Abstract
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.
Highlights
5-Fluorouracil (5-FU) is a pyrimidine analogue used to treat a wide range of solid tumors [1]
In cases of the 5-FU accidentally penetrating into the anterior chamber of the eye, the drug may damage corneal endothelial cells and impair physiological functions such as the pumping activity in the corneal endothelium
Evidence was presented that 5-FU (50 mg/ml) injected at the trabeculectomy site every five days for 15 days had the deleterious effects on corneal endothelial cells based on observations with confocal microscopy and scanning electron microscopy [8]
Summary
5-Fluorouracil (5-FU) is a pyrimidine analogue used to treat a wide range of solid tumors [1]. When 5-FU is administered repeatedly with subconjunctival injections, a certain amount of the drug may penetrate into the anterior chamber, where it causes toxicity to corneal endothelial cells. In cases of the 5-FU accidentally penetrating into the anterior chamber of the eye, the drug may damage corneal endothelial cells and impair physiological functions such as the pumping activity in the corneal endothelium. Subconjunctival 5-FU injections administered after glaucoma surgery led to apoptotic cell death in the conjunctival epithelium [12]. Apoptosis is a process of natural cell death, which is characterized by extreme heterogeneity of signal transduction pathways. To evaluate the potential chronic toxicity of 5-FU, cultured porcine corneal endothelial cells were used in the present study to investigate the apoptotic characteristics and mechanism involved in corneal damage induced by 5-FU
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