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CYP4X1 Inhibition by Flavonoid CH625 Normalizes Glioma Vasculature Through Reprogramming Tams Via CB2 and EGFR- STAT3 Axis S

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CYP4X1 Inhibition by Flavonoid CH625 Normalizes Glioma Vasculature Through Reprogramming Tams Via CB2 and EGFR- STAT3 Axis S Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2018/02/01/jpet.117.247130.DC1 1521-0103/365/1/72–83$35.00 https://doi.org/10.1124/jpet.117.247130 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 365:72–83, April 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics CYP4X1 Inhibition by Flavonoid CH625 Normalizes Glioma Vasculature through Reprogramming TAMs via CB2 and EGFR- STAT3 Axis s Chenlong Wang, Ying Li, Honglei Chen, Keqing Huang, Xiaoxiao Liu, Miao Qiu, Yanzhuo Liu, Yuqing Yang, and Jing Yang Department of Pharmacology and Hubei Province Key Laboratory of Allergy and Immune-related Diseases (C.W., Y.L., K.H., X.L., M.Q., Y.L., J.Y.), Experimental Teaching Center (J.Y.), and Department of Pathology and Pathophysiology (H.C.), School of Basic Medical Sciences, Wuhan University, Wuhan, China; Hubei Key Laboratory of Medical Information Analysis and Tumor Diagnosis & Treatment, South-central University for Nationalities, Wuhan, China (C.W.); and Department of Pharmaceutics, Ernest Mario Downloaded from School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey (Y.Y.) Received December 11, 2017; accepted January 29, 2018 ABSTRACT Tumor-associated macrophages (TAMs) are pivotal effector the TAMs. Furthermore, CH625 attenuated vascular abnormal- jpet.aspetjournals.org cells in angiogenesis. Here, we tested whether CYP4X1 in- ization and immunosuppression induced by coimplantation hibition in TAMs by flavonoid CH625 prolongs survival and of GL261 cells with CYP4X1high macrophages. In vitro TAM normalizes glioma vasculature. CH625 was selected against the polarization away from the M2 phenotype by CH625 inhibited CYP4X1 3D model by virtual screening and showed inhibitory proliferation and migration of endothelial cells, enhanced peri- activity on the CYP4X1 catalytic production of 14,15-EET-EA in cyte migration and T cell proliferation, and decreased VEGF and the M2-polarized human peripheral blood mononuclear cells TGF-b production accompanied with the downregulation of CB2 5 (IC50 16.5 mM). CH625 improved survival and reduced tumor and EGFR-dependent downstream STAT3 expression. These at ASPET Journals on September 23, 2021 burden in the C6 and GL261 glioma intracranial and subcutane- effects were reversed by overexpression of CYP4X1 and STAT3 ous model. In addition, CH625 normalized vasculature (evi- or exogenous addition of 14,15-EET-EA, VEGF, TGF-b, EGF, denced by a decrease in microvessel density and HIF-1a and CB2 inhibitor AM630. These results suggest that CYP4X1 expression and an increase in tumor perfusion, pericyte cover- inhibition in TAMs by CH625 prolongs survival and normalizes age, and efficacy of temozolomide therapy) accompanied with tumor vasculature in glioma via CB2 and EGFR-STAT3 axis and the decreased secretion of 14,15-EET-EA, VEGF, and TGF-b in may serve as a novel therapeutic strategy for human glioma. Introduction 2017). The challenge moving forward will be to overcome these limitations and expand the benefits of antiangiogenic Glioblastoma (GBM) is the most common primary malig- therapy. Vascular normalization induced by antiangiogenic nant brain tumor (Wick et al., 2017). Angiogenesis is integral treatment modestly enhances T cell infiltration and improves to the pathology of GBM (Turkowski et al., 2018). Inhibitors of the therapeutic efficacy of anticancer drugs and may represent vascular endothelial growth factor (VEGF) are commonly used a potential therapeutic strategy for human glioma (Goel et al., to block GBM angiogenesis in the clinic but fail to improve 2011; Peterson et al., 2016). overall survival (Wick et al., 2016). Moreover, antiangiogenic Tumor-associated macrophages (TAMs) constitute the ma- treatment with sunitinib, a multitargeted receptor tyrosine jority of the stromal cells within a GBM (Huijbers et al., 2016). kinase inhibitor, increases PD-L1 expression and fosters an TAMs are correlated with poor survival of patients with immunosuppressive microenvironment (Ramjiawan et al., recurrent glioblastoma (Huijbers et al., 2016; Kloepper et al., 2016). TAMs contribute to angiogenesis by indirect paracrine secretion of proangiogenic growth factors, including VEGF, This work was partly supported by the National Natural Science Foundation of China [Grant 81173089 (to J.Y.)] and Hubei Key Laboratory transforming growth factor (TGF-b) and hypoxia-inducible of Medical Information Analysis and Tumor Diagnosis & Treatment [Grants factor-1a (HIF-1a) (Huijbers et al., 2016). Conversely, de- PJS140011508 (to C.W.) and PJS140011707 (to Y.L.)]. https://doi.org/10.1124/jpet.117.247130. pletion of TAMs inhibits tumor growth and angiogenesis and s This article has supplemental material available at jpet.aspetjournals.org. improves the efficacy of chemotherapy as a result of vascular ABBREVIATIONS: AEA, anandamide; BMDM, bone marrow-derived macrophages; CB, cannabinoid receptors; CM, conditioned media; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; 14,15-EET-EA, 14,15-epoxyeicosatrienoic acid- ethanolamide; GBM, glioblastoma; HBVP, human brain vascular pericytes; HIF-1a, hypoxia-inducible factor-1a; HUVEC, human umbilical vein endothelial cells; IL, interleukin; PBMC, human peripheral blood mononuclear cells; a-SMA, a-smooth muscle actin; TAMs, tumor-associated macrophages; TGF, transforming growth factor; TMZ, temozolomide; VEGF, vascular endothelial growth factor. 72 CYP4X1 Inhibition Normalizes Glioma Vasculature 73 normalization (Guerriero et al., 2017; Wang et al., 2017). purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, Under different stimuli, macrophages can polarize into differ- MD). Antibodies against iNOS, CD206, arginase-1, p-VEGFR2, ent phenotypes: the two extremes of the phenotypic spectrum VEGFR2, VEGF, p-Smad2, Smad2, TGF-b, p-EGFR, EGFR, CB2, of TAMs are defined as the alternatively activated protumor caspase-3, Bcl-xL, Ki67, and b-actin were purchased from Abcam, Inc. (M2) versus classically activated antitumor (M1) states (Cambridge, MA). For the flow cytometric analysis, anti-CD8 was purchased from Abcam, Inc. Mouse recombinant IL-4 and IL-13 were (Peterson et al., 2016; Wang et al., 2017). Polarization of purchased from PeproTech (Rocky Hill, NJ). For in vivo experiment, TAMs is a novel strategy for vascular normalization and liposomal CH625 was prepared by mixing CH625, 1,2-dimyristoyl-sn- antitumor immunity (Huang et al., 2011). Our previous study glycero-3-phosphocholine and cholesterol at a mole ratio of 1:5:5 showed that reprogramming of M2-polarized macrophages according to a modification of Lim et al. (2000). The final products results in tumor growth inhibition and vascular normalization were stored at 220°C and warmed to room temperature just before in rat C6 glioma (Wang et al., 2017). Therefore, inhibiting use. For the in vitro experiment, CH625 was dissolved in dimethyl tumor angiogenesis by altering TAMs to an antitumoral sulfoxide (DMSO) as a stock solution (80 mM), stored at 220°C, and phenotype could represent a promising therapeutic strategy serial dilutions were made in DMSO. All culture reagents contained for glioblastoma. less than 0.125 endotoxin unit per milliliter as checked by Limulus The endocannabinoid system comprises endogenous ligands amebocyte lysate assay (BioWhittaker, Walkersville, MD). Homology Modeling. The homology model of human CYP4X1 such as anandamide (AEA), cannabinoid receptors (CB) 1/2, was automatically generated by the SWISS-MODEL program using and the proteins responsible for their synthesis and degrada- the crystal structure of human CYP4B1 (PDB id: 5T6Q) as a template Downloaded from tion (Snider et al., 2010). AEA markedly inhibits glioma (Bordoli et al., 2009). The heme was manually merged into the protein growth and angiogenesis through decreasing production of to occupy the same position as the heme of the template protein VEGF and TGF-b (Picardi et al., 2014; Ma et al., 2016). (CYP4B1) using the Coot 0.8.2 program (Emsley et al., 2010). Sub- Cytochrome P450 (CYP) 4X1, an orphan P450 protein, is sequently, a 5-ns dynamic simulation was performed using GRO- expressed in the brain and metabolizes AEA to 14,15- MACS 4.5.4 software (http://www.gromacs.org) (Pronk et al., 2013). epoxyeicosatrienoic acid-ethanolamide (14,15-EET-EA) (Snider The quality of the final model was validated by two programs, et al., 2010). Rhythmic regulation of Cyp4x1 gene expression Procheck and Verify_3D, both of which belong to the structure jpet.aspetjournals.org in the rat brain and vasculature may contribute to circadian analysis-validation online server sponsored by the UCLA-DOE In- stitute for Genomics and Proteomics (LaGier, 2017). changes in blood flow (Carver et al., 2014). CYP4X1 is over- Virtual Screening. Molecular docking studies were performed expressed in breast cancer and shows correlations with tumor using the Surflex-Dock26 of the SYBYL suite (SYBYL-X 2.0, Tripos) grade (Murray et al., 2010). Computational identification and (Joshi et al., 2016). For this purpose the homology model of human binding analysis of orphan human cytochrome P450 4X1 CYP4X1 was subjected to induced-fit docking studies. The active enzyme with substrates provide useful information on pocket of CYP4X1 for binding the inhibitors was generated above the structure-based drug design (Kumar, 2015). However, the heme-group of the homology model of CYP4X1 in
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