Studies on the cytotoxic mechanisms of ginkgetin in a human ovarian adenocarcinoma cell line.
The cytotoxic effects of ginkgetin, a natural biflavone isolated from Selaginella moellendorffii Hieron, were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in three different human cell lines: ovarian adenocarcinoma (OVCAR-3), cervical carcinoma (HeLa) and foreskin fibroblast (FS-5). The concentrations of ginkgetin required to induce 50% death (EC50) in OVCAR-3, HeLa, and FS-5 were 3.0, 5.2, and 8.3 microg/ml, respectively. Morphological changes in cells and their nuclei, DNA fragmentation with a characteristic pattern of inter-nucleosomal ladder, and double-stranded DNA breaks were detected following treatment with 3 microg/ml of this biflavone for 24 h. Incubation with 5 microg/ml ginkgetin led to increased intracellular levels of hydrogen peroxide as early as 30 min. The cytotoxicity of ginkgetin was partially inhibited by pretreating cells with vitamin C, vitamin E or catalase. Catalase not only afforded the best protective effect among three antioxidants, but also reduced both the DNA fragmentation and double-stranded DNA breakage induced by ginkgetin. Moreover, the involvement of caspase(s) in ginkgetin-induced apoptosis was demonstrated by the activation of caspase 3 after drug treatment and the suppression of cell death by a broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk). However, the protective effects of z-VAD-fmk and catalase were not additive. Taken together, our results indicated that the apoptosis induced by ginkgetin (especially at 5 microg/ml) is mediated mainly through the activation of caspase(s) by the hydrogen peroxide generated possibly through autooxidation of this biflavone.
Institute of Pharmacology, College of Life Science, National Yang-Ming University, Shih-Pai, Taipei, Taiwan, ROC.
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Chuang
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Chang
D000230:Adenocarcinoma / Q000473:pathology*; D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D044946:Biflavonoids; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D004249:DNA Damage; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000633:toxicity*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000473:pathology*; D016877:Oxidants / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D044946:Biflavonoids; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D005419:Flavonoids; D016877:Oxidants; C077458:ginkgetin; D004247:DNA; D006861:Hydrogen Peroxide; D020169:Caspases