title: The glycogen synthase kinase-3β/nuclear factor-kappa B pathway is involved in cinobufagin-induced apoptosis in cultured osteosarcoma cells.
abstract: Cinobufagin, a major component of cinobufacini (huachansu), is an important cardenolidal steroid. Several studies have suggested that cinobufagin has potent anti-cancer effects. The present study examines the apoptosis-inducing activity and the underlying mechanism of action of cinobufagin in osteosarcoma (OS) cells. Our results showed that cinobufagin potently inhibited the proliferation of U2OS, MG63 and SaOS-2 cells. Significant increases in G2/M cell-cycle arrest and apoptosis in OS cells were also observed. The expression levels of several apoptotic proteins were assessed after cinobufagin treatment in U2OS cells. Among them, xIAP, cIAP-1, survivin and Bcl-2 levels decreased remarkably, while the levels of Bax and cleaved-PARP increased. Furthermore, we validated the inhibition of GSK-3β/NF-κB signaling following cinobufagin treatment. Western blots showed a decrease in nuclear p65 protein expression after exposure to different concentrations of cinobufagin, while the phosphorylation of GSK-3β was simultaneously increased. Transduction with constitutively active forms of GSK-3β could protect against the downregulation of p65 and upregulation of cleaved-PARP that are induced by cinobufagin treatment. However, combined treatment with cinobufagin and SB216367 resulted in a significant reduction in p65 and an increase in cleaved-PARP in U2OS cells. Altogether, these results show that cinobufagin is a promising agent for the treatment of OS. These studies are the first to reveal the involvement of the GSK-3β/NF-κB pathway in cinobufagin-induced apoptosis.
authors.affiliation: Department of Orthopaedic Oncology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China.
authors.forename: Jun-Qiang
authors.initials: JQ
authors.lastname: Yin
authors.forename: Lili
authors.initials: L
authors.lastname: Wen
authors.forename: Liang-Cai
authors.initials: LC
authors.lastname: Wu
authors.forename: Zhen-Hua
authors.initials: ZH
authors.lastname: Gao
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Chang-Ye
authors.initials: CY
authors.lastname: Zou
authors.forename: Ping-Xian
authors.initials: PX
authors.lastname: Tan
authors.forename: Bi-Cheng
authors.initials: BC
authors.lastname: Yong
authors.forename: Qiang
authors.initials: Q
authors.lastname: Jia
authors.forename: Jing-Nan
authors.initials: JN
authors.lastname: Shen
mesh_terms: D000664:Amphibian Venoms / Q000032:analysis / Q000737:chemistry; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D002018:Bufanolides / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
chemical_list: D000664:Amphibian Venoms; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D002018:Bufanolides; D016328:NF-kappa B; C498143:RELA protein, human; D051996:Transcription Factor RelA; C545813:huachansu; D011065:Poly(ADP-ribose) Polymerases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; C002471:cinobufagin