title: 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol induces apoptosis in intestinal epithelial cells of rats via mitochondrial pathway.
abstract: BACKGROUND
  Euphorbia kansui is a traditional Chinese medicine widely used for the treatment of edema, ascite and asthma in China for centuries. However, its serious gastrointestinal toxicity restricted its safe clinical application. 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3EZ,20Ac-ingenol), a diterpenoid compound derived from kansui, has obvious gastrointestinal cytotoxicity in cells. Until now, its gastrointestinal cytotoxic mechanism is mostly unknown. This study focused on elucidating the cytotoxic mechanism of 3EZ,20Ac-ingenol in intestinal epithelial cells of rats (IEC-6 cells) to guide safer application of this herb in clinic.
  
  
  METHODS
  3EZ,20Ac-ingenol was isolated from the EtOAc extract of kansui. Cell morphology was detected by inverted phase contrast microscope and transmission electron microscope (TEM). Cell apoptosis was examined by Annexin V-FITC/PI dual-staining or Hoechst staining. ROS generation was detected with DCFH-DA staining by laser scanning confocal microscope. MMP change was examined with JC-1 staining by high content screening (HCS). Further, the release of cytochrome c, the expressions of Bax, Bcl-2, AIF and Apaf-1 were analyzed by western blot and the activities of caspase-3, 8, 9 were determined by ELISA. Additionally, cell cycle analysis was performed to detect the effects of 3EZ,20Ac-ingenol on cell cycle in IEC-6 cells by flow cytometry.
  
  
  RESULTS
  The study showed that 3EZ,20Ac-ingenol significantly reduced IEC-6 cells viability in a dose-dependent manner and the IC50 value was 5.74 μg/mL. Consistently, 3EZ,20Ac-ingenol could elevate reactive oxygen species (ROS), disrupt mitochondrial membrane potential (MMP), induce the release of cytochrome c from mitochondria to cytosol, enhance the expressions of Bax, AIF and Apaf-1, suppress the expression of Bcl-2, then activate caspase-3, 8, 9 cascade, and subsequently result in apoptosis. Additionally, 3EZ,20Ac-ingenol also could cause G2/M phase arrest in IEC-6 cells.
  
  
  CONCLUSIONS
  The results indicated that 3EZ,20Ac-ingenol induced the cytotoxicity of IEC-6 cells depends on induction of cell apoptosis via mitochondrial pathway and cell cycle arrest.
authors: Nanjing University of Chinese Medicine, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China.
authors: Jing
authors: J
authors: Gao
authors: Nanjing Jiangning Hospital of Chinese Medicine, Teaching Hospital of Nanjing University of Chinese Medicine, Nanjing 211100, China.
authors: Lan
authors: L
authors: Gao
authors: Nanjing University of Chinese Medicine, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China. Electronic address: zhangli@njucm.edu.cn.
authors: Li
authors: L
authors: Zhang
authors: Nanjing University of Chinese Medicine, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China.
authors: Weifeng
authors: W
authors: Yao
authors: Nanjing University of Chinese Medicine, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China.
authors: Yudan
authors: Y
authors: Cao
authors: Nanjing University of Chinese Medicine, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China.
authors: Beihua
authors: B
authors: Bao
authors: Nanjing University of Chinese Medicine, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China. Electronic address: awding105@163.com.
authors: Anwei
authors: A
authors: Ding
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004224:Diterpenes / Q000302:isolation & purification / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D028481:Euphorbia; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology
chemical_list: D004224:Diterpenes; C008125:ingenol
keywords: 3-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol (3EZ,20Ac-ingenol); Apoptosis; Caspase activation; Cell cycle arrest; Cytochrome c release; Mitochondria; Reactive oxygen species (ROS)