Human serum reduces mitomycin-C cytotoxicity in human tenon's fibroblasts
Jonathan G Crowston1, Xiao Y Wang, Peng T Khaw
1Center for Vision Research, Westmead Hospital and Westmead Millennium Research Institute, Sydney, NSW, Australia. jcrowston@eyecenter.ucsd.edu
Purpose:
To determine the effect of human serum factors on mitomycin-C (MMC) cytotoxicity in cultured human subconjunctival Tenon's capsule fibroblasts.
Methods:
Fibroblast monolayers were treated with 5-minute applications of mitomycin-C (0.4 mg/mL) and incubated in culture medium with or without additional human serum. Fibroblast apoptosis was quantified by direct cell counts based on nuclear morphology, flow cytometry with annexin-V/propidium iodide, and a lactate dehydrogenase release assay. The number of viable fibroblasts and fibroblast proliferation were measured with a colorimetric MTT assay and by bromodeoxyuridine (BrdU) labeling.
Results:
Mitomycin-C induced significant levels of fibroblast apoptosis. The addition of human serum resulted in a 40% reduction in MMC-induced fibroblast apoptosis (range, 31.3%-55.3%; P = 0.021) as determined by nuclear morphology and a 32.4% reduction measured by annexin-V/PI. There was a corresponding dose-dependent increase in the number of viable fibroblasts. Serum did not restore proliferation in MMC-treated fibroblasts.
Conclusions:
Factors present in human serum reduce MMC cytotoxicity in cultured human Tenon's fibroblasts. Human serum increased the number of viable fibroblasts by inhibiting MMC-induced fibroblast apoptosis. Serum factors access aqueous humor after trabeculectomy and may therefore influence the clinical outcome of MMC treatment.