Synergy With The Active Ingredients Of Toad Venom And Sorafenib Suppresses Hepatocellular Carcinoma HepG2 Cells Proliferation Through Down-regulating Akt/NF-κB Signaling Pathways

ObjectiveTo investigate the effect of the active ingredients of Toad Venom(bufalin and cinobufagin)combined with sorafenib on the proliferation and apoptosis of human hepatoma Hep G2 cells and the underlying mechanisms,and to provide a theoretical basis for the combined use of traditional Chinese medicine and molecular targeted drugs.MethodsThe rate of inhibition after treated with drugs 12 h,24 h,48 h were detected by MTT assay.The changes of cells morphology were detected by Hoechst33342 fluorescent staining.The changes of cell cycle were detected by flow cytometry.Western blot method was used to detect the expressions of proteins such as Akt,p-Akt,IκB,NF-κBp65,p-NF-κBp65,Bcl-2,Bax,Caspase3,Caspase8,Cyclin A,CDK2,PCNA,and detect the expression of proteins such as Akt,p-Akt in HCC Hep G2 cells treated with the monotherapy and combination of cinobufagin and sorafenib after LY294002 inhibited Akt signaling pathwayResultsBufalin,cinobufagin and sorafenib could inhibit the Hep G2 cells proliferation,and presenting in a does-and time-dependent manner.Meanwhile,it could significantly increase the inhibitory rate of cells compared with those of alone treatment and they played a synergistic activity in sorafenib combined with cinobufagin or bufalin by Jin Formula after 24 h treatment(P<0.01).The results of fluorescence staining showed that the morphological features of nuclear condensation were observed,and the combination trearments were significantly observed.Sorafenib induced the cell cycle G0/G1 phase arrest(P<0.05),and bufalin,cinobufagin and the combination trearments generated the cell cycle S phase arrest(P<0.05).The results of Western blot showed that the expressions of Akt,NF-κBp65 were not obviously changing between control and all other treatments(P>0.05).The expression levels of p-Akt,p-NF-κBp65,Bcl-2,PCNA,Cyclin A and CDK2 in combination trearments were significantly decreased as well were significantly increased expression levels of IκB,Bax,Caspase3 and Caspase8 compared to those in alone treatments(P<0.01).And after LY294002 inhibited Akt signaling pathway,the expression of Akt was not obviously changing between control and all other treatments(P>0.05),and the expression level of p-Akt in combination with two drugs was significantly decreased compared to those in alone treatments(P<0.01).ConclusionsThe active ingredients of Toad Venom(bufalin and cinobufagin)combined with sorafenib performed a synergetic effect on the anti-cancer of Hep G2 cells by down-regulating Akt/NF-κB signaling pathway.