Mechanism Study Of 1β-OH-arenobufagin In Apoptosis Induction In Hepatocellular Carcinoma Based On MTOR Signaling Pathway

Objective:Chansu,dried secretions from Bufonidae,has long been used for cancer treatment as a traditional Chinese medicine.In searching for effective anti-hepatoma agents from Chansu,our preliminary drug screening found that a bufadienolide,namely 1β-hydroxyl-arenobufagin(1βP-OH-ABF),displays anti-hepatoma activities.However,the anti-hepatoma effects and molecular mechanisms of 1β-OH-ABF have not been defined.In this study,we aim to evaluate the anti-hepatoma activity of 1β-OH-ABF against liver cancer Hep3B and HepG2 cells in vitro and in vivo,as well as explore the underlying mechanisms.Methods:(1)The anti-proliferative effects of 1β-OH-ABF on liver cancer Hep3B,HepG2,HuH7,SK-HEP-1 and normal hepatocyte LO2 cells were examined by MTT assay.The long-term proliferation inhibition of Hep3B and HepG2 was detected by cell clone formation method.(2)Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1β-OH-ABF.(3)The collapse of the mitochondrial membrane potential(ΔΨm)was detected by JC-1 staining assay.Western blotting was used to examine the expression levels of targeted proteins,including caspase-3,caspase-9,PARP,Bcl-2,Bax.(4)The role of mTOR in 1β-OH-ABF-induced apoptosis was investigated using small interfering RNA(siRNA)transfection.(5)Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1β-OH-ABF in vivo.Results:(1)We found that 1β-OH-ABF inhibits the proliferation of Hep3B,HepG2,HuH7,SK-HEP-1 cells but has little cytotoxicity in L02 cells.(2)1β-OH-ABF induces mitochondria dysfunction and triggers mitochondria apoptotic pathway,which is accompanied by the loss of ΔΨm,upregulation and translocation of Bax,as well as cleavages of caspase-9,caspase-3 and PARP.(3)1β-OH-ABF markedly decreases the expression level of p-Akt/Akt and p-mTOR(Ser2248 and Ser2481)/mTOR in a time-dependent manner.Inhibition of mTOR by siRNA strengthens 1β-OH-ABF-mediated apoptosis.(4)1β-OH-ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model.Conclusion:1β-OH-ABF induces mitochondrial apoptosis through the suppression of mTOR signaling,indicating that 1β-OH-ABF may serve as a potential agent for the treatment of liver cancer.