Study on the Cytotoxic Mechanisms of Ginkgetin, a Biflavonoid Isolated from Selaginella Moellendorffii Hieron, in OVCAR-3 cells

• Main Authors: Hao-Hsuan, Chuang, 莊皓宣
• Other Authors: Yeu, Su
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/31211678416873589116

碩士 === 國立陽明大學 === 藥理學研究所 === 89 === Ginkgetin, a biflavonoid compound isolated from Selaginella moellendorffii, was reported to possess a selectively inhibitory effect on the growth of human ovarian adenocarcinoma (OVCAR-3) cells. To examine whether Ginkgetin also has a selective cytotoxic effect, MTT assays were performed using three different human cell lines: OVCAR-3, HeLa (human cervical carcinoma cell lines) and FS-5 (human foreskin fibroblast) as targets. We found that Ginkgetin kills OVCAR-3 cells with an EC50 equals to 3.0 mg/ml, while EC50 of this compound on HeLa and FS-5 cells was 5.2 mg/ml and 8.3 mg/ml, respectively. To examine the possible involvement of apoptosis in the toxic effect of Ginkgetin on OVCAR-3 cells, their morphology was first examined by light microscopy. During drug treatment, cell shrinkage and detachment from dish were observed. Moreover, when cell lysates prepared from OVCAR-3 cells after incubating with 3 mg/ml and 5 mg/ml of Ginkgetin for 48 h were subjected to agarose gel electrophoretic analysis, and the characteristic pattern of inter-nucleosomal DNA fragmentation was also observed. These results suggest that Ginkgetin kills OVCAR-3 cells through an apoptotic pathway. Because Ginkgetin is a polyphenolic compound, whose apoptotic effect may mediate through inducing intracellular oxidative damage, we therefore examined whether antioxidants can protect cells against the toxicity of this compound. We found that pre-treatment with vitamin C, vitamin E and catalase increases the viability of cells, and pretreatment with catalase also reduces DNA fragmentation caused by Ginkgetin, especially when high dose (5 mg/ml) of Ginkgetin was used. Since DNA is a major target for reactive oxygen species (ROS), DNA damage induced by Ginkgetin was then examined by a sensitive fluorometric method. High degrees of double-stranded DNA breakage were detected after cells were treated with 3 mg/ml and 5 mg/ml of Ginkgetin for 24 h, and such damage could greatly be reduced by the addition of 1000 U/ml of catalase 1.5 h before treatment with Ginkgetin. These results indicate that the toxicity of Ginkgetin on OVCAR-3 cells is mediated at least in part by inducing oxidative stress. z-VAD-fmk, a caspase inhibitor with broad specificity, was shown to be able to increase cell survival significantly when added simultaneously with Ginkgetin. This result indicates the participation of caspase(s) in the apoptosis induced by Ginkgetin. However, co-addition of catalase with z-VAD-fmk did not further increase cell survival. Therefore, we concluded that in addition to oxidative damage, other mechanisms may also be involved in the apoptosis of OVCAR-3 cells induced by Ginkgetin. We also found that the protective effects of both anti-oxidants and z-VAD-fmk on cells treated with 3 mg/ml of Ginkgetin was lower than those treated with 5 mg/ml of similar compound. These results indicate that oxidative damage is the major cause of apoptosis when cells treated with high dose (5 mg/ml). On the other hand, oxidative damage seems to play a very minor role in apoptosis induced by low dose of Ginkgetin, suggesting other mechanism(s) is involved in this apoptosis process.