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Wei Tang, Yujie Zhang, Chaoxu Qian, Zhilan Yuan, Jun Du; Induction and Mechanism of Apoptosis by Hydroxycamptothecin in Human Tenon's Capsule Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2012;53(8):4874-4880. doi: 10.1167/iovs.11-8968.
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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.
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.
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.
Caspase-3 and caspase-9 are important elements in regulating HCPT-induced apoptosis in HTFs.
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