Abstract 1328: Arenobufagin induces apoptosis, autophagy and cell cycle arrest in hepatocellular carcinoma cells

Abstract

Abstract Backgrounds: Hepatocellular carcinoma (HCC) is a deadly form of cancer without effective chemotherapy so far. Thus, there is an urgent need for novel chemotherapeutic agents for the treatment of HCC. Toad venom is frequently used in the treatment of liver cancer in traditional Chinese medicine. However, the exact component and the precise underlying mechanisms against tumor cells remain unclear. In the present research, we sought to study the antitumor mechanism of arenobufagin, a major active ingredient isolated from toad venom, in HCC cells. Methods and Results: The in vitro and in vivo antitumor activity was measured by colonic formation and HepG2/ADM tumor xenograft, respectively. Arenobufagin-induced HepG2 and HepG2/ADM cell apoptosis was detected by Annexin V-FITC staining and transmission electron microscope. The mitochondrial membrane potential measured by JC-1 staining combined with the changes of expression of Caspase9, Caspase3, PARP, Bax and Bcl-2 in HepG2 and HepG2/ADM cells suggested arenobufagin induced apoptosis via the mitochondrial pathways. Increasing autophagosomes that identified by Cyto-ID® staining and transmission electron microscope were observed in HepG2/ADM cells after arenobufagin treatment. Autophagy-specific inhibitors (e.g. 3-methyladenine, chloroquine and bafilomycin A1), small interfering RNAs target Beclin1 and Atg5 enhance arenobufagin-induced apoptosis, indicated that arenobufagin-mediated autophagy may protects HepG2/ADM cells from undergoing apoptotic cell death. We also demonstrated that inhibition of PI3K/Akt/mTOR pathway promotes the development of both autophagy and apoptosis caused by arenobufagin treatment. In addition, arenobufagin arrested HCC cells in G2 phase was determined by flow cytometry and p-Histone H3 (Ser10) staining. Arenobufagin caused double-strand DNA breaks and triggered the DNA damage response was evaluated by comet assay andγH2AX staining, and these effects were partly via the ATM/ATR-Chk1/Chk2-Cdc25C signaling pathway. We used a synthetic biotinylated arenobufagin-conjugated chemical probe in live cells to show that arenobufagin accumulated mainly in the nucleus. The microscopic thermodynamic parameters measured using isothermal titration calorimetry (ITC) also demonstrated that arenobufagin directly bound to DNA in vitro. The spectral characteristics of DNA (e.g. UV-visible absorption spectrum, circular dichroism spectrum and fluorescence intensity of the ethidium bromide-DNA system) indicated that arenobufagin bound to DNA by intercalation. Molecular modeling suggested arenobufagin intercalated with DNA via hydrogen bonds between arenobufagin and GT base pairs. Conclusion: This study provides novel insights into arenobufagin-induced HCC cells apoptosis, autophagy and cell cycle arrest. It is valuable for the further investigation of the use of arenobufagin in clinical anticancer chemotherapy. Citation Format: Min-Feng Chen, Li-Juan Deng, Wen-Cai Ye, Dong-Mei Zhang. Arenobufagin induces apoptosis, autophagy and cell cycle arrest in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1328.