title: Timosaponin A-III induces autophagy preceding mitochondria-mediated apoptosis in HeLa cancer cells.
abstract: Timosaponin A-III (TAIII), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. Here, we provide evidence that TAIII induces autophagy in HeLa cells followed by apoptotic cell death. TAIII-induced autophagy was morphologically characterized by the formation of membrane-bound autophagic vacuoles recognizable at the ultrastructural level. TAIII-treated cells expressing green fluorescent protein (GFP)-labeled microtubule-associated protein 1 light chain 3 (LC3) displayed punctate fluorescence indicative of LC3 recruitment to the autophagosome. This was associated with the conversion of LC3-I (the cytosolic form) into LC3-II (the lipidated form located on the autophagosome membrane). TAIII treatment also induced mitochondrial dysfunction involving overproduction of reactive oxygen species and reduction of mitochondrial membrane potential accompanied by induction of mitochondrial permeability transition. Prolonged exposure to TAIII resulted in cytochrome c release and caspase-3 activation, events that signified the onset of apoptotic cell death. TAIII-induced autophagy preceded apoptosis, as evidenced by early autophagic vacuole formation, GFP-LC3 translocation, and LC3-II increase in the absence of caspase-3 cleavage. Notably, TAIII-mediated apoptotic cell death was potentiated by treatment with autophagy inhibitor 3-methyladenine or small interfering RNA against the autophagic gene beclin 1. These findings suggest that TAIII-elicited autophagic response plays a protective role that impedes the eventual cell death. In terms of structure-activity relationship, the sugar chain in TAIII is indispensable to the drug action, as the sugar-lacking aglycone sarsasapogenin did not induce autophagy and exhibited weaker cytotoxicity.
authors.affiliation: Department of Chemistry, Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Hong Kong, China.
authors.forename: Lai-King
authors.initials: LK
authors.lastname: Sy
authors.forename: Siu-Cheong
authors.initials: SC
authors.lastname: Yan
authors.forename: Chun-Nam
authors.initials: CN
authors.lastname: Lok
authors.forename: Ricky Y K
authors.initials: RY
authors.lastname: Man
authors.forename: Chi-Ming
authors.initials: CM
authors.lastname: Che
mesh_terms: D031403:Anemarrhena / Q000737:chemistry; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D000071186:Beclin-1; D053148:Caspase 3 / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018384:Oxidative Stress / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D012503:Saponins; D016572:Cyclosporine; D045304:Cytochromes c; D002374:Catalase; D013482:Superoxide Dismutase; D053148:Caspase 3