Mechanism of mitomycin-induced apoptosis in cultured corneal endothelial cells.
OBJECTIVE
Previous studies have indicated that improper use of mitomycin C (MMC) caused cytotoxicity in corneal endothelial cells. The aim of the present study was to investigate whether MMC induces cellular apoptosis in corneal endothelial cells and to determine the mechanism by which this may occur.


METHODS
Porcine corneal endothelial cells were acquired from primary culture. Cellular damage and caspase pathway were estimated with a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. The apoptotic characteristics were detected by means of flow cytometry, the TUNEL (terminal deoxyribonucleotidyl-transferase-(TdT)-mediated deoxyuridine-5'-triphosphate-digoxigenin (dUTP) nick-end labeling) test, immunofluorescent staining, and western blotting.


RESULTS
The results indicated that MMC was toxic to corneal endothelial cells in a time-dependent 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 MMC-induced cellular damage. Following exposure to MMC, a change in the mitochondrial membrane potential was positively detected by flow cytometric assay with MitoLight dye while cellular cytochrome c that was released from the mitochondria to the cytoplasm was detected by immunofluorescent staining. A positive TUNEL test revealed that cellular DNA apoptosis had occurred following exposure to 0.001 and 0.01 mg/ml MMC for 24 h. Positive annexin V-FITC, and negative propidium iodide (PI) staining indicated that the cellular plasma membrane underwent apoptosis following 0.001 mg/ml MMC exposure for 24 h. Western blot assay demonstrated down-regulation of the Bcl-2 protein and upregulation of the p53 and p21 proteins, which were all involved in apoptosis induced by MMC.


CONCLUSIONS
These results indicate that mitomycin-induced cellular apoptosis in corneal endothelial cells may be mediated through caspase-8, caspase-9, and the mitochondrial regulated pathways as well as through upregulation of p53-dependent and p21-dependent signal transduction pathways.
Wu
Kwou-Yeung
KY
Department of Ophthalmology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Wang
Hwei-Zu
HZ
Hong
Show-Jen
SJ
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D004728:Endothelium, Corneal / Q000166:cytology* / Q000201:enzymology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D020287:In Situ Nick-End Labeling; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016685:Mitomycin / Q000494:pharmacology*; D011419:Propidium / Q000378:metabolism; D013552:Swine
D017304:Annexin A5; D011419:Propidium; D016685:Mitomycin; D004247:DNA; D020169:Caspases; D016650:Fluorescein-5-isothiocyanate