Nie Daotai, Krishnamoorthy Sriram, Jin Rongxian, Tang Keqin, Chen YuChyu, Qiao Yan, Zacharek Alex, Guo Yande, Milanini Julie, Pages Gilles, Honn Kenneth V
Department of Radiation Oncology, Wayne State University School of Medicine, Karmanos Cancer Institute, Detroit, Michigan 48202, USA.
J Biol Chem. 2006 Jul 7;281(27):18601-9. doi: 10.1074/jbc.M601887200. Epub 2006 Apr 25.
12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.
12-脂氧合酶利用花生四烯酸合成12(S)-氢过氧化二十碳四烯酸，该物质可转化为终产物12(S)-羟基二十碳四烯酸，这是一种促进肿瘤发生和转移的类花生酸。12-脂氧合酶在多种癌症中表达增加。当在人前列腺癌或乳腺癌中过表达时，12-脂氧合酶可促进体内肿瘤血管生成和生长。本研究旨在阐明12-脂氧合酶增强血管生成的机制。在此我们报告，脂氧合酶的泛抑制剂去甲二氢愈创木酸和12-脂氧合酶的选择性抑制剂黄芩苷可降低人前列腺癌PC-3细胞中VEGF的表达。与载体对照细胞相比，PC-3细胞中12-脂氧合酶的过表达导致VEGF蛋白水平增加了3倍。在12-LOX转染的PC-3细胞中发现PI 3-激酶活性增加，而LY294002对PI 3-激酶的抑制显著降低了VEGF的表达。Northern印迹和实时PCR分析显示，用12-脂氧合酶表达构建体转染的PC-3细胞中VEGF转录水平升高。使用VEGF启动子荧光素酶构建体(-1176/+54)，我们发现12-脂氧合酶转染的PC-3细胞中VEGF启动子活性增加了10倍。使用基于VEGF启动子的荧光素酶测定法，将VEGF启动子-88至-66之间的区域鉴定为对12-脂氧合酶有反应。用突变构建体进行的进一步分析表明，Sp1是12-脂氧合酶刺激VEGF所需的转录因子。用功能阻断抗体中和VEGF可显著降低12-脂氧合酶转染的PC-3细胞刺激内皮细胞迁移的能力，这表明VEGF是12-脂氧合酶介导的肿瘤血管生成刺激的重要效应因子。
