title: 5-Fluorouracil-Induced Apoptosis Changes in Cultured Corneal Epithelial Cells.
abstract: OBJECTIVE
  Repeated subconjunctival injections with 5-fluorouracil (5-FU) after trabeculectomy are used in glaucoma patients for the inhibition of overproliferation in wound site. Thus, a certain amount of the drug may penetrate into epithelial layer, where it causes toxicity to corneal epithelial cells. The aim of this study was to evaluate the toxic effects of 5-FU and mechanisms of drug-induced apoptosis in cultured corneal epithelial cells.
  
  
  METHODS
  Cellular damage and the caspase pathway were estimated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic characteristics were detected by flow cytometry, a TUNEL test, and western blotting in cultured corneal epithelial cells.
  
  
  RESULTS
  The results indicated that 5-FU was toxic to corneal epithelial cells in a time- and dose-dependent manner. Pretreatment with a general caspase inhibitor (Z-VAD-FMK), a caspase-8 inhibitor (Z-IETD-FMK), and a caspase-9 inhibitor (Z-LEHD-FMK) reversed 5-FU-induced cellular damage. Following exposure to 5-FU, a flow cytometric assay with MitoLight dye demonstrated the significant loss of mitochondrial membrane potential. A positive TUNEL test revealed that cellular DNA apoptosis occurred following exposure to 0.5, 1, and 5 mg/mL 5-FU for 15 h. Positive annexin V-FITC and negative propidium iodide (PI) staining indicated that the cell membrane exhibited apoptosis upon exposure to 1 and 5 mg/mL 5-FU for 15 h. The western blot assay demonstrated upregulation of the p21 protein but downregulation of the Bcl-2 proteins induced by 5-FU.
  
  
  CONCLUSIONS
  These data reveal that 5-FU-induced cellular apoptosis in corneal epithelial cells may be mediated through caspase-8, caspase-9, and mitochondria-regulated pathways, as well as by upregulation of p21 and downregulation of Bcl-2-dependent signal transduction pathways.
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan .|2 Department of Ophthalmology, Kaohsiung Medical University Hospital , Kaohsiung, Taiwan .
authors.forename: Kwou-Yeung
authors.initials: KY
authors.lastname: Wu
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan .|2 Department of Ophthalmology, Kaohsiung Medical University Hospital , Kaohsiung, Taiwan .
authors.forename: Yu-Hung
authors.initials: YH
authors.lastname: Lai
authors.affiliation: 3 Department of Pharmacology, College of Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan .
authors.forename: Yu-Chiao
authors.initials: YC
authors.lastname: Yang
authors.affiliation: 1 Department of Ophthalmology, College of Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan .|2 Department of Ophthalmology, Kaohsiung Medical University Hospital , Kaohsiung, Taiwan .
authors.forename: Wen-Chuan
authors.initials: WC
authors.lastname: Wu
authors.affiliation: 3 Department of Pharmacology, College of Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan .
authors.forename: Show-Jen
authors.initials: SJ
authors.lastname: Hong
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000187:drug effects / Q000473:pathology*; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*
chemical_list: D000964:Antimetabolites, Antineoplastic; D020169:Caspases; D005472:Fluorouracil