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Mitomycin-C Induces the Apoptosis of Human Tenon’s Capsule Fibroblast by Activation of c-Jun N-Terminal Kinase 1 and Caspase-3 Protease

¹From the Institute of Vision Research and the Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea; and the ³Vestibulocochlear Research Center and ⁴Departments of Microbiology and ⁵Ophthalmology, Wonkwang University School of Medicine, Iksan, Jeonbuk, Korea.

PURPOSE. To investigate whether mitochondrial dysfunction and mitogen-activated protein kinase family proteins are implicated in apoptotic signaling of human Tenon’s capsule fibroblasts (HTCFs) by mitomycin-C.

METHODS. Apoptosis was determined by Hoechst nuclei staining, agarose gel electrophoresis, and flow cytometry in HTCFs treated with 0.4 mg/mL mitomycin-C for 5 minutes. Enzymatic digestion of florigenic biosubstrate assessed the catalytic activity of caspase proteases, including caspase-3, caspase-8, and caspase-9. Phosphotransferase activity of c-Jun N-terminal kinase (JNK) 1 was measured by in vitro immune complex kinase assay using c-Jun_1–79 protein as a substrate. Mitochondrial membrane potential transition (MPT) was measured by flow cytometric analysis of JC-1 staining.

RESULTS. Mitomycin-C (0.4 mg/mL) induced the apoptosis of HTCFs, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G₀/G₁ fraction of cell cycle increase. The catalytic activity of caspase-3 and caspase-9 was significantly increased and was accompanied by cytosolic release of cytochrome c and MPT in response to mitomycin-C. Treatment with mitomycin-C resulted in the increased expression of Fas, FasL, Bad, and phosphorylated p53 and a decreased level of phosphorylated AKT. Treatment with mitomycin-C also increased the phosphotransferase activity and tyrosine phosphorylation of JNK1, whose inhibitor significantly suppressed the cytotoxicity of mitomycin-C.

CONCLUSIONS. Mitomycin-C induced the apoptosis of HTCFs through the activation of intrinsic and extrinsic caspase cascades with mitochondrial dysfunction. It also activated Fas-mediated apoptotic signaling of fibroblasts. Furthermore, the activation of JNK1 played a major role in the cytotoxicity of mitomycin-C.

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