Kelleher Michael O, McMahon Michael, Eggleston Ian M, Dixon Mark J, Taguchi Keiko, Yamamoto Masayuki, Hayes John D
Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Scotland, UK.
Carcinogenesis. 2009 Oct;30(10):1754-62. doi: 10.1093/carcin/bgp182. Epub 2009 Jul 24.
Epithionitriles represent a previously unrecognized class of cancer chemopreventive phytochemical generated from alkenyl glucosinolates in cruciferous vegetables. In rat liver RL-34 epithelial cells, 1-cyano-2,3-epithiopropane (CETP), 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent) were shown to induce cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione (GSH) S-transferase A3 and the glutamate-cysteine ligase modifier subunit; CETP was more potent in this regard than were either CETB or CETPent, with 50 microM CETP eliciting a remarkable approximately 10-fold induction of NQO1. Furthermore, 50 microM CETP stimulated a 2.0-fold overproduction of GSH in RL-34 cells. Transfection experiments demonstrated that epithionitriles induced gene expression through an antioxidant response element (ARE) and that transactivation of an Nqo1-luciferase reporter plasmid was dependent on NF-E2 p45-related factor 2 (Nrf2), a cap'n'collar basic region leucine zipper transcription factor. Evidence is presented that CETP affected Nrf2-mediated induction of ARE-driven transcription by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase substrate adaptor that negatively regulates Nrf2. We found that Nqo1 was expressed constitutively at high levels in Keap1(-/-) mouse embryonic fibroblasts (MEFs) and it was not further induced by CETP. However, knock-in of mouse Keap1 or zebrafish Keap1a into Keap1(-/-) MEFs repressed Nqo1-luciferase reporter gene activity, but repression by the murine or zebrafish proteins was antagonized by CETP. Pre-treatment of Nrf2(+/+) MEFs, but not Nrf2(-/-) MEFs, with 15 microM CETP for 24 h conferred 2.4-fold resistance against subsequent exposure to the alpha,beta-unsaturated aldehyde acrolein, indicating that the phytochemical exerts chemopreventive properties against genotoxic xenobiotics.
环硫腈代表了一类以前未被认识的癌症化学预防植物化学物质，它由十字花科蔬菜中的链烯基芥子油苷生成。在大鼠肝脏RL - 34上皮细胞中，1 - 氰基 - 2,3 - 环硫丙烷（CETP）、1 - 氰基 - 3,4 - 环硫丁烷（CETB）和1 - 氰基 - 4,5 - 环硫戊烷（CETPent）被证明能诱导细胞保护酶，包括NAD(P)H：醌氧化还原酶1（NQO1）、谷胱甘肽（GSH）S - 转移酶A3和谷氨酸 - 半胱氨酸连接酶修饰亚基；在这方面，CETP比CETB或CETPent更有效，50微摩尔的CETP能显著诱导NQO1增加约10倍。此外，50微摩尔的CETP能刺激RL - 34细胞中GSH过量产生2.0倍。转染实验表明，环硫腈通过抗氧化反应元件（ARE）诱导基因表达，并且Nqo1 - 荧光素酶报告质粒的反式激活依赖于NF - E2 p45相关因子2（Nrf2），一种帽状衣领碱性区域亮氨酸拉链转录因子。有证据表明，CETP通过抑制Kelch样ECH相关蛋白1（Keap1）影响Nrf2介导的ARE驱动转录的诱导，Keap1是一种对Nrf2起负调节作用的泛素连接酶底物衔接蛋白。我们发现，Nqo1在Keap1(-/-)小鼠胚胎成纤维细胞（MEF）中组成性高表达，并且它不会被CETP进一步诱导。然而，将小鼠Keap1或斑马鱼Keap1a敲入Keap1(-/-) MEF中会抑制Nqo1 - 荧光素酶报告基因活性，但CETP能拮抗小鼠或斑马鱼蛋白的抑制作用。用15微摩尔的CETP对Nrf2(+/+) MEF进行预处理24小时，而不是对Nrf2(-/-) MEF进行预处理，能使细胞对随后暴露于α,β - 不饱和醛丙烯醛产生2.4倍的抗性，这表明这种植物化学物质对遗传毒性异源生物具有化学预防特性。
