The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2011; 286(51):44045-56. DOI: 10.1074/jbc.M111.278754
Source: PubMed


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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    • "Recognition of high-mannose structures by mannose receptors in macrophages and dendritic cells is critical to the innate immune response responsible for elimination of bacteria and viruses and for initiation of organ-specific autoimmunity [11] [12]. Other work established that mannoserich oligosaccharides can suppress the immune response [13], ligand binding [14], and intracellular signal transduction [15]. Integrins are transmembrane receptors which mediate adhesive events critical to an effective immune response [16] [17] [18] [19]. "
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    ABSTRACT: N-glycosylation plays an important role in the majority of physiological and pathological processes occurring in the immune system. Alteration of the type and abundance of glycans is an element of lymphocyte differentiation; it is also common in the development of immune-mediated inflammatory diseases. The N-glycosylation process is very sensitive to different environmental agents, among them the pharmacological environment of immunosuppressive drugs. Some results show that high-mannose oligosaccharides have the ability to suppress different stages of the immune response. We evaluated the effects of cyclosporin A (CsA) and rapamycin (Rapa) on high-mannose/hybrid-type glycosylation in human leukocytes activated in a two-way mixed leukocyte reaction (MLR). CsA significantly reduced the number of leukocytes covered by high-mannose/hybrid N-glycans, and the synergistic action of CsA and Rapa led to an increase of these structures on the remaining leukocytes. This is the first study indicating that β 1 and β 3 integrins bearing high-mannose/hybrid structures are affected by Rapa and CsA. Rapa taken separately and together with CsA changed the expression of β 1 and β 3 integrins and, by regulating the protein amount, increased the oligomannose/hybrid-type N-glycosylation on the leukocyte surface. We suggest that the changes in the glycosylation profile of leukocytes may promote the development of tolerance in transplantation.
    Analytical cellular pathology (Amsterdam) 09/2015; 2015(1):324980. DOI:10.1155/2015/324980 · 0.85 Impact Factor
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    • "O-linked glycosylation is linked to the hydroxyl group of serine or threonine to form only one to four sugar residues and occurs in the lumen of the Golgi cisternae [30]. Importantly, Nacev et al. [31] have indicated that hypoglycosylated VEGFR2 accumulates in rough ER and is not further transported into the cell membrane in HUVECs, as detected using immunofluorescence staining. Herein, we also observed that 2-DG treatment resulted in accumulation of VEGFR2 in ER and that mannose reversed this effect of 2-DG, suggesting that the interference with N-linked glycosylation of VEGFR2 may be involved in the anti-angiogenic actions of 2-DG. "
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    ABSTRACT: 2-Deoxyglucose (2-DG) is a glucose analogue and has been shown to inhibit angiogenesis in human umbilical vascular endothelial cells (HUVECs) through interference with N-linked glycosylation. However, the anti-angiogenic mechanisms of 2-DG are not fully elucidated. We first employed an ex vivo rat aortic rings model to substantiate the anti-angiogenic action of 2-DG and then used HUVECs to investigate the molecular mechanism underlying such an action. Results reveal that 2-DG (0.05-1.0 mM) significantly inhibited tube formation in both rat aortic rings and HUVECs. 2-DG (0.1-1.0 mM) also significantly inhibited cell invasion and migration, as well as the activity and mRNA and protein expression of matrix metalloproteinase (MMP)-2 in HUVECs. In addition, 2-DG (1.0 mM) significantly inhibited mRNA and protein expression of vascular endothelial growth receptor 2 (VEGFR2) in a time-dependent manner. 2-DG also significantly inhibited the phosphorylation of the focal adhesion kinase (FAK) and mitogen-activated protein kinase (p38), the downstream molecules of VEGFR2. The effects of 2-DG on tube formation, MMP-2 activity, and VEGFR2 protein expression in HUVECs were reversed by mannose, an N-linked glycosylation precursor. Mannose also reversed 2-DG-induced accumulation of VEGFR2 in endoplasmic reticulum. This ex vivo and in vitro study demonstrates that 2-DG inhibits angiogenesis with an action involving attenuation of VEGFR2 signaling and MMP-2 expression, possibly resulting from interference with N-linked glycosylation of VEGFR2. Further studies are needed to show that 2-DG inhibits VEGF-mediated angiogenesis or that the actual status of N-glycosylation of VEGFR2 is affected by the treatment. Copyright © 2015. Published by Elsevier Inc.
    Life sciences 08/2015; 139. DOI:10.1016/j.lfs.2015.08.002 · 2.70 Impact Factor
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    • "ITZ is a well-known antifungal drug that inhibits CYP51, a cytochrome P450 required for sterol biosynthesis (Lestner and Hope, 2013). In addition, ITZ exerts anticancer activity by inhibiting angiogenesis—through disturbing mTOR signaling and vascular endothelial growth factor receptor 2 (VEGFR2) trafficking—and the Hedgehog (Hh) signaling pathway (Kim et al., 2010; Nacev et al., 2011; Xu et al., 2010). ITZ has been found to be efficacious in patients with several cancer types in multiple phase 2 clinical studies (Antonarakis et al., 2013; Kim et al., 2014; Rudin et al., 2013). "
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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; 10(4). DOI:10.1016/j.celrep.2014.12.054 · 8.36 Impact Factor
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