title: Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells.
abstract: Silibinin, as the major active constituent of silymarin, has its various biological effects. Here, we investigated the inhibitory effects of silibinin on HeLa cell growth in relation to autophagy and apoptosis induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Silibinin dose and time-dependently decreased cell growth cultured in medium containing 10% fetal bovine serum or in serum free media (SFM) with an IC(50) of approximately 80-100 and 40-60 μM at 24 h, respectively. Silibinin induced autophagy at 12 h, confirmed by monodansylcadervarine (MDC) staining and up-regulation of beclin-1, and induced apoptosis at 24 h, detected by observation of apoptotic bodies and activation of caspase-3. 3-methyladenine (3-MA) inhibited silibinin-induced autophagy and attenuated the silibinin's inhibitory effect on cell viability, suggesting that autophagy enhanced silibinin-induced cell death. Silibinin increased ROS levels at 12 h, and ROS scavenger, N-acetylcysteine (NAC), significantly reversed the cytotoxicity of silibinin through inhibiting both autophagy and apoptosis. Specific antioxidants were applied and results indicated that hydroxyl radical (·OH) was the major ROS induced by silibinin, and OH scavenger glutathione (GSH) inhibited apoptosis and autophagy. Silibinin also generated RNS production in the cells at 12 h. High concentration of N omega-nitro-l-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor attenuated the cytotoxicity of silibinin by decreasing ROS levels, leading to down-regulation of apoptosis. Silibinin also could interrupt the respiring functions of mitochondria, leading to ROS production and oxidative damage.
authors.affiliation: China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors.forename: Simiao
authors.initials: S
authors.lastname: Fan
authors.forename: Lihua
authors.initials: L
authors.lastname: Li
authors.forename: Shengeng
authors.initials: S
authors.lastname: Chen
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.forename: Min
authors.initials: M
authors.lastname: Qi
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Tashiro
authors.forename: Satoshi
authors.initials: S
authors.lastname: Onodera
authors.forename: Takashi
authors.initials: T
authors.lastname: Ikejima
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D053148:Caspase 3 / Q000032:analysis; D002470:Cell Survival; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000077385:Silybin; D012838:Silymarin / Q000037:antagonists & inhibitors / Q000494:pharmacology*
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D012838:Silymarin; D017665:Hydroxyl Radical; D000077385:Silybin; C025946:3-methyladenine; D019001:Nitric Oxide Synthase; D053148:Caspase 3; D005978:Glutathione; D000225:Adenine; D019331:NG-Nitroarginine Methyl Ester; D000111:Acetylcysteine