Apoptosis of Keratocytes Induced by Mitomycin C

ABSTRACT

PURPOSE: The purpose of this study was to evaluate the effect of mitomycin C on rabbit keratocytes for their potential to modulate corneal stromal wound healing. We also investigated the pathway on which the modulation occurs. METHODS: Keratocytes were isolated from New Zealand White Rabbits and cultured. We used Hoechst stain and flowcytometric analysis with Annexin V to identify the kind of response that mitomycin C induced from the keratocytes. After cultured keratocytes were exposed to 0.005%, 0.01%, 0.02%, 0.04%, and 0.06% mitomycin C, we evaluated the response with LDH assay. Next, after exposing the keratocytes to 0.01% mitomycin C, we evaluated the responses with LDH assay at 6, 12, and 24 hours. Keratocytes were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK(R)), specific caspase-8 inhibitor (Z-IETD-FMK(R)), and specific caspase-9 inhibitor (Z-LEHD-FMK(R)), then treated with 0.01% mitomycin C. Twelve hours later, an LDH assay was performed. Cytochrome C immunostain was done after exposure to 0.01% mitomycin C. RESULTS: We observed shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation on Hoechst staining. In flowcytometric analysis, the cells showed apoptotic change. LDH activities increased significantly at a concentration of 0.005% and greater and were time-dependent until 24 hours. CPP32-like protease inhibitor decreased the LDH activity, but there was no statistical significance. Specific caspase-8 and -9 inhibitors significantly reduced the LDH activities that were induced by mitomycin C. The keratocytes which had been pretreated with mitomycin C were stained with cytochrome C antibody. CONCLUSIONS: Mitomycin C induces apoptosis, rather than necrosis, in cultured corneal keratocytes. This apoptosis occurs via the caspase pathway, and is especially related to the mitochondrial pathway, and caspases 8, and 9.