Induction and mechanism of apoptosis by hydroxycamptothecin in human Tenon's capsule fibroblasts.
OBJECTIVE
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.
Department of Ophthalmology, the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province, China.
Wei
W
Tang
Yujie
Y
Zhang
Chaoxu
C
Qian
Zhilan
Z
Yuan
Jun
J
Du
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism*; D053181:Caspase 8 / Q000187:drug effects / Q000378:metabolism*; D053453:Caspase 9 / Q000187:drug effects / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D058475:Tenon Capsule / Q000187:drug effects / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D000972:Antineoplastic Agents, Phytogenic; D015853:Cysteine Proteinase Inhibitors; D012333:RNA, Messenger; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C028098:10-hydroxycamptothecin; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D002166:Camptothecin