title: Cinobufagin induces autophagy-mediated cell death in human osteosarcoma U2OS cells through the ROS/JNK/p38 signaling pathway.
abstract: The main objective of this study was to explore whether autophagy could be triggered by cinobufagin, and to clarify the role of autophagy in the antitumor effects of cinobufagin on U2OS cells and the underlying mechanisms. U2OS cells were exposed to 15, 30, 60 and 120 mg/l cinobufagin for 0, 12, 24 and 48 h. An MTT assay was used to measure cell viability. FITC-Annexin Ⅴ/PI staining and flow cytometry were used to analyze the apoptotic ratio, while apoptotic morphological changes were assessed by PI and Hoechst 33258 viable cell staining. The effects of autophagy on the cells were investigated with GFP-LC3b green fluorescence plasmid transfection and transmission electron microscopy. The levels of caspase-3, -8, - 9, cleaved PARP, LC3-II/LC3-I, p62 and the activation of JNK/p-38 were detected by western blot analysis. Reactive oxygen species (ROS) fluorescence intensity was examined under fluorescence microscopy with an analysis software system. Cell proliferation was obviously inhibited by cinobufagin in a dose- and time-dependent manner. The apoptosis ratio was gradually increased with treatment time as evidenced by flow cytometric analysis and Hoechst 33258 staining. Exposure to cinobufagin resulted in the activation of caspase-3, -8, -9, as well as cleaved PARP which indicated that cinobufagin induced caspase-dependent apoptosis. Autophagy was confirmed in the cinobufagin-treated cells as evidenced by formation of autophagosomes, accumulation of GFP-LC3 fluorescence particles as well as the upregulation of LC3-II/LC3-I levels. Inhibition of autophagy diminished apoptosis as detected by the MTT assays. Moreover the percentage of apoptotic cells decreased following pretreatment with 3-MA, CQ and si-beclin-1. Cinobufagin also induced phosphorylation of the JNK and p38 signaling pathway as well as ROS generation. The JNK and p38 inhibitors significantly attenuated coexistence of apoptosis and autophagy-related proteins. The ROS scavenger also prevented phosphorylation of the JNK and p38 signaling pathway. Our research proved that cinobufagin triggered apoptosis and autophagic cell death via activation of the ROS/JNK/p-38 axis.
authors.affiliation: Luoyang Orthopaedic-Traumatological Hospital and Henan Orthopaedic Hospital, Luoyang, Henan 471002, P.R. China.
authors.forename: Kun
authors.initials: K
authors.lastname: Ma
authors.affiliation: Luoyang Orthopaedic-Traumatological Hospital and Henan Orthopaedic Hospital, Luoyang, Henan 471002, P.R. China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Luoyang Orthopaedic-Traumatological Hospital and Henan Orthopaedic Hospital, Luoyang, Henan 471002, P.R. China.
authors.forename: Man-Yu
authors.initials: MY
authors.lastname: Huang
authors.affiliation: Luoyang Orthopaedic-Traumatological Hospital and Henan Orthopaedic Hospital, Luoyang, Henan 471002, P.R. China.
authors.forename: Wu-Yin
authors.initials: WY
authors.lastname: Li
authors.affiliation: College of Pharmacy, Henan University, Kaifeng, Henan 475000, P.R. China.
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Hu
mesh_terms: D000664:Amphibian Venoms / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D001859:Bone Neoplasms / Q000188:drug therapy / Q000473:pathology*; D002018:Bufanolides / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D012516:Osteosarcoma / Q000188:drug therapy / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000664:Amphibian Venoms; D000970:Antineoplastic Agents; D002018:Bufanolides; C118585:P62 protein, human; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; D011065:Poly(ADP-ribose) Polymerases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; C002471:cinobufagin