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Objective: To reveal the mechanisms of hellebrigenin-induced G2/M arrest and apoptosis in HepG2 cells in vitro.Methods: (1)In vitro the cytotoxic effect of hellebrigenin were investigated by MTT assay and colony formation assay; (2) DNA content, apoptosis raito and mitochondrial membrane potential were determined by flow cytometry; (3) DNA damage was evaluated by comet assay; (4) γH2A.X were evaluated by immunofluorescence; (5) Morphology of apoptotic cells was observed using Hoechst 33258 staining assay; (6) The expression level of molecules in ATM-Chk2-Cdc25C-CDK1/cyclin B1 signaling pathways, PARP, cytochrome c,caspase-9, caspase-3, Bax, Bcl-2, and Akt were analyzed by western blot.Results: Hellebrigenin potently reduced the viability of HepG2 cells in a dose-and time-dependent manner, with IC50 values of 0.40 ± 0.05 μ mol/L, 0.13 ± 0.01 μmol/L and 0.10 ± 0.01 μmol/L after 24 h, 48 h and 72 h treatment respectively.Colony formation assay was further confirmed the cytotoxic effect of hellebrigenin on HepG2 cells.Cell cycle analysis showed that hellebrigenin triggered G2/M arrest.Hellebrigenin decreased CDK1 expression level, while p-CDK1 (Tyr15) and Cyclin B1 expression levels were increased.It was found that treatment with hellebrigenin significantly increased Tail Length, Tail DNA (%) and Olive DNA Moments when compared with that of untreated cells.Further study found a cumulation of p-H2A.X (Ser139) and the activation of ATM-Chk2-Cdc25C signal pathway in hellebrigenin-treated cells.It was also found that hellebrigenin induced mitochondrial apoptosis, characterized by Bax translocation to mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c into cytosol and sequential activation of caspases and PARP.In addition, hellebrigenin markedly down-regulated Akt and p-Akt (Thr308 and Ser473) in a dose-dependent manner.Activation of Akt by pretreatment with hIGF-Ⅰ remarkably attenuated the hellebrigenin-induced cytotoxicity.Combined treatment of hellebrigenin with Akt siRNA significantly decreased the proportion of G2/M arrest when compared with cells treated with hellebrigenin alone.Silencing Akt with siRNA enhanced hellebrigenin-induced apoptosis as indicated by the substantial increase in the population of subG1 phase in cell cycle profile, apoptotic cells and specific cleavage of PARP.Conclusion: Hellebrigenin induces DNA damage to initiate the ATM-Chk1/Chk2-CDC25 DNA damage response pathway, thereby inhibiting CDK1/Cyclin B1 kinase, subsequently leading to cell cycle arrest at G2/M phase and finally initiates mitochondrial apoptosis.In addition, Akt may play an indispensable role in the process of cell cycle arrest and apoptosis in response to hellebrigenin induced DNA damage.