Synergistic Anti-cancer Effect Of Bufalin And Sorafenib On Human Hepatocellular Carcinoma Cell Lines

ObjectiveTo investigate the mechanism(s) by which the multikinase inhibitor sorafenib and a cardiotonic steroid bufalin interact to inhibit human hepatocellular carcinoma cell lines, PLC/PRF/5 (p53 mutant) and HepG-2(p53 wild type).Materials and MethodsEffects of sorafenib and bufalin as single and combinatorial agents were examined on cultured cells using CCK-8 assay. CalcuSyn software was used to assess synergistic or additive inhibition. Western blotting measured cooperative effects on MAPK and PI3K/Akt signaling pathways.ResultsConcurrent exposure of both drugs at fixed ratio (bufalin:sorafenib,1:25) for 48h resulted in synergistic interactions in both cell lines. The IC50 of Bufalin in PLC/PRF/5 and HepG-2 were 157.87nM,33.65nM. The IC50 of Sorafenib in PLC/PRF/5 and HepG-2 were 3565.7nM,5719.21nM. The IC50 of combination group in PLC/PRF/5 and HepG-2 were 105.83 nM (Bufalin 53.02nM+Sorafenib 1325.41nM) and 27nM (Bufalin 13.52nM+Sorafenib 338.03nM)Sorafenib inhibited cell growth by down-regulating pERK, While bufalin could slightly up-regulate pAKT. Sorafenib and bufalin synergistically inhibited cultured cells by cooperatively up-regulating pAKT, down-regulating FoxO3a and pERK1/2. Compared with the combination group treated with the PI3K/Akt inhibitor, LY294002, pAKT was not significantly increased in the group without LY294002, while pERK expression was up-regulated.Conclusion This study showed that bufalin and sorafenib causing synergistic inhibition at low dose. PLC/PRF/5 was more sensitive in combination group than HepG-2.The possible mechanism involved were a) Sorafenib and bufalin synergistically inhibit the expression of pERK; b) Akt activation could induce ROS-mediated apoptosis by increasing intracellular ROS through increased oxygen consumption and by inhibiting the expression of ROS scavengers downstream of FoxO3a. Up-regulation of ROS then induced cell senescence (HepG-2 p53+) or death (PLC/PRF/5 p53-); c) Meanwhile, the crosstalk between MAPK signaling pathway and PI3K/Akt signaling pathway occurred. The up-regulated pAKT could down-regulate the expression of pERK.