The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells
ABSTRACT Itraconazole is a safe and widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Currently, there are four active clinical trials evaluating itraconazole as a cancer therapeutic. Tumor growth is dependent on angiogenesis, which is driven by the secretion of growth factors from the tumor itself. We report here that itraconazole significantly inhibited the binding of vascular endothelial growth factor (VEGF) to VEGF receptor 2 (VEGFR2) and that both VEGFR2 and an immediate downstream substrate, phospholipase C γ1, failed to become activated after VEGF stimulation. These effects were due to a defect in VEGFR2 trafficking, leading to a decrease in cell surface expression, and were associated with the accumulation of immature N-glycans on VEGFR2. Small molecule inducers of lysosomal cholesterol accumulation and mammalian target of rapamycin (mTOR) inhibition, two previously reported itraconazole activities, failed to recapitulate itraconazole's effects on VEGFR2 glycosylation and signaling. Likewise, glycosylation inhibitors did not alter cholesterol trafficking or inhibit mTOR. Repletion of cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, was also able to restore VEGFR2 glycosylation and signaling. This suggests that the new effects of itraconazole occur in parallel to those previously reported but are downstream of a common target. We also demonstrated that itraconazole globally reduced poly-N-acetyllactosamine and tetra-antennary complex N-glycans in endothelial cells and induced hypoglycosylation of the epidermal growth factor receptor in a renal cell carcinoma line, suggesting that itraconazole's effects extend beyond VEGFR2.
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ABSTRACT: Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.Cell Reports 01/2015; DOI:10.1016/j.celrep.2014.12.054 · 7.21 Impact Factor
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ABSTRACT: Sialic acid (NeuAc) is a major anion on endothelial cells (ECs) that regulates different biological processes including angiogenesis. NeuAc is present in the oligosaccharidic portion of integrins, receptors that interact with extracellular matrix components and growth factors regulating cell adhesion, migration, and proliferation. Tat is a cationic polypeptide that, once released by HIV-1(+) cells, accumulates in the extracellular matrix, promoting EC adhesion and proangiogenic activation by engaging α(v)β(3). By using two complementary approaches (NeuAc removal by neuraminidase or its masking by NeuAc-binding lectin from Maackia amurensis, MAA), we investigated the presence of NeuAc on endothelial α(v)β(3) and its role in Tat interaction, EC adhesion, and proangiogenic activation. α(v)β(3) immunoprecipitation with biotinylated MAA or Western blot analysis of neuraminidase-treated ECs demonstrated that NeuAc is associated with both the α(v) and the β(3) subunits. Surface plasmon resonance analysis demonstrated that the masking of α(v)β(3)-associated NeuAc by MAA prevents Tat/α(v)β(3) interaction. MAA and neuraminidase prevent α(v)β(3)-dependent EC adhesion to Tat, the consequent FAK and ERK1/2 phosphorylation, and EC proliferation, migration, and regeneration in a wound-healing assay. Finally, MAA inhibits Tat-induced neovascularization in the ex vivo human artery ring sprouting assay. The inhibitions are specific because the NeuAc-unrelated lectin from Ulex europaeus is ineffective on Tat. Also, MAA and neuraminidase affect only weakly integrin-dependent EC adhesion and proangiogenic activation by fibronectin. In conclusion, NeuAc is associated with endothelial α(v)β(3) and mediates Tat-dependent EC adhesion and proangiogenic activation. These data point to the possibility to target integrin glycosylation for the treatment of angiogenesis/AIDS-associated pathologies.Journal of Biological Chemistry 04/2012; 287(24):20456-66. DOI:10.1074/jbc.M111.337139 · 4.60 Impact Factor
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ABSTRACT: Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disorder involving mainly synovial joints. It can progress to a severely debilitating form with pulmonary, renal and cardiovascular involvement. Currently, disease-modifying antirheumatic drugs (DMARDs) remain the gold standard pharmacological therapy for RA (along with nonsteroidal anti-inflammatory drugs and corticosteroids). However, DMARDs are more or less ineffective in the late phase of the disease and adverse effects often limit their use. Studies show that serum levels of vascular endothelial growth factor (VEGF) remain elevated throughout the course of RA. In experimental models, the administration of pro-angiogenic cytokines, such as VEGF or FGF, has been shown to increase the severity of the disease. Therefore, anti-angiogenic drugs such as bevacizumab (which is already being used as an anti-tumor agent) may play a significant role in longstanding RA. However, adverse effects such as hypertension, gastro-intestinal perforation and the high cost of bevacizumab are major concerns. A recent study suggests that itraconazole, an antifungal drug, has a role in selectively inhibiting angiogenesis and growth of tumor in non-small cell lung cancer. Hence, this drug may be beneficial in the treatment of RA, especially in the later phase when other modalities have failed, or as an adjuvant. To test our hypothesis, we propose a randomized, double-blinded trial in patients with longstanding RA. The control group receives the standard DMARD therapy plus placebo, while the case group receives itraconazole in addition to DMARD therapy. Serum and synovial VEGF levels, in both the control group and the case group, are compared and their correlation with the symptoms is judged. If the VEGF levels are lower and/or the symptoms are less severe in the case group, our hypothesis will be confirmed. Multi-institutional efforts are needed to confirm this hypothesis, as it is relatively new and trial data is limited.Medical Hypotheses 06/2012; 79(3):313-4. DOI:10.1016/j.mehy.2012.05.019 · 1.15 Impact Factor