Studies on the cytotoxic mechanisms of ginkgetin in a human ovarian adenocarcinoma cell line

The cytotoxic effects of ginkgetin, a natural biflavone isolated from Selag inella 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 for eskin 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 mu g/ml , respectively. Morphological changes in cells and their nuclei, DNA fragme ntation with a characteristic pattern of inter-nucleosomal ladder, and doub le-stranded DNA breaks were detected following treatment with 3 mu g/ml of this biflavone for 24 h. Incubation with 5 mu g/ml ginkgetin led to increas ed intracellular levels of hydrogen peroxide as early as 30 min. The cytoto xicity of ginkgetin was partially inhibited by pretreating cells with vitam in C. vitamin E or catalase. Catalase not only afforded the best protective effect among three antioxidants, but also reduced both the DNA fragmentati on and double-stranded DNA breakage induced by ginkgetin. Moreover, the inv olvement of caspase(s) in ginkgetin-induced apoptosis was demonstrated by t he 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-f mk and catalase were not additive. Taken together our results: indicated th at the apoptosis induced by ginkgetin (especially at 5 mu g/ml) is mediated mainly through the activation of caspase(s) by the hydrogen peroxide gener ated possibly through autooxidation of this biflavone.