Journal of Neuroscience Research, 2010, 88(4):877-86.

Genistein induces receptor and mitochondrial pathways and increases apoptosis during BCL-2 knockdown in human malignant neuroblastoma SK-N-DZ cells

Joseph George, Naren L. Banik, and Swapan K. Ray

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

The potent antiapoptotic molecule Bcl-2 is markedly up-regulated in a majority of cancers, including neuroblastoma. Genistein is an isoflavone with antitumor properties. The present study sought to elucidate the molecular mechanism of genistein-induced apoptosis and also to examine the effect of genistein in increasing apoptosis during Bcl-2 knockdown in human malignant neuroblastoma SK-N-DZ cells. The cells were transfected with Bcl-2 siRNA plasmid vector, treated with 10 microM genistein, or the combination, and subjected to TUNEL staining and FACS analysis. Semiquantitative and real-time RT-PCR experiments were performed for examining expression of Fas ligand (FasL), tumor necrosis factor-alpha (TNF-alpha), Fas-associated death domain (FADD), and TNFR-1-associated death domain (TRADD). The cell lysates were analyzed by Western blotting for levels of molecules involved in both receptor- and mitochondria-mediated apoptotic pathways. Treatment with the combination of Bcl-2 siRNA and genistein resulted in more than 80% inhibition of cell proliferation. TUNEL staining and FACS analysis demonstrated apoptosis in 70% of cells after treatment with the combination of both agents. Apoptosis was associated with increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and activation of caspases through the mitochondria-mediated apoptotic pathway. Genistein triggered the receptor-mediated apoptotic pathway through upregulation of TNF-alpha, FasL, TRADD, and FADD and activation of caspase-8. Combination of Bcl-2 siRNA and genistein triggered a marked increase in cleavage of DFF45 and PARP that resulted in enhanced apoptosis. Our study demonstrates that Bcl-2 knockdown during genistein treatment effectively induced apoptosis in neuroblastoma cells. Therefore, this strategy could serve as a potential therapeutic regimen to inhibit the growth of human malignant neuroblastoma.

美国《神经科学研究杂志》，2010年3月

在人类恶性神经母细胞瘤 SK-N-DZ细胞中，经过Bcl-2表达降低染料木黄酮诱导受体和线粒体通路并且增加细胞凋亡

约瑟夫  乔治，那仁达莱 L 班尼克 和瓦潘 K 雷

哥伦比亚南卡罗莱纳州医科大学病理学、 微生物和免疫学院以及神经科学院

强效抗凋亡分子 Bcl-2 在大多数癌症包括神经母细胞瘤中明显上调。金雀异黄素是一种具有抗癌特性的异黄酮。本研究力求阐明染料木黄酮诱导细胞凋亡的分子机制，同时也探究了染料木黄酮在人类恶性神经母细胞瘤 SK-N-DZ 细胞中使Bcl-2表达降低促进细胞凋亡的影响。这些细胞被Bcl-2的siRNA 质粒载体转染，用10 uM 的染料木素或复合物来处理，然后用 TUNEL 染色和流式细胞仪来分析。进行半定量和实时 RT-PCR 实验来测试 Fas 配体 (FasL)、肿瘤坏死因子-α （TNF-α）、 Fas 相关死亡结构域 (FADD) 和 TNFR-1- 相关死亡结构域 (TRADD) 的 表达。用分子水平的免疫印迹法分析参与了受体和线粒体介导的凋亡通路的细胞裂解液。用 Bcl-2的 siRNA 和金雀异黄素的联合治疗会引起超过 80%的细胞增殖抑制作用。TUNEL 染色和流式细胞仪表明在经过两种药物联合治疗以后表现出 70 %的细胞凋亡。细胞凋亡与通过线粒体介导的细胞凋亡通路增加的 Bax:Bcl-2 比、细胞色素 c线粒体的释放和半胱氨酸蛋白酶的激活有关。金雀异黄素通过上调 TNF-α、 FasL、 TRADD，FADD 和激活半胱氨酸蛋白酶-8引发受体介导的细胞凋亡途径。Bcl-2 siRNA 和金雀异黄素的组合物诱导了DFF45和 PARP 卵裂的显著增加，这导致了细胞凋亡的增强。我们的研究表明，在神经母细胞瘤中染料木黄酮治疗期间Bcl-2表达减小有效地诱导细胞凋亡。因此，这种策略可以作为潜在的治疗方案来抑制人类恶性神经母细胞瘤的生长。