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Published on: July 17, 2018
Apoptosis in keratocytes caused by mitomycin C
Tae-im Kim1, Hungwon Tchah, Seung-ah Lee
1Department of Ophthalmology, University of Ulsan, College of Medicine, Asan Medical Center, Seoul, Korea.
Purpose:
The purpose of this study was to quantify the effect of mitomycin C on rabbit keratocytes, with a view to determining its potential in modulating corneal stromal wound healing. In addition, the pathway by which this regulation occurs was investigated.
Methods:
Keratocytes were isolated from New Zealand White rabbits and cultured. Hoechst staining and flow cytometric analyses with annexin V were used to identify the nature of the keratocyte response to mitomycin C. The response of cultured keratocytes to 0.005%, 0.01%, 0.02%, 0.04%, and 0.06% mitomycin C was evaluated with the lactate dehydrogenase (LDH) assay. In addition, after exposure of keratocytes to 0.01% mitomycin C, the LDH assay was performed at different times of 6, 12, and 24 hours. Keratocytes were preincubated with various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) and treated with 0.01% mitomycin C. The LDH assay was performed after 12 hours. Cytochrome c immunostain was performed after exposure to 0.01% mitomycin C.
Results:
Hoechst staining revealed shrinkage of the cytoplasm, formation of apoptotic bodies, and nuclear fragmentation. Apoptotic changes in cells were detected by flow cytometry. LDH activities increased significantly at concentrations of 0.005% mitomycin C or greater and were time dependent until 24 hours. Treatment with a CPP32-like protease inhibitor caused a decrease in LDH activity, although the results were not statistically significant. Specific inhibitors of caspase-8 and -9 significantly reduced the LDH activity induced by mitomycin C. Cytochrome c immunostaining of keratocytes pretreated with mitomycin C showed strongly positive findings.
Conclusions:
Mitomycin C induced apoptosis, not necrosis, in cultured corneal keratocytes through the caspase pathway-specifically, caspase-8 and -9-related to the mitochondrial pathway.
Insights
Mitomycin C induces apoptosis in rabbit corneal keratocytes via the caspase pathway, specifically involving caspase-8 and caspase-9, which are linked to the mitochondrial pathway.
Area of Science:
- Ophthalmology
- Cell Biology
- Pharmacology
Background:
- Corneal stromal wound healing is a complex process involving keratocytes.
- Understanding the cellular mechanisms that modulate this healing is crucial for developing effective treatments.
- Mitomycin C is a drug with antiproliferative properties that has been used in ophthalmic procedures.
Purpose of the Study:
- To quantify the effect of mitomycin C on rabbit corneal keratocytes.
- To determine mitomycin C's potential in modulating corneal stromal wound healing.
- To investigate the specific cellular pathway through which mitomycin C exerts its effects.
Main Methods:
- Rabbit keratocytes were cultured and exposed to varying concentrations of mitomycin C.
- Apoptosis was assessed using Hoechst staining and flow cytometry with annexin V.
- Cell viability was measured using the lactate dehydrogenase (LDH) assay.
- Involvement of caspases and the mitochondrial pathway was investigated using specific inhibitors and cytochrome c immunostaining.
Main Results:
- Mitomycin C induced characteristic apoptotic changes in keratocytes, including nuclear fragmentation.
- LDH activity, an indicator of cell damage, increased significantly with mitomycin C exposure in a dose- and time-dependent manner.
- Inhibitors of caspase-8 and caspase-9 significantly reduced mitomycin C-induced LDH activity.
- Cytochrome c release into the cytoplasm was observed, indicating mitochondrial pathway activation.
Conclusions:
- Mitomycin C induces apoptosis, not necrosis, in cultured corneal keratocytes.
- The apoptotic process is mediated through the caspase pathway, involving caspase-8 and caspase-9.
- Mitochondrial pathways are implicated in mitomycin C-induced apoptosis of corneal keratocytes.
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