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AIM Daphnoretin is a bicoumarin compound isolated from natural product, which is used to treat many diseases in the history.Based on the anti-tumor activity of daphnoretin, we tested the pro-apoptotic effect of daphnoretin and investigated the molecular mechanism involved.METHODS The inhibitory effects of daphnoretion on viabilityand proliferation of HeLa cells were determined by MTT assay and EdUincorporation assay.Daphnoretin-induced apoptotic morphological changes were analyzed by Hoechst staining.The number and stage of apoptotic Hela cells were determined by flow cytometry.Gene expression was determined by reverse transcription polymerase chain reaction.Protein expression was determined by Western blot.The caspase activity of HeLa cells was detected by caspase 3 colorimetric assay kit and caspase 9 colorimetric assay kit.RESULTS Daphnoretin significantly inhibited viability and proliferation of HeLa cells by its pro-apoptotic activity.The apoptotic ceils exhibited strong, nuclear blue fluorescence after the Hoechst treatment, and lost the function of tubule formation in Matrigel.Unlike carboplatin treatment, daphnoretin mainly induced cell apoptosis at early stage.Daphnoretinhas no effect on mRNA level of bax, bcl-2 and p53.However, it could significandy promoted protein expression of Bax, caspase 3 and easpase 9, decreased protein expression of bcl-2.Besides, daphnoretin induced both caspase 3 and caspase 9 activity significandy, suggesting a possible mitochondia-mediated pathway.CONCLUSION We proved the pro-apoptotic effect of daphnoretin and investigated the possible mitochondria pathway in the daphnorotin-induced apoptosis in HeLa cells.