title: P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK.
abstract: Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.
authors.affiliation: China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning, China.
authors.forename: Simiao
authors.initials: S
authors.lastname: Fan
authors.forename: Min
authors.initials: M
authors.lastname: Qi
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.forename: Lihua
authors.initials: L
authors.lastname: Li
authors.forename: Guodong
authors.initials: G
authors.lastname: Yao
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: D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000502:physiology*; D052160:Benzothiazoles / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D045744:Cell Line, Tumor / Q000187:drug effects / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016158:Genes, p53; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology*; D020935:MAP Kinase Signaling System; D053078:Membrane Potential, Mitochondrial; D018384:Oxidative Stress; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000077385:Silybin; D012838:Silymarin / Q000494:pharmacology; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*; D014846:Vulvar Neoplasms / Q000473:pathology
chemical_list: D000873:Anthracenes; D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D052160:Benzothiazoles; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D012838:Silymarin; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C432165:pyrazolanthrone; D014050:Toluene; D000077385:Silybin; C121565:pifithrin; D048031:JNK Mitogen-Activated Protein Kinases