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ObjectiveTo study the roles and potential mechanism of bufalin-mediated radiosensitization of hepatocellular carcinoma cell linesMethods1. Human hepatocellular carcinoma cell lines PLC/PRF/5, HepG2, smmc7721 and sk-hepl had been treated with bufalin for 24 hours. Cells growth inhibition was analyzed by WST (water-soluble tetrazolium salt) assay.2. Cell clonogenic survival assay was utilized to detect the bufalin-mediated radiosensitization of hepatocellular carcinoma cell lines.3. Cell cycle of the PLC/PRF/5 cell line before and after irradiation was detected by Flow Cytometry (FCM).4. The expression of some proteins in MAPK signal transduction pathway、STAT3 signal transduction pathway、NFκB signal transduction pathway were detected by Western Blot (WT).Results1. The hepatocellular carcinoma cell lines had been treated with various concentration of bufalin for 24h, the OD (optical density) value of each well was detected by WST-8. The IC50 of bufalin in PLC/PRF/5, HepG2, smmc7721 and sk-hepl were 25.40nM,122.30nM,70.38nM and 34.76nM, respectively.2. Through cell clonogenic survival assay, it was indicated that bufalin can improve the radiosensitivity of PLC/PRF/5, HepG2, smmc7721 and sk-hepl. The SER (sensitizing enhancement ratio) of PLC/PRF/5, HepG2, smmc7721 and sk-hepl was 1.041,1.255,1.462 and 1.298, respectively. 3. There was little different in cell cycle between the group of irradiation combining with bufalin and the group of irradiation alone through FCM. While after the irradiation the combining group was more easily blocked at the stages of G2/M than the group of irradiation alone.4. The expression of ERK1/2, JNK, p38, STAT3, NFκB, p-ERK1/2, p-JNK, p-p38, p-STAT3 and p-NFκB was detected by WT. After irradiation the level of activated p-STAT3 began to increase at the point of 30min while the other proteins with little changed in the group of irradiation alone. In contrast the level of activated p-STAT3 in the group of irradiation combining with bufalin was decreased in the beginning. The levels of activated p-JAK1, p-JAK2, p-Src were also detected between the combined group and the group of irradiation alone after irradiation. We found that only the level of p-JAK2began to increase at the point of 30minafter irradiation in the irradiation alone group which was reversed in the combined group. SD-1008, the JAK2/STAT3 inhibitor, was also inhibited the levels of activated p-JAK2 andp-STAT3 after irradiation. And the improved radiosensitivity of PLC/PRF/5 by SD-1008 was also detected by the cell clonogenic survival assay. This was indicated that the mechanism of bufalin-mediated radiosensitization of hepatocellular carcinoma cell lines may be by inhibition of the JAK2/STAT3 signal transduction pathway which was similar to the JAK2/STAT3 inhibitor SD-1008.Conclusion1. Bufalin could improve the radiosensitivity of PLC/PRF/5, HepG2, smmc7721 and sk-hepl in vitro.2. The mechanism of bufalin-mediated radiosensitization of hepatocellular carcinoma cell lines may be by inhibitingthe JAK2/STAT3 signal transduction pathway and by blocking the cell at the stages of G2/M.
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