Cancer, 2010, 116(1):164–176.

Flavonoids activated caspases for apoptosis in human glioblastoma T98G and U87MG cells but not in human normal astrocytes

Arabinda Das, Naren L. Banik, and Swapan K. Ray

Department of  Neurosciences, Medical University of South Carolina, Charleston, South Carolina; Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina

BACKGROUND: Human glioblastoma is a deadly brain cancer that continues to defy all current therapeutic strategies. The authors induced apoptosis in human glioblastoma T98G and U87MG cells following treatment with apigenin, (-)-epigallocatechin, (-)-epigallocatechin-3-gallate (EGCG), and genistein that did not induce apoptosis in human normal astrocytes.

METHODS: Induction of apoptosis was examined using Wright staining and ApopTag assay. Production of reactive oxygen species (ROS) and increase in intracellular free Ca²⁺ were measured by fluorescent probes. Analysis of mRNA and Western blotting indicated increases in expression and activities of the stress kinases and cysteine proteases for apoptosis. JC-1 showed changes in mitochondrial membrane potential (ΔΨ_m) and use of specific inhibitors confirmed activation of kinases and proteases in apoptosis.

RESULTS: Treatment of glioblastoma cells with apigenin, (-)-epigallocatechin, EGCG, or genistein triggered ROS production that induced apoptosis with phosphorylation of p38 mitogen-activated protein kinase(MAPK) and activation of the redox-sensitive c-Jun N-terminal kinase 1 pathway. Pretreatment of cells with ascorbic acid attenuated ROS production and p38 MAPK phosphorylation. Increases in intracellular free Ca²⁺ and activation of caspase-4 indicated involvement of endoplasmic reticulum stress in apoptosis. Other events in apoptosis included overexpression of Bax, loss of ΔΨ_m, mitochondrial release of cytochrome c and Smac into the cytosol, down regulation of baculoviral inhibitor-of-apoptosis repeat -containing proteins, and activation of calpain, caspase-9, and caspase-3.  (-)-epigallocatechin and EGCG also induced caspase-8 activity. Apigenin, (-)-epigallocatechin, EGCG, and genistein did not induce apoptosis in human normal astrocytes.

CONCLUSION: Results strongly suggest that flavonoids are potential therapeutic agents for induction of apoptosis in human glioblastoma cells.

美国《癌症》，2010年1月

黄酮类化合物在人体恶性胶质瘤 T98G 和 U87MG 细胞中激活半胱氨酸蛋白酶使其凋亡而不会影响人体正常的星形胶质细胞

阿拉宾达 达斯、 那仁达莱 L 班尼克 和瓦潘  k  雷

南卡罗来纳州南卡罗莱纳医科大学神经科学学院以及病理学、微生物学和免疫学学院

背景：人体恶性胶质瘤是一种致命的脑癌，当前的所有治疗策略均对它束手无策。本文作者分别用芹菜素、（-）-表没食子儿茶素、（-） -儿茶素-3-没食子酸酯 (EGCG) 和金雀异黄素等来诱导人体恶性胶质瘤 T98G 和 U87MG 细胞的凋亡，并且不会引起人体正常星形胶质细胞的凋亡。

方法：使用瑞氏染色和 ApopTag 含量测定进行诱导细胞凋亡的研究。活性氧(ROS)的产生和细胞内游离 Ca^{2 +} 的增加通过荧光探针来测定。mRNA 和蛋白印迹的分析表明应激激酶和半胱氨酸蛋白酶对细胞凋亡的表达和活动有所增加。JC-1 表明在线粒体膜电位(ΔΨm)的变化 并且使用特定的抑制剂来确认激酶和蛋白酶在细胞凋亡中的活动。

结果：用芹菜素、（-）-表没食子儿茶素、EGCG 和金雀异黄素等治疗胶质母细胞瘤细胞时，会引发活性氧的产生进而诱导细胞凋亡，以及p38 有丝分裂原激活的蛋白激酶(MAPK) 的磷酸化和氧化还原敏感的 c-Jun N-末端激酶 信号通路1的活动。用抗坏血酸预处理细胞可以减弱活性氧的产生和 p38 MAPK的 磷酸化。细胞内游离 Ca^{2 +} 的增多以及半胱氨酸蛋白酶-4的活动 表明内质网应激参与了细胞凋亡。在细胞凋亡中的其他变动包括 凋亡蛋白的过表达， ΔΨ_m损失、细胞色素 c释放线粒体以及 Smac 进入胞浆，杆状病毒凋亡抑制剂重复蛋白质的下调，和钙蛋白酶、 半胱氨酸蛋白酶-9，和半胱氨酸蛋白酶-3 的激活。（-）-表儿茶素和 EGCG 还会诱导半胱氨酸蛋白酶-8 激活。芹菜素，（-）-表儿茶素、 EGCG 和金雀异黄素不会引起人类正常星形胶质细胞凋亡。