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 mu g/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 mu g/ml of this biflavone for 24 h. Incubation with 5 mu g/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 mu g/ml) is mediated mainly through the activation of caspase(s) by the hydrogen peroxide generated possibly through autooxidation of this biflavone.