhibition were examined by molecular docking and verified by Western blot analysis. Results: Among 3 purified flavonoids, pinocembrin, pinostrobin, and chrysin, and 1 indole alkaloid (3-farnesylindole), only pinocembrin showed inhibitory effects on the proliferation of 2 HCC cell lines, HepG2 and Li-7, whereas chrysin showed specific toxicity to HepG2. Pinocembrin was then selected for further study. Flow cytometric analyses revealed that pinocembrin arrested the HCC cell cycle at the G1 phase with a minimal effect on cell death induction. Pinocembrin exerted the suppression of STAT3, as shown by the molecular docking on STAT3 with a better binding affinity than stattic, a known STAT3 inhibitor. Pinocembrin also suppressed STAT3 phosphorylation at both Tyr705 and Ser727. Cell cycle regulatory proteins under the modulation of STAT3, namely cyclin D1, cyclin E, CDK4, and CDK6, are substantially suppressed in their expression levels. Conclusion: Pinocembrin extracted from A. dulcis exerted a significant growth inhibition on HCC cells via suppressing STAT3 signaling pathways and its downstream-regulated genes.