Intracellular Reactive Oxygen Species Scavenging Research Articles

Chemical Nanotherapy: Nitroxyl radical-containing Nanoparticle Protects Neuroblastoma SH-SY5Y Cells From Aβ-induced Oxidative Stress

Excessive accumulation of beta-amyloid (Abeta) has been proposed as a pivotal event in the pathogenesis of Alzheimer's disease. Possible mechanisms underlying Abeta-induced neuronal cytotoxicity include excess production of reactive oxygen species (ROS) and apoptosis. We have designed novel nanoparticles, nitroxyl radical-containing nanoparticles (RNPs), which possess nitroxyl radical in the core and chemically scavenges ROS. This study aimed to determine the potential neuroprotective role of RNPs on Abeta-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were preincubated with 0.1-1 mM RNP for 24 h and then incubated with 20 microM Abeta1-42, for 48 h. In every group, cell viability, apoptotic rate, ROS levels including superoxide anion radicals and hydroxyl radicals, ROS production including lipid peroxidation, protein oxidation and DNA oxidation were measured. SH-SY5Y cells preincubated with 0.1-2 mM RNP for 24 h were protected from Abeta-induced damage. SH-SY5Y cells preincubated with more than 2 mM RNPfor 24 h showed cytotoxicity. From the quantitative analyses, it was observed that RNPs reduced intracellular oxidative stress. RNP treatment significantly reduced the amount of oxidized lipids, proteins and DNA. It also reduced DNA fragmentations, which caused lower apoptosis levels. RNPs are promising intracellular ROS scavengers.

Protective effect of kobophenol A on nitric oxide-induced cell apoptosis in human osteoblast-like MG-63 cells: Involvement of JNK, NF-κB and AP-1 pathways

Nitric oxide (NO) is a multifunctional signaling molecule and the cytotoxic species responsible for a variety of pathologic disorders including bone destruction. High NO levels induce the apoptosis of osteoblasts and decrease the bone mineral density. We investigated the influence of kobophenol A (kob A) on apoptosis in cultured human osteoblast-like MG-63 cells. Direct NO donor sodium nitroprusside (SNP) that has been recognized as an inducer of apoptosis in various cell lines significantly induced cell death and NO production in MG-63 cells. Coincubation of kob A in SNP-treated MG-63 cells resulted in a significant protection against NO-induced cell death. This is associated with increase in intracellular reactive oxygen species (ROS) scavenging activity and the inhibition of decrease in mitochondrial membrane potential (MMP) by kob A. We also found that kob A inhibited the down-regulation of Bcl-2 and Bcl-XL, whereas the level of Bax expression was decreased by kob A treatment in SNP-treated MG-63 cells. Furthermore, kob A inhibited SNP-induced phosphorylation of JNK and c-Jun, and SNP-induced reduction in NF-κΒ and AP-1 activities, implicating that protective effect of kob A may occur through the regulation of JNK, NF-κΒ and AP-1 signaling pathways. Together, these findings suggest that kob A has a protective effect against NO-mediated osteoblast apoptosis and might be a plausible candidate for treatment of inflammatory bone diseases relevant to osteoblast cell death.

Antioxidant Effects of the Ethanol Extract from Flower of Camellia japonica via Scavenging of Reactive Oxygen Species and Induction of Antioxidant Enzymes

The aim of this study was to investigate the antioxidant properties of the ethanol extract of the flower of Camellia japonica (Camellia extract). Camellia extract exhibited 1,1-diphenyl-2-picrylhydrazyl radical and intracellular reactive oxygen species (ROS) scavenging activity in human HaCaT keratinocytes. In addition, Camellia extract scavenged superoxide anion generated by xanthine/xanthine oxidase and hydroxyl radical generated by the Fenton reaction (FeSO4 + H2O2) in a cell-free system, which was detected by electron spin resonance spectrometry. Furthermore, Camellia extract increased the protein expressions and activity of cellular antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase. These results suggest that Camellia extract exhibits antioxidant properties by scavenging ROS and enhancing antioxidant enzymes. Camellia extract contained quercetin, quercetin-3-O-glucoside, quercitrin and kaempferol, which are antioxidant compounds.

Tetramethylpyrazine Inhibits Hypoxia-Induced Pulmonary Vascular Leakage in Rats via the ROS-HIF-VEGF Pathway

Tetramethylpyrazine (TMP) is a reactive oxygen species (ROS) antagonist that has potent properties for the treatment of a variety of vascular diseases, such as ischemic stroke and pulmonary hypertension secondary to chronic obstructive pulmonary diseases. However, there are few data about the role of TMP in hypoxia-induced pulmonary vascular leakage. This study examined the effect of TMP on hypoxia-induced pulmonary vascular leakage and the underlying mechanisms. Rat pulmonary microvascular endothelial cells (RPMVECs) treated with TMP or not were subjected to hypoxic or normoxic conditions for 24 h, and the monolayer permeability, intracellular ROS, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) proteins levels were determined. Additionally, rats administrated TMP were exposed to hypobaric hypoxia to evaluate the effect of TMP in vivo by measuring lung water content, pulmonary vascular leakage into the lungs and immunohistochemistry for HIF-1α and VEGF. Hypoxia was found to cause a significant increase in RPMVEC monolayer permeability and intracellular ROS, HIF-1α and VEGF protein levels. Treatment with TMP decreased the hypoxia-induced RPMVEC monolayer permeability and attenuated the elevation of ROS, HIF-1α and VEGF protein levels. TMP-treated animals showed less pulmonary vascular leakage and HIF-1α and VEGF expression compared with those exposed to hypoxia alone. These observations supported that TMP inhibited the increase in pulmonary vascular permeability induced by hypoxia. The underlying mechanisms may be related to the scavenging of intracellular ROS and the suppression of hypoxia-induced upregulation of HIF-1α and VEGF proteins.

Isolation and identification of an antioxidant flavonoid compound from citrus-processing by-product

Large amounts of citrus by-products are released from juice-processing plants every year. Most bioactive compounds are found in the peel and inner white pulp. Flavonoids are a widely distributed group of bioactive compounds. The methanolic extract of citrus peel powder has been shown to possess strong antioxidant activity. Therefore the aim of this study was to isolate the major antioxidant flavonoid compound from Citrus unshiu (satsuma) peel as citrus by-product and evaluate its antioxidant activity. The major flavonoid isolated from C. unshiu peel was identified as quercetagetin. The structure of the compound was determined by tandem mass spectrometry and ultraviolet spectroscopy. Its antioxidant activity was assessed by assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl radical and intracellular reactive oxygen species (ROS) scavenging and DNA damage inhibition. Quercetagetin showed strong DPPH radical-scavenging activity (IC₅₀ 7.89 µmol L⁻¹) but much lower hydroxyl radical-scavenging activity (IC₅₀ 203.82 µmol L⁻¹). Furthermore, it significantly reduced ROS in Vero cells and showed a strong protective effect against hydrogen peroxide-induced DNA damage. The results of this study suggest that quercetagetin could be used in the functional food, cosmetic and pharmaceutical industries.

Evaluation of antioxidant properties of a new compound, pyrogallol-phloroglucinol-6,6'-bieckol isolated from brown algae,Ecklonia cava

In this study, antioxidant and free radical scavenging activities of the natural antioxidative compound, pyrogallol-phloroglucinol-6,6'-bieckol (PPB) isolated from brown algae, Ecklonia cava was assessed in vitro by measuring the radical scavenging activities (DPPH, alkyl, hydroxyl, and superoxide) using electron spin resonance (ESR) spectrometry, intracellular reactive oxygen species (ROS) scavenging activity, and DNA damage assay. According to the results of these experiments, the scavenging activity PPB against difference radicals was in the following order: DPPH, alkyl, hydroxyl, and superoxide radicals (IC50; 0.90, 2.54, 62.93 and 109.05 µM). The antioxidant activities of PPB were higher than that of the commercial antioxidant, ascorbic acid. Furthermore, PPB effectively inhibited DNA damage induced by H2O2. These results suggest that the natural antioxidative compound, PPB, can be used by the natural food industry.

Comparative effects of dietary flavanols on antioxidant defences and their response to oxidant-induced stress on Caco2 cells

Flavanols are an important fraction of our diet both for their antioxidant capacity and because they are constituents of greatly accepted foodstuffs such as tea, wine and cocoa. In addition to their antioxidant activity by directly scavenging intracellular reactive oxygen species (ROS), flavanols have been recently shown to enhance protective enzymes. The objective was to evaluate the antioxidant response of colon-derived Caco2 cells to dietary flavanols. Four representative flavanols were selected: epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and procyanidin B2 (PB2). Cell viability, concentration of ROS and reduced glutathione (GSH), and activity of antioxidant/detoxification enzymes and caspase 3 were determined. Treatment of Caco2 cells with flavanols decreased ROS production but did not affect GSH content. ECG induced glutathione peroxidase (GPx), whereas PB2 evoked a dose-dependent increase in GPx, glutathione reductase and glutathione-S-transferase. Enhancement of the antioxidant defences implies an improved cell response to an oxidative challenge. Hence, Caco2 cells treated 20 h with the flavanols, especially PB2, and then submitted to an oxidative stress induced by a pro-oxidant, tert-butyl-hydroperoxide, showed a reduced ROS production, restricted activation of caspase 3 and higher viability than cells plainly submitted to the stressor. Flavanols protect Caco2 cells against an induced oxidative stress and subsequent cellular death by reducing ROS production and preventing caspase-3 activation. In particular, PB2 increases the activity of antioxidant/detoxification enzymes and thus protects Caco2 cells by directly counteracting free radicals and also by activating the antioxidant defence system.

Non-thermal plasma induces apoptosis in melanoma cells via production of intracellular reactive oxygen species.

Non-thermal atmospheric pressure dielectric barrier discharge (DBD) plasma may provide a novel approach to treat malignancies via induction of apoptosis. The purpose of this study was to evaluate the potential of DBD plasma to induce apoptosis in melanoma cells. Melanoma cells were exposed to plasma at doses that did not induce necrosis, and cell viability and apoptotic activity were evaluated by Trypan blue exclusion test, Annexin-V/PI staining, caspase-3 cleavage, and TUNEL® analysis. Trypan blue staining revealed that non-thermal plasma treatment significantly decreased the viability of cells in a dose-dependent manner 3 and 24 h after plasma treatment. Annexin-V/PI staining revealed a significant increase in apoptosis in plasma-treated cells at 24, 48, and 72 h post-treatment (p < 0.001). Caspase-3 cleavage was observed 48 h post-plasma treatment at a dose of 15 J/cm(2). TUNEL® analysis of plasma-treated cells demonstrated an increase in apoptosis at 48 and 72 h post-treatment (p < 0.001) at a dose of 15 J/cm(2). Pre-treatment with N-acetyl-L: -cysteine (NAC), an intracellular reactive oxygen species (ROS) scavenger, significantly decreased apoptosis in plasma-treated cells at 5 and 15 J/cm(2). Plasma treatment induces apoptosis in melanoma cells through a pathway that appears to be dependent on production of intracellular ROS. DBD plasma production of intracellular ROS leads to dose-dependent DNA damage in melanoma cells, detected by γ-H2AX, which was completely abrogated by pre-treating cells with ROS scavenger, NAC. Plasma-induced DNA damage in turn may lead to the observed plasma-induced apoptosis. Since plasma is non-thermal, it may be used to selectively treat malignancies.

Puerarin protects Alzheimer's disease neuronal cybrids from oxidant-stress induced apoptosis by inhibiting pro-death signaling pathways

Mitochondrial oxidative stress induced by reactive oxygen species (ROS) has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease (AD). We used mitochondrial transgenic neuronal cell cybrid models of sporadic AD (SAD), which overproduce ROS compared to control cybrids, to investigate the protective effects of puerarin, an isoflavone purified from Chinese herb radix of Pueraria lobata, on viability, endogenous ROS and intracellular signaling pathways. SAD cybrids had increased apoptosis and increased accumulation of ROS that was inhibited by puerarin. Western blotting demonstrated that SAD cybrids had increased basal activation of the caspase-3, p38 and c-Jun N-terminal kinase (JNK) that were inhibited by puerarin. Puerarin was also found to decrease Bax/Bcl-2 ratio. These results suggest that expression of SAD mitochondrial genes in cybrids activates oxidative-stress-related signaling pathways and reduces viability, and that the protective effects of puerarin inhibit oxidative-stress-induced apoptosis through down-regulation of Bax/Bcl-2 ratio, which blocks the activation of JNK, p38 and caspase-3. Therefore, puerarin may act as an intracellular ROS scavenger, and protect neurons against oxidative-stress-induced apoptosis.

Intracellular GSH Depletion Triggered Mitochondrial Bax Translocation to Accomplish Resveratrol-Induced Apoptosis in the U937 Cell Line

We have previously demonstrated that resveratrol (Resv)-induced cellular apoptosis occurs after formation of reactive oxygen species (ROS) but the role of GSH has not been well defined. Our experimental data enumerated that Resv treatment (50 μm) induced apoptosis in human leukemic monocyte lymphoma cells, which was preceded by cellular GSH efflux. High concentration of extracellular thiol (GSH, N-acetyl cysteine) and two specific inhibitors of carrier-mediated GSH extrusion, methionine or cystathionine, prevented the process of oxidative burst and cell death. This proved that GSH efflux could be a major molecular switch to modulate Resv-induced ROS generation. Spectrofluorometric data depicted that after 6 h of Resv treatment, ROS generation was evident. Pretreatment of cells with intracellular ROS scavenger (polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase) efficiently reduced ROS generation but failed to prevent intracellular GSH depletion. Thus, it suggested that intracellular GSH depletion was independent of ROS production but dependent on GSH extrusion. Furthermore, to bridge the link between GSH efflux and ROS generation, we carried out confocal microscopy of the localization of Bax protein. Microscopic analysis and small interfering RNA treatment emphasized that cellular GSH efflux triggered Bax translocation to mitochondria, which resulted in the loss of mitochondrial membrane potential, ROS generation, and caspase 3 activation and thus triggered apoptosis.

Hispidin produced from Phellinus linteus protects pancreatic β-cells from damage by hydrogen peroxide

Phellinus linteus, which is a traditional medicinal mushroom used in Asian countries for the treatment of various diseases, has attracted a lot of attention due to its antioxidant, anti-inflammatory, anti-mutagenicity, and cell-mediated immunity properties in addition to its ability to inhibit tumor growth and metastasis. However, the antidiabetic efficacy of P. linteus has not yet been examined. In this study, hispidin from P. linteus exhibited quenching effects against DPPH radicals, superoxide radicals, and hydrogen peroxide in a dose-dependent manner. Intracellular reactive oxygen species scavenging activity of hispidin was approximately 55% at a concentration of 30 microM. In addition, hispidin was shown to inhibit hydrogen peroxide-induced apoptosis and increased insulin secretion in hydrogen peroxide-treated cells. These combined results indicate that hispidin may act as an antidiabetic and that this property occurs through preventing beta-cells from the toxic action of reactive oxygen species in diabetes.

Oxidative Stress Partially Contributes to Iron-Induced Alpha-Synuclein Aggregation in SK-N-SH Cells

Intracellular Lewy body formation is one of the hallmarks of Parkinson's disease (PD). As its main component, aggregated α-synuclein is presented in the substantia nigra, the same region iron accumulation occurs. In this study, the relationship between iron and α-synuclein aggregation was investigated. In the remaining cells, 1 mmol/l ferric and ferrous iron could induce cell loss in SK-N-SH cells and α-synulein aggregation. Pretreatment with 5 μmol/l vitamin E, a potent intracellular reactive oxygen species (ROS) scavenger could totally abolish ROS formation and cell viability reduction induced by ferric and ferrous iron treatment. However, the intracellular α-synuclein aggregation could only be partially alleviated. Due to the predicted iron responsive element (IRE) in the 5'-untranslated region of the human α-synuclein mRNA contains, we observed that α-synuclein mRNA level was up-regulated in SK-N-SH cells with iron regulatory protein (IRP) knockdown and more α-synuclein aggregations were observed in cells. The results suggest that iron-induced intracellular aggregated α-synuclein is partially dependent on oxidative stress and iron might also regulate α-synuclein aggregation through the IRE/IRP system.

A COMPARATIVE STUDY OF THE POTENTIAL ANTIOXIDANT ACTIVITIES OF GINSENOSIDES

ABSTRACT Ginseng roots are believed to contain over 20 different types of ginsenosides. However, no reports exist on the antioxidant activity of ginsenosides according to their various structures. The present study involves a comparison of the various forms of ginsenosides, a series of derivatives originating from the attachment of different sugar moieties to triterpene dammarane, with respect to their intracellular reactive oxygen species scavenging activity. Among the ginsenosides, Rb2 and Rc showed the strongest antioxidant activity, followed by (in decreasing order) Rg2, Rh2, Rh1, Rf, Rg3, Rg1, Rb1, Re and Rd. Furthermore, the antioxidant activity ranks of the various forms of ginsenosides were influenced by the types of dammarane, as well as the number of sugar moieties, and substitutive groups. To the best of our knowledge, this is the first report evaluating the antioxidant properties of ginsenosides with the goal of determining their structure–activity relationship. PRACTICAL APPLICATIONS Ginseng (Panax ginseng C.A. Meyer), a member of the Araliaceae family, is traditionally considered as one of the most important medicinal plants with high ginsenoside content. Ginsenoside is a triterpenoid glycoside, which is known to have diverse physiological and pharmacological activities. However, the correlation of structure and antioxidant activity has never been studied thus far in ginsenosides. A relationship exists between the type and position of the sugar moieties in ginsenoside. From these results, ginsenosides Rb2 and Rc might be very useful for the development of functional food and raw materials of medicine for antioxidants preventing oxidative stress-related diseases. And structure and antioxidant relationship may be potential for evaluating the structure and function relationship of other ginsenosides in order to elucidate which part of ginsenoside is essential with regards to increasing antioxidant activity and the development of novel antioxidants to treat diseases associated with free radicals.

The Neurogenic Basic Helix-Loop-Helix Transcription Factor NeuroD6 Confers Tolerance to Oxidative Stress by Triggering an Antioxidant Response and Sustaining the Mitochondrial Biomass

Preserving mitochondrial mass, bioenergetic functions and ROS (reactive oxygen species) homoeostasis is key to neuronal differentiation and survival, as mitochondria produce most of the energy in the form of ATP to execute and maintain these cellular processes. In view of our previous studies showing that NeuroD6 promotes neuronal differentiation and survival on trophic factor withdrawal, combined with its ability to stimulate the mitochondrial biomass and to trigger comprehensive antiapoptotic and molecular chaperone responses, we investigated whether NeuroD6 could concomitantly modulate the mitochondrial biomass and ROS homoeostasis on oxidative stress mediated by serum deprivation. In the present study, we report a novel role of NeuroD6 as a regulator of ROS homoeostasis, resulting in enhanced tolerance to oxidative stress. Using a combination of flow cytometry, confocal fluorescence microscopy and mitochondrial fractionation, we found that NeuroD6 sustains mitochondrial mass, intracellular ATP levels and expression of specific subunits of respiratory complexes upon oxidative stress triggered by withdrawal of trophic factors. NeuroD6 also maintains the expression of nuclear-encoded transcription factors, known to regulate mitochondrial biogenesis, such as PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α), Tfam (transcription factor A, mitochondrial) and NRF-1 (nuclear respiratory factor-1). Finally, NeuroD6 triggers a comprehensive antioxidant response to endow PC12-ND6 cells with intracellular ROS scavenging capacity. The NeuroD6 effect is not limited to the classic induction of the ROS-scavenging enzymes, such as SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1) and PRDX5 (peroxiredoxin 5), but also to the recently identified powerful ROS suppressors PGC-1α, PINK1 (phosphatase and tensin homologue-induced kinase 1) and SIRT1. Thus our collective results support the concept that the NeuroD6–PGC-1α–SIRT1 neuroprotective axis may be critical in co-ordinating the mitochondrial biomass with the antioxidant reserve to confer tolerance to oxidative stress.

Differential cytoprotective effect of copper- and iron-containing chlorophyllins against oxidative stress-mediated cell death

Chlorophyllin (CHL), a copper-containing chlorophyll derivative, was previously reported to possess reactive oxygen species (ROS) scavenging activity. However, the antioxidant property of iron-containing chlorophyllin, iron chlorin e6 (FeCe6), has not been explored in detail. This study systematically investigated the antioxidant capacity of two chlorophyllins, CHL and FeCe6, and their cytoprotective effects against ROS toxicity in human Jurkat T-cells. Both CHL and FeCe6 exhibited a wide range of antioxidant activities to ABTS radical, singlet oxygen, peroxynitrite, and peroxyl radical. Notably, FeCe6 exerted a significant suppressive effect on hydrogen peroxide-dependent cytotoxicity and apoptosis in Jurkat T-cells, while negligible or much weaker inhibition was observed by CHL. This differential cytoprotective effect is likely due to the different hydrogen peroxide scavenging capacity of two chlorophyllins. Moreover, FeCe6 attenuated menadione-induced cell death and tumour necrosis factor (TNF)-α-induced IκB phosphorylation via scavenging of intracellular ROS. Taken together, the results suggest that FeCe6 is a promising cytoprotective antioxidant against oxidative stress-mediated cellular toxicity.

Cytotoxicity and mechanism of action of a new ROS-generating microsphere formulation for circumventing multidrug resistance in breast cancer cells

Multidrug resistance (MDR) is one of the main challenges in the treatment of breast cancer. A new microsphere formulation able to generate reactive oxygen species (ROS) locally was thus investigated for circumventing MDR in breast cancer cells in this work. Glucose oxidase (GOX) was encapsulated in alginate/chitosan hydrogel microspheres (ACMS-GOX). The in vitro cytotoxicity of ACMS-GOX to murine breast cancer EMT6/AR1.0 cells, which overexpress P-glycoprotein (P-gp), was evaluated by a clonogenic assay. The mechanism of the cytotoxicity of ACMS-GOX was investigated by using various extracellular and intracellular ROS scavengers and antioxidant enzyme inhibitors. The effect of lipid peroxidation and cellular uptake of GOX was also evaluated. ACMS-GOX exhibited similar dose and time-dependent cytotoxicity to EMT6/AR1.0 cells as to their wild-type EMT6/WT parent cells, in effect circumventing the MDR phenotype of EMT6/AR1.0 cells. Extracellular H(2)O(2) and intracellular hydroxyl radical were found to play critical roles in the cytotoxicity of ACMS-GOX. Cellular uptake of GOX was negligible and thus not responsible for intracellular ROS generation. Combining ACMS-GOX with intracellular antioxidant inhibitors-enhanced cytoxicity. This work demonstrates that the ACMS-GOX are effective in circumventing P-gp-mediated MDR in breast cancer cells.

Crataegus Special Extract WS 1442 Causes Endothelium-dependent Relaxation via a Redox-sensitive Src- and Akt-dependent Activation of Endothelial NO Synthase but Not via Activation of Estrogen Receptors

This study determined whether the Crataegus (Hawthorn species) special extract WS 1442 stimulates the endothelial formation of nitric oxide (NO), a vasoprotective factor, and characterized the underlying mechanism. Vascular reactivity was assessed in porcine coronary artery rings, reactive oxygen species (ROS) formation in artery sections by microscopy, and phosphorylation of Akt and endothelial NO synthase (eNOS) in endothelial cells by Western blot analysis. WS 1442 caused endothelium-dependent relaxations in coronary artery rings, which were reduced by N-nitro-L-arginine (a competitive inhibitor of NO synthase) and by charybdotoxin plus apamin (two inhibitors of endothelium-derived hyperpolarizing factor-mediated responses). Relaxations to WS 1442 were inhibited by intracellular ROS scavengers and inhibitors of Src and PI3-kinase, but not by an estrogen receptor antagonist. WS 1442 stimulated the endothelial formation of ROS in artery sections, and a redox-sensitive phosphorylation of Akt and eNOS in endothelial cells. WS 1442 induced endothelium-dependent NO-mediated relaxations of coronary artery rings through the redox-sensitive Src/PI3-kinase/Akt-dependent phosphorylation of eNOS.

Denbinobin inhibits nuclear factor-κB and induces apoptosis via reactive oxygen species generation in human leukemic cells

Denbinobin, a 1,4-phenanthrenequinone firstly isolated from the stems of Dendrobium moniliforme (Shi-Hu in Chinese medicine), has been reported to exhibit anti-tumoral and anti-inflammatory activities through mechanism(s) not yet fully understood. Because of the critical role of the transcription factor NF-κB and of ROS-induced activation of stress regulated kinases in tumorigenesis, we have investigated the effect of denbinobin on these pathways. We found that denbinobin is a potent inhibitor of TNFα and PMA-induced NF-κB activation, and that it can block the phosphorylation and degradation of IκBα by inhibiting TAK1 activity, an event lying upstream of IKK activation. Moreover, treatment with denbinobin not only elicited apoptotic signalling, including mitochondrial membrane dysfunction, activation of caspases and cleavage of poly(ADP-ribose) polymerase, but also induced intracellular reactive oxygen species (ROS) generation and sustained activation of the mitogen-activated kinases (MAPKs) ERK1+2, p38 and JNK 1+2. The apoptotic effects of denbinobin could be prevented by pre-treatment with the intracellular ROS scavenger N-acetyl- l-cysteine, but not by pharmacological inhibition of MAPKs, suggesting that intracellular ROS generation underlies denbinobin-induced apoptosis, and that this effect takes place in an MAPKs-independent pathway. To define the structural elements critical for these activities, a series of phenanthrenequinones with different substituents in the phenanthrene- and/or in the quinone ring were prepared and assayed for NF-κB inhibition and ROS production. In this way, the major structure–activity relationships and the structural elements critical for the activity of denbinobin could be established.

Preventive Effect of 7,8-Dihydroxyflavone against Oxidative Stress Induced Genotoxicity

We elucidated the protective effect of 7,8-dihydroxyflavone against hydrogen peroxide (H(2)O(2))-induced DNA damage. We found that 7,8-dihydroxyflavone scavenges 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and intracellular reactive oxygen species (ROS). 7,8-Dihydroxyflavone with antioxidant effect prevented the H(2)O(2)-induced cellular DNA damage, as evidenced by comet tail, 8-hydroxy-2'-deoxyguanosine (8-OHdG) content, and phospho-histone H2A.X protein expression. Hence, 7,8-dihydroxyflavone was shown to protect cell via the inhibition of apoptosis induced by H(2)O(2). This was substantiated by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population, and decreased DNA fragmentation. Furthermore, 7,8-dihydroxyflavone activated the protein kinase B (PKB, Akt) signal pathway, which is a major survival signal pathway. In addition, LY294002, which is phosphatidylinositol 3 kinase (PI3K, upstream of Akt) inhibitor, attenuated the protective effect of 7,8-dihydroxyflavone against H(2)O(2)-induced cell damage. In conclusion, 7,8-dihydroxyflavone was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing Akt activity.

In Vitro Antioxidant Activity of 5-HMF Isolated from Marine Red Alga Laurencia undulata in Free-Radical-Mediated Oxidative Systems

Marine red algae of genus Laurencia are becoming the most important resources to produce unique natural metabolites with wide bioactivities. However, reports related to Laurencia undulata, an edible species used as folk herb, are rarely found to date. In this research, 5-hydroxymethyl- 2-furfural (5-HMF) was isolated and characterized by nuclear magnetic resonance (NMR) from Laurencia undulata as well as other marine algae. The following characteristics of 5-HMF were systematically evaluated: its antioxidant activities, such as typical free-radicals scavenging in vitro by electron spin resonance spectrometry (ESR) and intracellular reactive oxygen species (ROS) scavenging; membrane protein oxidation; oxidative enzyme myeloperoxidase (MPO) inhibition; as well as expressions of antioxidative enzymes glutathione (GSH) and superoxide dismutase (SOD) on the gene level, using the polymerase chain reaction (PCR) method. The results demonstrated that 5-HMF could be developed as a novel marine natural antioxidant or potential precursor for practical applications in the food, cosmetic, and pharmaceutical fields.