title: Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells.
abstract: Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho‑mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.
authors.affiliation: Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan, R.O.C.
authors.forename: Shih-Chang
authors.initials: SC
authors.lastname: Tsai
authors.forename: Jai-Sing
authors.initials: JS
authors.lastname: Yang
authors.forename: Shu-Fen
authors.initials: SF
authors.lastname: Peng
authors.forename: Chi-Cheng
authors.initials: CC
authors.lastname: Lu
authors.forename: Jo-Hua
authors.initials: JH
authors.lastname: Chiang
authors.forename: Jing-Gung
authors.initials: JG
authors.lastname: Chung
authors.forename: Meng-Wei
authors.initials: MW
authors.lastname: Lin
authors.forename: Jen-Kun
authors.initials: JK
authors.lastname: Lin
authors.forename: Sakae
authors.initials: S
authors.lastname: Amagaya
authors.forename: Cinderella
authors.initials: C
authors.lastname: Wai-Shan Chung
authors.forename: Theng-Thang
authors.initials: TT
authors.lastname: Tung
authors.forename: Wen-Wen
authors.initials: WW
authors.lastname: Huang
authors.forename: Michael T
authors.initials: MT
authors.lastname: Tseng
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D002018:Bufanolides / Q000008:administration & dosage* / Q000737:chemistry; D002023:Bufo bufo; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D059447:Cell Cycle Checkpoints / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051057:Proto-Oncogene Proteins c-akt / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000494:pharmacology; D014688:Venoms / Q000737:chemistry
chemical_list: D002018:Bufanolides; D014688:Venoms; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; C022777:bufalin