title: Miltirone induced mitochondrial dysfunction and ROS-dependent apoptosis in colon cancer cells.
abstract: OBJECTIVE
  To study the characteristics of miltirone-induced anti-colon cancer effects.
  
  
  METHODS
  Cell viability was detected using MTT assay. LDH (lactate dehydrogenase) leakage was detected using CytoTox96® non-radioactive cytotoxicity kit. Apoptosis was detected by FCM (flow cytometry). Caspase activation was determined by chemiluminescence or western blotting. AIF (apoptosis-inducing factor) expression in the cell fraction was determined by western blotting. ROS (reactive oxygen species), MMP (mitochondrial membrane potential) and mitochondrial mass were determined by confocal microscope. Intracellular calcium was detected by both FCM and confocal microscope. To determine the roles of ROS and Ca(2+) in the pro-apoptotic activity of miltirone, colon cancer cells were pretreated with kinds of antioxidants, dicoumarol, calpeptin or BAPTA-AM in some cases.
  
  
  RESULTS
  Miltirone exhibited potent cytotoxicity on colon cancer cells with a better selectivity than that of dihydrotanshinone. The pro-apoptotic activity of miltirone was p53- and ROS-dependent. In detail, miltirone induced direct mitochondrial damage, including significant decrease of mitochondrial ROS, MMP, mass and increase of intracellular ROS and Ca(2+). NQO1 (quinone oxidoreductase1) was supposed to be a defender for the cytotoxicity induced by miltirone in colon cancer cells. Furthermore, miltirone induced time- and concentration-dependent translocation of AIF and activation of caspases.
  
  
  CONCLUSIONS
  In this study, ROS- and p53-dependent apoptosis induced by miltirone on colon cancer cells was firstly revealed. Strong positive feedback between mitochondrial dysfunction and accumulation of intracellular Ca(2+) was suggested to be the characteristic of the anti-colon cancer activity of miltirone.
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China; Beijing Key Laboratory of Drug Targets Research and New Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100050, China. Electronic address: wanglinster@gmail.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Shen
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Long-Fei
authors.initials: LF
authors.lastname: Li
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Ruby Lok-Yi
authors.initials: RL
authors.lastname: Chan
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Ming-Xing
authors.initials: MX
authors.lastname: Li
authors.affiliation: Department of Anaesthesia and Intensive Care, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: William Ka-Kei
authors.initials: WK
authors.lastname: Wu
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China. Electronic address: chcho@cuhk.edu.hk.
authors.forename: Chi-Hin
authors.initials: CH
authors.lastname: Cho
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D001728:Dicumarol / Q000494:pharmacology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000473:pathology*; D016660:NAD(P)H Dehydrogenase (Quinone); D010616:Phenanthrenes / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D051033:Apoptosis Inducing Factor; D010616:Phenanthrenes; D017382:Reactive Oxygen Species; C068880:miltirone; D001728:Dicumarol; D007770:L-Lactate Dehydrogenase; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D020169:Caspases; D002118:Calcium
keywords: Apoptosis; Colon cancer; Miltirone; Mitochondria; ROS; p53