Repeated subconjunctival injections with 5-fluorouacil (5-FU) after trabeculectomy are widely used in glaucoma patients for the inhibition of excess scar formation in wound site. The aim of this study was to evaluate the toxic effects of 5-FU and mechanisms of drug-induced apoptosis in cultured porcine corneal endothelial cells. Cellular damage and the caspase pathway were estimated with a MTT assay. The apoptotic characteristics were detected with flow cytometry, a TUNEL test and Western blotting. The results indicated that 5-FU was toxic to corneal endothelial 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 demon-strated the loss of mitochondrial membrane potential. A positive TUNEL test revealed that cellular DNA apoptosis occurred following exposure to 0.05, 0.1, and 0.5 mg/ml 5-FU for 15 h. Annexin V-FITC and negative propidium iodide (PI) staining indicated that the cell membrane underwent apoptosis upon exposure to 0.1 and 0.5 mg/ml 5-FU for 15 h. The Western blot assay demonstrated up-regulation of the Bax, p53 and p21 proteins induced by 5-FU. Taken together, these data reveal that 5-FU-induced cellular apoptosis in corneal endothelial cells may be mediated through caspase-8, caspase-9 and mitochondrial regulated pathways, as well as by up-regulation of Bax-, p53-, and p21-dependent signal transduction pathways.
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