title: Novel multiple apoptotic mechanism of shikonin in human glioma cells.
abstract: BACKGROUND
  Shikonin is the main naphthoquinone compound of the root of Lithospermum erythrorhizon. Our previous study demonstrated that shikonin possesses anticancer activity in human hepatoma cells. However, the anticancer mechanism of shikonin in human glioma cells is unclear at present. In the present study, we demonstrated that shikonin induces apoptosis in three human glioma cell lines: U87MG, Hs683, and M059K cells.
  
  
  METHODS
  Cell cycle, generation of reactive oxygen species (ROS), depletion of glutathione (GSH), and disruption of mitochondrial transmembrane potential in shikonin-treated cells were determined by flow cytometry. Apoptosis-related proteins, catalase, and superoxide dismutase-1 (SOD-1) were determined by Western blot testing. N-acetylcysteine (NAC), pifithrin-α (PFT-α), or cyclosporin A were applied to evaluate the molecular mechanism of shikonin in apoptosis.
  
  
  RESULTS
  Shikonin induces the generation of ROS, depletion of GSH, disruption of mitochondrial transmembrane potential, upregulation of p53, and cleavage of PARP [poly(ADP-ribose) polymerase] in U87MG glioma cells. Moreover, shikonin causes catalase downregulation and SOD-1 upregulation as well as decreased Bcl-2 and increased Bax expression. Pretreatment with NAC, PFT-α, or cyclosporin A causes the recovery of shikonin-induced apoptosis. The ROS generation and GSH depletion induced by shikonin trigger mitochondrial transmembrane potential disruption. ROS production was partially dependent on the upregulation of p53 upon shikonin treatment.
  
  
  CONCLUSIONS
  These studies are the first to show that shikonin-induced apoptosis occurs through multiple pathways in human glioma cells. We conclude that shikonin may be used as a potential chemotherapeutic agent against human glioma.
authors.affiliation: Department of Microbiology, Immunology and Biopharmaceuticals, College of Life Sciences, National Chiayi University, Chiayi, Taiwan, ROC.
authors.forename: Ching-Hsein
authors.initials: CH
authors.lastname: Chen
authors.forename: Miao-Ling
authors.initials: ML
authors.lastname: Lin
authors.forename: Ping-Lin
authors.initials: PL
authors.lastname: Ong
authors.forename: Jen-Tsung
authors.initials: JT
authors.lastname: Yang
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D052160:Benzothiazoles / Q000494:pharmacology; D002374:Catalase / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000494:pharmacology; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D005910:Glioma / Q000378:metabolism*; D005978:Glutathione / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009285:Naphthoquinones / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects; D011065:Poly(ADP-ribose) Polymerases / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000187:drug effects / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000187:drug effects / Q000378:metabolism
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; C494826:BAX protein, human; D052160:Benzothiazoles; D009285:Naphthoquinones; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; C000606290:SOD1 protein, human; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D014050:Toluene; C016101:shikonin; D016572:Cyclosporine; C121565:pifithrin; D002374:Catalase; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D011065:Poly(ADP-ribose) Polymerases; D005978:Glutathione; D000111:Acetylcysteine