Synthesis and Evaluations of Novel Apocynin Derivatives as Anti-Glioma Agents
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Apocynin (4-hydroxy-3-methoxyacetophenone) is a natural polyphenolic compound with multiple biological activities. In the present study, a series of apocynin derivatives were designed and synthesized. The in silico ADMET prediction, blood-brain barrier (BBB) penetration assay, anti-NADPH oxidase activity, reactive oxygen species (ROS) levels, and anti-glioma effects of these apocynin derivatives were evaluated. The anti-glioma mechanisms of candidate compounds were studied by flow cytometer and Western blot. The results showed that D31 exhibited higher BBB penetration, increased ROS generations and significant anti-glioma effects both in vitro and in vivo. Further studies showed that D31 inhibited the activations of NF-κB pathway. Overall, our data demonstrated that D31 inhibited growth and induced apoptosis of glioma, which might be caused by ROS-related NF-κB activation. The current study suggested that D31 could be further explored for its potential use in anti-glioma therapy.Keywords:
Apocynin
Reactive oxygen species (ROS) play a central role in the pathogenesis of many cardiovascular diseases, such as atherosclerosis and hypertension. Endothelial NADPH oxidase is the major source of intracellular ROS. The present study investigated the role of endothelial NADPH oxidase-derived ROS in angiopoietin-1 (Ang-1)-induced angiogenesis. Exposure of porcine coronary artery endothelial cells (PCAECs) to Ang-1 (250 ng/ml) for periods up to 30 min led to a transient and dose-dependent increase in intracellular ROS. Thirty minutes of pretreatment with the NADPH oxidase inhibitors diphenylene iodinium (DPI, 10 microM) and apocynin (200 microM) suppressed Ang-1-stimulated ROS. Pretreatment with either DPI or apocynin also significantly attenuated Ang-1-induced Akt and p44/42 MAPK phosphorylation. In addition, inhibition of NADPH oxidase significantly suppressed Ang-1-induced endothelial cell migration and sprouting from endothelial spheroids. Using mouse heart microvascular endothelial cells from wild-type (WT) mice and mice deficient in the p47(phox) component of NADPH oxidase (p47(phox-/-)), we found that although Ang-1 stimulated intracellular ROS, Akt and p42/44 MAPK phosphorylation, and cell migration in WT cells, the responses were strikingly suppressed in cells from the p47(phox-/-) mice. Furthermore, exposure of aortic rings from p47(phox-/-) mice to Ang-1 demonstrated fewer vessel sprouts than WT mice. Inhibition of the Tie-2 receptor inhibited Ang-1-induced endothelial migration and vessel sprouting. Together, our data strongly suggest that endothelial NADPH oxidase-derived ROS play a critical role in Ang-1-induced angiogenesis.
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Recent studies suggest reactive oxygen species (ROS) induced in mammalian cells exposed to multi-walled carbon nanotubes (MWCNTs) could mediate the cytotoxicity. This study was conducted to determine the mechanisms responsible for MWCNTs-induced ROS production in human primary macrophages. Our results showed that superoxide levels were significantly increased in a time-dependent manner in blood monocyte-derived macrophages treated with 100 microg/ml MWCNTs for 12 h. Concomitantly, MWCNTs induced membrane translocation of the NADPH oxidase subunits p47phox and p67phox, a signature event for NADPH oxidase activation. Pre-incubation with apocynin, a selective inhibitor of NADPH oxidase, prevented both membrane translocation of p47phox and superoxide production. Treatment with MWCNTs also resulted in an increased cytotoxicity in human primary macrophages that was significantly attenuated by both apocynin and antioxidants. These findings demonstrate that MWCNTs activate NADPH oxidase in human macrophages, which may contribute to ROS generation in MWCNTs treated-macrophages.
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The methoxy‐substituted catechol, apocynin is a powerful inhibitor of NADPH oxidase. Recently apocynin has been shown to prevent the aggregation of platelets in response to agonists such as collagen and thrombin. We examined whether NADPH oxidase plays a role in collagen‐induced aggregation, elucidating the mechanism(s) of action of apocynin on this enzyme complex. Platelets were isolated from 10 week‐old male C57BL6/J and Nox2‐deficient (Nox2 −/− ) mice to assess platelet aggregation (aggregometry) and NADPH oxidase expression (immunoblotting for Nox2). Collagen (1‐30 μg/ml) caused a concentration‐dependent increase in platelet aggregation (maximum response ~35.35 ± 3.18%; n=6). This response was inhibited by apocynin (P<0.05, n=6), albeit at higher concentrations (=100µM). Immunoblotting with an anti‐Nox2 antibody from platelets isolated from wild‐type mice revealed strong bands at 58kDa, 65kDa and 91kDa. Collagen was as effective at eliciting aggregation in platelets from Nox2 −/− and wild‐type mice. Moreover, the superoxide scavengers, SOD (300U/ml) and PEG‐SOD (300U/ml) had no effect on platelet aggregation in response to collagen. These data suggest that while platelets express NADPH oxidase, it is not responsible for mediating collagen‐induced aggregation. Apocynin's anti‐platelet actions are likely to be due to an off target effect of the drug which remains to be determined. (NHMRC 384136)
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Oxidative stress has been linked to the origin and progression of cardiovascular diseases. Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase is a multi-component, NADPH-dependent enzyme that generates superoxide anion in the presence of molecular oxygen. The enzyme has been identified and characterized in all 3 vascular wall cell types and represents the major source of reactive oxygen species (ROS) production in the vascular wall. Inhibition of NADPH oxidase activation appears to suppress the sequence of cellular events that leads to a variety of cardiovascular diseases, including atherosclerosis. The naturally occurring methoxyphenol apocynin has been found to inhibit NADPH oxidase upon activation by peroxidases (e.g. soybean peroxidase, myeloperoxidase) or ROS under mild reaction conditions. Upon peroxidase-catalyzed activation, the apocynin oxidation products act to block the assembly and activation of NADPH oxidase. Although the mechanism of inhibition of NADPH oxidase remains largely unknown, apocynin's high effectiveness and low toxicity makes it a promising lead compound in the development of new therapeutic agents for cardiovascular diseases.
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Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex in experimental studies. NADPH oxidase was originally identified immune cells as playing an important microbicidal role. In cerebral ischemia, inflammation is increasingly being recognized as contributing negatively to neurological outcome, with NADPH-oxidase as an important source of superoxide. Recently, several forms of this oxidase have been found in a variety of non-immune cells. Neuronal NADPH oxidase is thought to participate in long-term potentiation and intercellular signaling. However, excessive superoxide production is damaging and has been shown to play an important role in the progression of brain injury. NADPH oxidase is a multisubunit complex composed of membrane-associated gp91 phox and p22 phox subunits and cytosolic subunits, p47 phox , p67 phox , and p40 phox and Rac. When NADPH oxidase is activated through phosphorylatoin of p47 phox , cytosolic subunits translocate to the cell membrane and fuse with the catalytic subunit, gp91 phox . The activated enzyme complex transports electrons to oxygen, thus producing the superoxide anion (O2 � - ), a precursor of reactive oxygen species. An NADPH oxidase assembly inhibitor, apocynin, has been shown alleviate oxidative stress and improves neurological outcome after cerebral ischemia. There is recent interest in the role of NADPH oxidase and apocynin as neuroprotective strategies against ischemia. This review will focus on therapeutic effects of NADPH oxidase assembly and its inhibitor apocynin in stroke and other brain injuries.
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Oscillatory fluid shear stress (OSS) increases vascular oxidative stress and induces endothelial cell dysfunction. In our previous study, we demonstrated that OSS induced superoxide generation via activation of NADPH oxidase enzyme system. In this study, we examined the effect of OSS on mitochondrial dysfunction. Using chemical inhibitors against NADPH oxidase (Apocynin) and JNK (SP600125), we demonstrated that OSS induced JNK activation via NADPH oxidase in bovine aortic endothelial cells (BAEC). JNK activation peaked at 1h following treatment of OSS (±3dyn/cm 2 ) by western blot analysis. Apocynin inhibited this induction and also inhibited OSS‐induced superoxide generation measured by dihydroethidium. We then examined the role of JNK activation in OSS‐induced mtO 2 ·− production by using MitoSox Red dye specific for mtO 2 ·− . We found that SP600125 inhibited ·− production as quantified by flow cytometry. OSS‐stimulated mtO 2 Our data suggests that OSS‐activated NADPH oxidase induces intra‐cellular superoxide production and activates JNK, which in turn induces mtO 2 ·− production.. This study is supported by NIH HL083015 and HL091302.
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Of the multiple sources of reactive oxygen species (ROS) in the blood vessel, NADPH oxidases are the primary source. Whereas several studies have implicated NADPH oxidases in the initiation of atherosclerosis, their roles in disease progression are incompletely understood. Our objective was to determine the potential clinical relevance of inhibiting NADPH oxidase in established atherosclerosis. Using a hypercholesteremic murine model of atherosclerosis (ApoE−/−/LDLR−/− (AS) mice on normal chow diet), we first established a time-dependent relationship between superoxide levels and lesion size in AS mice. Next, we identified NADPH oxidase as the primary source of ROS in atherosclerotic lesions. Treatment of aortic segments from AS mice with apocynin, which interferes with NADPH oxidase activation in part by preventing translocation of the subunit p47phox, significantly reduced superoxide levels. Moreover, addition of apocynin to the drinking water of AS mice produced a decrease in lesion size as compared to untreated AS mice, with the effect most pronounced in the thoracoabdominal aorta but absent from the aortic arch. Granulocyte function in AS+apocynin mice was suppressed, confirming efficacy of apocynin treatment. We conclude that apocynin attenuates the progression of atherosclerosis in hypercholesterolemic mice, potentially by its ability to inhibit generation of superoxide by NADPH oxidase.
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Objective(s) Increased reactive oxygen species (ROS) production is implicated in the pathogenesis of arterial hypertension and the development of endothelial dysfunction. NADPH oxidase type enzyme family has been suggested to form ROS and to interfere with endothelium-dependent relaxation. However, the specific isoform of NADPH oxidases that may predominantly contribute to these events remains to be clarified. Materials and Methods Here we investigated the expressional regulation of NADPH oxidase isoforms (NOX1, NOX2 and NOX4) in aorta of aged spontaneously hypertensive rats (SHR) in comparison to age matched Wistar Kyoto rats (WKY). Moreover, we examined the effect of in vitro inhibition of NADPH oxidase by apocynin or the novel NADPH oxidase inhibitor, VAS2870 on the vascular reactivity and ROS production. Results Our results showed that ROS formation was largely increased in aorta of SHR as measured by dihydroethidine (DHE) fluorescence and inhibited by apocynin or VAS2870. NADPH oxidase activity, measured by lucigenin-enhanced chemiluminescence and of NOX1 and NOX2 protein levels were increased in aortic homogenates from SHR compared to WKY. However, NOX4 protein expression was not significantly changed. Furthermore, the impaired acetylcholine-induced relaxation of SHR aorta was significantly improved in the presence of either apocynin or VAS2870. Conclusion Collectively, our data suggest that NADPH oxidases, particularly NOX1 and NOX2 are relevant sources of ROS in the aorta of aged SHR thereby cause endothelial dysfunction, and VAS2870 is effective as apocynin in reversing these consequences.
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Endothelial Dysfunction
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