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

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 (1β-OH-ABF), displays anti-hepatoma activities. However, the anti-hepatoma effects and molecular mechanisms of 1β-OH-ABF have not been defined. 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. 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 and colony formation assay. Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1β-OH-ABF. 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. The role of mTOR in 1β-OH-ABF-induced apoptosis was investigated using small interfering RNA (siRNA) transfection. Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1β-OH-ABF in vivo. We found that 1β-OH-ABF inhibits the proliferation of Hep3B, HepG2, HuH7, SK-HEP-1 cells but has little cytotoxicity towards LO2 cells. 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. Mechanistically, 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. Critically, 1β-OH-ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model. 1β-OH-ABF induces mitochondrial apoptosis through the suppression of mTOR signaling in vitro and in vivo, indicating that 1β-OH-ABF may serve as a potential agent for the treatment of liver cancer. Copyright © 2020 Elsevier B.V. All rights reserved.

Citation

Li-Juan Deng, Yu-He Lei, Jing-Yu Quan, Bao-Jing Li, Dong-Mei Zhang, Hai-Yan Tian, Ye Chen, En-Xin Zhang, Lei Chen, Wen-Cai Ye, Wei-Min Ning, Lin-Zhong Yu, Jun-Shan Liu. 1β-OH-arenobufagin induces mitochondrial apoptosis in hepatocellular carcinoma through the suppression of mTOR signaling pathway. Journal of ethnopharmacology. 2021 Feb 10;266:113443

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PMID: 33022344

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