title: Crosstalk of ROS/RNS and autophagy in silibinin-induced apoptosis of MCF-7 human breast cancer cells in vitro.
abstract: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in regulating cell survival and death. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with anti-tumor activities, but it was found to induce cytoprotective ROS/RNS in human breast cancer MCF-7 cells. Furthermore, treatment with silibinin down-regulates ERα expression in MCF-7 cells, and inducing both autophagy and apoptosis. In this study we explored the relationship between ER-associated pathways and RNS/ROS in MCF-7 cells. We also investigated the molecular mechanisms underlying the reciprocal regulation between ROS/RNS levels and autophagy in the death signaling pathways in silibinin-treated MCF-7 cells. Silibinin (100-300 μmol/L) dose-dependently increased ROS/RNS generation in MCF-7 cells (with high expression of ERα and low expression of ERβ) and MDA-MB-231 cells (with low expression of ERα and high expression of ERβ). Scavenging ROS/RNS significantly enhanced silibinin-induced death of MCF-7 cells, but not MDA-MB231 cells. Pharmacological activation or blockade of ERα in MCF-7 cells significantly enhanced or decreased, respectively, silibinin-induced ROS/RNS generation, whereas activation or block of ERβ had no effect. In silibinin-treated MCF-7 cells, exposure to the ROS/RNS donators decreased the autophagic levels, whereas inhibition of autophagy with 3-MA significantly increased ROS/RNS levels. We further showed that increases in ROS/RNS generation, ERα activation or autophagy down-regulation had protective roles in silibinin-treated MCF-7 cells. Under a condition of ERα activation, scavenging ROS/RNS or stimulating autophagy enhanced the cytotoxicity of silibinin. These results demonstrate the existence of two conflicting pathways in silibinin-induced death of MCF-7 cells: one involves the down-regulation of ERα and thereby augmenting the pro-apoptotic autophagy downstream, leading to cell death; the other involves the up-regulation of pro-survival ROS/RNS; and that the generation of ROS/RNS and autophagy form a negative feedback loop whose balance is regulated by ERα.
authors.affiliation: China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Zheng
authors.affiliation: China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Liu
authors.affiliation: China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors.forename: Wei-Wei
authors.initials: WW
authors.lastname: Liu
authors.affiliation: China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Li
authors.affiliation: China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: Department of Medical Education & Primary Care, Kyoto Prefectural University of Medicine, Kajiicho 465, Kamikyo-ku, Kyoto City, Kyoto 602-8566, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Tashiro
authors.affiliation: Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Onodera
authors.affiliation: China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ikejima
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D047628:Estrogen Receptor alpha / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000077385:Silybin; D012838:Silymarin / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D012838:Silymarin; D000077385:Silybin; C025946:3-methyladenine; D000225:Adenine