Abstract:

BACKGROUND: Chrysin, a dietary flavonoid or natural product, widely distributed in health food and Chinese herbal medicine, has promising cancer-preventive and cancer-therapeutic activities. Some problems remain poorly understood: whether chrysin is beneficial to liver cancer prevention and treatment? What are the underlying mechanisms? Whether chrysin promotes the differentiation of liver cancer stem cells?
OBJECTIVE: By using human hepatocellular carcinoma cells as the subject, the study aimed to uncover the subcellular and molecular mechanisms of actions for chrysin.
METHODS: The acid phosphatase assay, scanning electron microscopy and immunoblotting were used to investigate the effects of chrysin on the BEL-7402 cells, and to reveal the underlying anticancer mechanisms.
RESULTS AND CONCLUSION: Chrysin has potent antiproliferative activity against cultured BEL-7402 cells, with an IC50 of  24.9 mol/L or 6.3 g/mL. When administered together with LY294002, a specific inhibitor of the PI3K-AKT pathway, chrysin cooperatively and significantly augmented the anticancer effect. However, chrysin alone did not lead to changes in the expression levels of AKT, phosphorylated AKT, and AKT’s downstream molecule GSK-3 and phospho-GSK-3, though β-catenin reduced slightly under a higher dosage. In chrysin-treated cells, the density of the microvillus-like protrusions on the cell surfaces was drastically increased; Membrane nanoparticles were emerging from the protrusions. Cells were severely retracted, leaving wide gaps between cultured cells. There were also dying or dead cells that were morphologically different from apoptotic cells. Numerous phospho-Ser/Thr bands were changed in chrysin-treated cells. Chrysin also caused extensive dephosphorylation of tyrosine on proteins, which was also seen in positive control or epirubicin-treated cells. No significant changes were observed in the levels of CDK1, phospho-CDK1/2, CDC25A, CDC25B and CD133. However, dose-dependent induction of PARP-1 fragmentation was observed upon chrysin treatment. Taken together, the antiproliferative effect of chrysin on the BEL-7402 cells was not resulted from blocking of the PI3K-AKT or Wnt/β-catenin pathway, nor from blocking of the key regulatory factors in cell cycle, but was likely resulted from ultrastructural alterations on the cell surfaces due to malfunction of biomembranes, and widespread modifications of protein phosphorylation. As a consequence, non-classical cell death was likely induced in chrysin-treated liver cancer cells.