1β-OH-arenobufagin induces mitochondrial apoptosis in hepatocellular carcinoma through the suppression of mTOR signaling pathway

摘要

Ethnopharmacological relevance: 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 1 beta-hydroxyl-arenobufagin (1 beta-OH-ABF), displays anti-hepatoma activities. However, the anti-hepatoma effects and molecular mechanisms of 1 beta-OH-ABF have not been defined. @@@ Aim of the study: To evaluate the anti-hepatoma activity of 1 beta-OH-ABF against liver cancer Hep3B and HepG2 cells in vitro and in vivo, as well as explore the underlying mechanisms. @@@ Materials and methods: The anti-proliferative effects of 1 beta-OH-ABF on liver cancer Hep3B, HepG2, HuH7, SK-HEP-1 and normal hepatocyte LO2 cells were examined by MTT assay and colony formation assay. Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1 beta-OH-ABF. The collapse of the mitochondrial membrane potential (Delta Psi m) was detected by JC-1 staining assay. Western blotting was used to examine the expression levels of targeted proteins. The role of mTOR in 1 beta-OH-ABF-induced apoptosis was investigated using small interfering RNA (siRNA) transfection. Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1 beta-OH-ABF in vivo. @@@ Results: We found that 1 beta-OH-ABF inhibits the proliferation of Hep3B, HepG2, HuH7, SK-HEP-1 cells but has little cytotoxicity towards LO2 cells. 1 beta-OH-ABF induces mitochondria dysfunction and triggers mitochondria apoptotic pathway, which is accompanied by the loss of Delta Psi m, upregulation and translocation of Bax, as well as cleavages of caspase-9, caspase-3 and PARP. Mechanistically, 1 beta-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 1 beta-OH-ABF-mediated apoptosis. Critically, 1 beta-OH-ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model. @@@ Conclusion: 1 beta-OH-ABF induces mitochondrial apoptosis through the suppression of mTOR signaling in vitro and in vivo, indicating that 1 beta-OH-ABF may serve as a potential agent for the treatment of liver cancer.

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