title: Luteolin induces cholangiocarcinoma cell apoptosis through the mitochondrial-dependent pathway mediated by reactive oxygen species.
abstract: OBJECTIVE
  To investigate the apoptosis-inducing effect and underlying mechanisms of luteolin in cholangiocarcinoma (CCA) cells.
  
  
  METHODS
  Cell viability was determined by sulphorhodamine B. Apoptosis was detected using acridine orange/ethidium bromide dye staining and annexin V/PI staining followed by flow cytometry. The effect of luteolin on the oxidative status of CCA cells was evaluated by measuring intracellular reactive oxygen species (ROS) levels using the dihydroethidium method and quantifying glutathione levels. The mitochondria transmembrane potential (ΔΨm) was examined through JC-1 staining. The protein levels were determined by Western blot. Caspase activity was determined using specific fluorogenic substrates.
  
  
  RESULTS
  Luteolin decreased KKU-100 CCA cells' viability by induction of apoptosis. Luteolin treatment increased ROS production and decreased glutathione levels. These changes were associated with the decrease of Nrf2, γ-glutamylcysteine ligase and heme oxygenase-1 proteins. Moreover, luteolin induced mitochondrial depolarization, which was accompanied by the release of cytochrome c and a decrease of Bcl-2 and Bcl-XL proteins. Pretreatment with antioxidants, 4-hydroxy-TEMPO and N-acetyl-L-cysteine significantly prevented luteolin-induced CCA cell death and loss of ΔΨm. In addition, luteolin induced the activation of caspase-9 and caspase-3.
  
  
  CONCLUSIONS
  Luteolin exerts its pro-apoptotic action partly through generating intracellular ROS that then contributes to the activation of mitochondria-mediated apoptotic cell death.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Natthawan
authors.initials: N
authors.lastname: Kittiratphatthana
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.|Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Veerapol
authors.initials: V
authors.lastname: Kukongviriyapan
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.|Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Auemduan
authors.initials: A
authors.lastname: Prawan
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.|Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Laddawan
authors.initials: L
authors.lastname: Senggunprai
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis; D001650:Bile Duct Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D018281:Cholangiocarcinoma / Q000188:drug therapy / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006801:Humans; D047311:Luteolin / Q000494:pharmacology* / Q000627:therapeutic use; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D008517:Phytotherapy; D010936:Plant Extracts / Q000494:pharmacology* / Q000627:therapeutic use; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D010936:Plant Extracts; D017382:Reactive Oxygen Species; D045304:Cytochromes c; D051547:Heme Oxygenase-1; D053148:Caspase 3; D053453:Caspase 9; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D047311:Luteolin
keywords: apoptosis; cholangiocarcinoma; luteolin; mitochondria; reactive oxygen species