title: Mitomycin-C induces the apoptosis of human Tenon's capsule fibroblast by activation of c-Jun N-terminal kinase 1 and caspase-3 protease.
abstract: OBJECTIVE
  To investigate whether mitochondrial dysfunction and mitogen-activated protein kinase family proteins are implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by mitomycin-C.
  
  
  METHODS
  Apoptosis was determined by Hoechst nuclei staining, agarose gel electrophoresis, and flow cytometry in HTCFs treated with 0.4 mg/mL mitomycin-C for 5 minutes. Enzymatic digestion of florigenic biosubstrate assessed the catalytic activity of caspase proteases, including caspase-3, caspase-8, and caspase-9. Phosphotransferase activity of c-Jun N-terminal kinase (JNK) 1 was measured by in vitro immune complex kinase assay using c-Jun(1-79) protein as a substrate. Mitochondrial membrane potential transition (MPT) was measured by flow cytometric analysis of JC-1 staining.
  
  
  RESULTS
  Mitomycin-C (0.4 mg/mL) induced the apoptosis of HTCFs, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G(0)/G(1) fraction of cell cycle increase. The catalytic activity of caspase-3 and caspase-9 was significantly increased and was accompanied by cytosolic release of cytochrome c and MPT in response to mitomycin-C. Treatment with mitomycin-C resulted in the increased expression of Fas, FasL, Bad, and phosphorylated p53 and a decreased level of phosphorylated AKT. Treatment with mitomycin-C also increased the phosphotransferase activity and tyrosine phosphorylation of JNK1, whose inhibitor significantly suppressed the cytotoxicity of mitomycin-C.
  
  
  CONCLUSIONS
  Mitomycin-C induced the apoptosis of HTCFs through the activation of intrinsic and extrinsic caspase cascades with mitochondrial dysfunction. It also activated Fas-mediated apoptotic signaling of fibroblasts. Furthermore, the activation of JNK1 played a major role in the cytotoxicity of mitomycin-C.
authors.affiliation: Institute of Vision Research and the Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Gong Je
authors.initials: GJ
authors.lastname: Seong
authors.forename: Channy
authors.initials: C
authors.lastname: Park
authors.forename: Chan Yoon
authors.initials: CY
authors.lastname: Kim
authors.forename: Young Jae
authors.initials: YJ
authors.lastname: Hong
authors.forename: Hong-Seob
authors.initials: HS
authors.lastname: So
authors.forename: Sang-Duck
authors.initials: SD
authors.lastname: Kim
authors.forename: Raekil
authors.initials: R
authors.lastname: Park
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D018929:Cell Culture Techniques; D002470:Cell Survival; D003239:Connective Tissue Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D004587:Electrophoresis, Agar Gel; D004789:Enzyme Activation; D053222:Fas Ligand Protein; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D016685:Mitomycin / Q000494:pharmacology*; D010766:Phosphorylation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism
chemical_list: D000903:Antibiotics, Antineoplastic; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor; D014443:Tyrosine; D016685:Mitomycin; D048055:Mitogen-Activated Protein Kinase 8; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases