Abstract
BackgroundArachidonic 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.MethodsA 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.ResultsThe 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 EC50 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 (PGE2). 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.ConclusionsReprogramming 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.
Highlights
Arachidonic acid (AA) metabolic enzymes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 and cytochrome P450 (CYP) 4A11 play important roles in glioma angiogenesis
Structure-based virtual screening against COX-2, microsomal prostaglandin E synthase-1 (mPGES-1) and CYP4A11 and zebrafish screening Given that the crystal structure of CYP4A11 is not included in the Protein Data Bank, and CYP4A11 had the highest sequence identity (56%) with CYP4B1 (Fig. 1a), the CYP4B1 (PDB id: 5T6Q) was used as a template to build the 3D structure of CYP4A11
20-hydroxyeicosatetraenoic acid (20-HETE) or prostaglandin E2 (PGE2) reversed the antiangiogenic effects of the ISL in the zebrafish embryo and rabbit cornea We investigated whether the flavonoid ISL inhibits angiogenesis in the zebrafish embryo angiogenesis model through the downregulation of CYP4A and COX-2/ microsomal PGE synthase (mPGES)-1 signaling, and found that exogenous addition of 20-HETE or PGE2 partly reversed the antiangiogenic activities of ISL (Fig. 2)
Summary
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. Multiple angiogenesis inhibitors have been therapeutically validated in both preclinical and clinical settings, but concomitantly elicit tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness and increased metastasis [3]. It appears that clinical application of antiangiogenic therapy is more complex than originally thought [4]. The combination inhibition of COX-2, mPGES-1 and CYP4A could represent a promising therapeutic strategy for tumor angiogenesis
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