Apoptosis of Keratocytes Induced by Mitomycin C.

Choi, Jaewan; Kim, Tae Im; Tchah, Hungwon

Original Article

Journal of the Korean Ophthalmological Society 2004;45(3):490-499.
Published online March 1, 2004.

Apoptosis of Keratocytes Induced by Mitomycin C.

Department of Ophthalmology, University of Ulsan, College of Medicine, Asan Medical Center, Seoul, Korea. hwtchah@amc.seoul.kr

Mitomycin C에 의해 유도된 각막간질세포의 Apoptosis

최재완 ( Choe Jae Wan ) , 김태임 ( Kim Tae Im ) , 차흥원 ( Cha Heung Won )

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.

Key Words: Apoptosis;Caspase 8;Caspase 9;Keratocytes;Mitochondria;Mitomycin C