類黃酮素化合物對砷誘導細胞凋亡之活性研究

Effects of Flavonoids on Arsenic-induced Apoptosis in Human Keratinocyte

郭佳儀(Jia-Yi Kuo)

指導教授 ： 陳彥州

臺北醫學大學/藥學院/生藥學研究所/碩士(2005年)

摘要

摘 要 雖然三氧化二砷（As2O3）能在多種癌細胞誘導細胞凋亡，但是不同細胞對As2O3敏感度卻大不一樣。而活性氧分子在As2O3所誘導的細胞凋亡扮演重要角色。類黃酮素化合物為植物中衍生的抗氧化劑，普遍存在水果蔬菜當中，對多種癌細胞具有抗細胞增生和誘導細胞凋亡的作用。在我們的研究中，我們探討As2O3在有類黃酮素的存在下對於人類角質細胞(HaCaT)的毒殺作用及其可能的機轉。我們利用MTT assay，LDH release和DNA階梯狀斷裂來探討其毒殺作用。且利用西方點墨法來檢測蛋白p53，p21，bax，bad，bcl-xL的表現，caspase-3的活化和PARP及D4GDI的裂解。結果顯示As2O3在有hesperetin 和quercetin的存在下，可以增強As2O3對人類角質細胞的毒殺作用，反之hesperidin和rutin則沒有任何增強作用。As2O3在有hesperetin 和quercetin的存在下可以誘導caspase-3的活化，藉由其分子量為17KDa的活化態還有其作用蛋白質PARP和D4GDI的裂解( 85KDa及23KDa band的產生)。而p53的基因表現則明顯受到抑制。ROS的產生則是藉由2`,7`-dichlorodihydrofluorescein diacetate (DCHF-DA)來檢測，我們發現As2O3在有hesperetin 和quercetin的存在下，能在人類角質細胞增加ROS的產生。NAC的前處理可以減輕As2O3在有hesperetin 和quercetin的存在下所引發的細胞毒殺作用和蛋白caspase-3的活化。因此我們推論As2O3在有hesperetin 和quercetin的存在下在人類角質細胞所誘導的細胞凋亡，可能是透過ROS的產生以及p21蛋白的增加和caspase-3的活化。

關鍵字

砷 ； 類黃酮素

並列摘要

Abstract Although arsenic trioxide (As2O3) induced apoptosis in a relatively wide species of tumors, the sensitivity of different cell types to this treatment varies to a great extent. Because reactive oxygen species ( ROS) are critically involved in As2O3-induced apoptosis. Flavonoids, which are plant-derived antioxidants, occur in fruits and vegetables and have been examined extensively for their anti-proliferative and pro-apoptotic effects in various cancer cell lines. In this study, we investigate the cytotoxic effect of As2O3 plus flavonoids in human immortalized keratinocyte (HaCaT) and its possible mechanism. Cytotoxicity was determined by MTT assay, LDH release, and DNA fragmentation. Protein levels of p53, p21, bax, bad, bcl-XL, caspase-3 and the cleavage of poly(ADP)-ribose polymerase(PARP) and D4GDI were detected by western blotting. The results demonstrated that coadministration of hesperetin or quercetin with As2O3 enhanced As2O3-rendered cytotoxicity in HaCaT cells, whereas the coadministration with hesperidin or rutin cause no detectable apoptotic effects in HaCaT cells. As2O3 coadministration of hesperetin or quercetin induced caspase-3 expression with appearance of its 17 KDa peptide fragment, and cleavage of PARP and D4GDI, with appearance of the 85 KDa and 23KDa cleavage products.Apparently decrease in p53 protein level also found in this treatment. The production of ROS that was examined by 2`,7`-dichlorodihydrofluorescein diacetate (DCHF-DA), increased significantly after the treatment with As2O3 plus hesperetin or quercetin in HaCaT cells. The cytotoxicity and caspase-3 activation enhancement by hesperetin or quercetin could be mildly abolished by the antioxidant N-acetyl-L-cysteine(NAC). Therefore, we concluded that coadministration of hesperetin or quercetin induced apoptosis in HaCaT cells might be through the generation of ROS and p21, caspase-3 cascade were involved in this apoptotic mechanisms.

並列關鍵字

arsenic ； flavonoids

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