Department of Pharmacy, Zhongnan Hospital of Wuhan University, Donghu Road 169, Wuhan, 430071, China.
Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Donghu Road 185, Wuhan, 430071, China.
J Exp Clin Cancer Res. 2019 Aug 22;38(1):371. doi: 10.1186/s13046-019-1361-2.
Arachidonic acid (AA) metabolic enzymes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1) and cytochrome P450 (CYP) 4A11 play important roles in glioma angiogenesis. Thus, there is an urgent need to identify the underlying mechanisms and develop strategies to overcome them.
A homology model of human CYP4A11 was constructed using SYBYL-X 2.0. Structure-based virtual screening against COX-2, mPGES-1 and CYP4A11was performed using the Surflex-Dock of the SYBYL suite. The candidates were further evaluated their antiangiogenic activities in a zebrafish embryo and rabbit corneal angiogenesis model. Laser doppler analysis was used to measure tumor perfusion. The expression of CD31 and α-SMA was measured by immunofluorescence. Western blot was used to measure the expression of HIF-1, Akt and p-Akt. The gene expression of FGF-2, G-CSF, PDGF, TGF-β, Tie-2, VEGF, lncRNA NEAT1 and miR-194-5p were determined using qPCR. The production of FGF-2, TGF-β and VEGF were analyzed using ELISA. Bioinformatic analysis and luciferase reporter assays confirmed the interaction between lncRNA NEAT1 and miR-194-5p.
The nearly 36,043 compounds from the Traditional Chinese Medicine (TCM) database were screened against COX-2, mPGES-1 and CYP4A11 3D models, and the 17 top flavonoids were identified. In zebrafish screening, isoliquiritigenin (ISL) exhibited the most potent antiangiogenic activities with the EC values of 5.9 μM. Conversely, the antiangiogenic effects of ISL in the zebrafish and rabbit corneal models were partly reversed by 20-hydroxyeicosatetraenoic acid (20-HETE) or prostaglandin E2 (PGE). ISL normalized glioma vasculature and improved the efficacy of temozolomide therapy in the rat C6 glioma model. Inhibition of COX-2, mPGES-1 and CYP4A by ISL decreased FGF-2, TGF-β and VEGF production in the C6 and U87 glioma cells with p-Akt downregulation, which was reversed by Akt overexpression. Furthermore, ISL downregulated lncRNA NEAT1 but upregulated miR-194-5p in the U87 glioma cell. Importantly, lncRNA NEAT1 overexpression reversed ISL-mediated increase in miR-194-5p expression, and thereby attenuated FGF-2, TGF-β and VEGF production.
Reprogramming COX-2, mPGES-1 and CYP4A mediated-AA metabolism in glioma by flavonoid ISL inhibits the angiogenic Akt- FGF-2/TGF-β/VEGF signaling through ceRNA effect of miR-194-5p and lncRNA NEAT1, and may serve as a novel therapeutic strategy for human glioma.
花生四烯酸（AA）代谢酶，包括环氧化酶-2（COX-2）、微粒体前列腺素 E 合酶-1（mPGES-1）和细胞色素 P450（CYP）4A11，在神经胶质瘤血管生成中起重要作用。因此，迫切需要确定潜在的机制并开发克服这些机制的策略。
使用 SYBYL-X 2.0 构建人 CYP4A11 的同源模型。使用 SYBYL 套件中的 Surflex-Dock 对 COX-2、mPGES-1 和 CYP4A11 进行基于结构的虚拟筛选。候选物进一步在斑马鱼胚胎和兔角膜血管生成模型中评估其抗血管生成活性。激光多普勒分析用于测量肿瘤灌注。通过免疫荧光测定 CD31 和α-SMA 的表达。使用 Western blot 测量 HIF-1、Akt 和 p-Akt 的表达。使用 qPCR 测定 FGF-2、G-CSF、PDGF、TGF-β、Tie-2、VEGF、lncRNA NEAT1 和 miR-194-5p 的基因表达。使用 ELISA 分析 FGF-2、TGF-β 和 VEGF 的产生。生物信息学分析和荧光素酶报告基因测定证实了 lncRNA NEAT1 和 miR-194-5p 之间的相互作用。
从中药（TCM）数据库中筛选了近 36043 种化合物以针对 COX-2、mPGES-1 和 CYP4A11 的 3D 模型，鉴定出 17 种最有效的黄酮类化合物。在斑马鱼筛选中，异甘草素（ISL）表现出最强的抗血管生成活性，EC 值为 5.9μM。相反，ISL 在斑马鱼和兔角膜模型中的抗血管生成作用部分被 20-羟二十碳四烯酸（20-HETE）或前列腺素 E2（PGE）逆转。ISL 使神经胶质瘤血管正常化，并改善了替莫唑胺治疗大鼠 C6 神经胶质瘤模型的疗效。ISL 抑制 COX-2、mPGES-1 和 CYP4A 的作用降低了 C6 和 U87 神经胶质瘤细胞中 FGF-2、TGF-β 和 VEGF 的产生，同时下调了 p-Akt，而过表达 Akt 则逆转了这一作用。此外，ISL 在 U87 神经胶质瘤细胞中下调了 lncRNA NEAT1 但上调了 miR-194-5p。重要的是，lncRNA NEAT1 的过表达逆转了 ISL 介导的 miR-194-5p 表达增加，并因此减弱了 FGF-2、TGF-β 和 VEGF 的产生。
通过黄酮类化合物 ISL 重塑神经胶质瘤中 COX-2、mPGES-1 和 CYP4A 介导的 AA 代谢，通过 miR-194-5p 和 lncRNA NEAT1 的 ceRNA 效应抑制了血管生成 Akt-FGF-2/TGF-β/VEGF 信号通路，可能为人类神经胶质瘤提供一种新的治疗策略。
