Induction and mechanism of apoptosis by hydroxycamptothecin in human Tenon's capsule fibroblasts.

PURPOSE: We investigated apoptosis induced by hydroxycamptothecin (HCPT) in human Tenon's capsule using fibroblasts cultured from human Tenon's capsule (HTFs), and the mechanism of induction.

METHODS: HTFs were treated with 0-4 mg/L HCPT for 24 hours. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptotic cells were analyzed by Hoechst 33258 stain. The mRNA and protein levels of caspase-3, -8, and -9 were detected by RT-PCR and Western blotting.

RESULTS: By MTT assay, HCPT induced apoptosis in HTFs in a concentration- and time-dependent manner. Hoechst 33258 staining and transmission electron microscopy showed typical apoptotic morphology, such as condensed chromatin, irregular nuclei, and apoptotic body formation. The mRNA and protein levels of caspase-3 and caspase-9 were upregulated, while caspase-8 was unchanged. Z-VAD-FMK, a caspase inhibitor, inhibited the apoptosis of fibroblasts induced by HCPT. The expression levels of caspase-3 and caspase-9 were down-regulated after Z-VAD-FMK treatment.

CONCLUSIONS: Caspase-3 and caspase-9 are important elements in regulating HCPT-induced apoptosis in HTFs.