Reactive Oxygen Species Production Activity Research Articles

Reduction of Neutrophil Activation by Phosphodiesterase 4 Blockade in Behçet's Disease.

Behçet's disease (BD) is a systemic vasculitis with inflammatory lesions mediated by cytotoxic T cells and neutrophils. Apremilast, an orally available small-molecule drug that selectively inhibits phosphodiesterase 4 (PDE4), has been recently approved for the treatment of BD. We aimed to investigate the effect of PDE4 inhibition on neutrophil activation in BD. We studied surface markers and reactive oxygen species (ROS) production by flow cytometry, and neutrophil extracellular traps (NETs) production and molecular signature of neutrophils by transcriptome analysis before and after PDE4 inhibition. Activation surface markers (CD64, CD66b, CD11b, and CD11c), ROS production, and NETosis were up-regulated in BD patient neutrophils compared to healthy donor neutrophils. Transcriptome analysis revealed 1,021 significantly dysregulated neutrophil genes between BD patients and healthy donors. Among dysregulated genes, we found a substantial enrichment for pathways linked to innate immunity, intracellular signaling, and chemotaxis in BD. Skin lesions of BD patients showed increased infiltration of neutrophils that colocalized with PDE4. Inhibition of PDE4 by apremilast strongly inhibited neutrophil surface activation markers as well as ROS production, NETosis, and genes and pathways related to innate immunity, intracellular signaling, and chemotaxis. We highlight key biologic effects of apremilast on neutrophils in BD.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential “nitrite stress–Duox activation–ROS production” pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for “nitrite stress–Duox activation–ROS production–Dorsal activation–AMP synthesis” that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

New In Vitro Model of Oxidative Stress: Human Prostate Cells Injured with 2,2-diphenyl-1-picrylhydrazyl (DPPH) for the Screening of Antioxidants.

The antioxidant activity of natural compounds consists in their ability to modulate gene and protein expression, thus inducing an integrated cell protective response and repair processes against oxidative stress. New screening tools and methodologies are crucial for the actual requirement of new products with antioxidant activity to boost endogenous oxidative stress responsive pathways, Reactive Oxygen Species (ROS) metabolism and immune system activity, preserving human health and wellness. In this study, we performed and tested an integrated oxidative stress analysis, using DPPH assay and PNT2 cells injured with DPPH. We firstly investigated the mechanism of action of the oxidising agent (DPPH) on PNT2 cells, studying the variation in cell viability, oxidative stress genes, inflammatory mediator and ROS levels. The results reveal that DPPH activated ROS production and release of Prostaglandin E2 in PNT2 at low and intermediate doses, while cells switched from survival to cell death signals at high doses of the oxidising agent. This new in vitro oxidative stress model was validated by using Trolox, β-carotene and total extract of the green microalga Testraselmis suecica. Only the T. suecica extract can completely counteract DPPH-induced injury, since its chemical complexity demonstrated a multilevel protecting and neutralising effect against oxidative stress in PNT2.

Regulation of Kappa Opioid Receptor Inactivation Depends on Sex and Cellular Site of Antagonist Action.

The prototypical member of the receptor-inactivating kappa opioid receptor (KOR) antagonists, norbinaltorphimine (norBNI), produces prolonged receptor inactivation by a cJun kinase mechanism. These antagonists have potential therapeutic utility in the treatment of stress disorders; however, additional preclinical characterization is necessary to understand important aspects of their action. In this study, we report that norBNI does not work as effectively in female mice as in males because of estrogen regulation of G protein receptor kinase (GRK); pretreatment of ovary-intact female mice with the selective GRK2/3 inhibitor, Compound 101, made females equally sensitive to norBNI as males. Prior observations suggested that in vivo treatment with norBNI does not produce long-lasting inhibition of KOR regulation of dopamine release in the nucleus accumbens. We assessed the persistence of norBNI receptor inactivation in subcellular compartments. Fast-scan cyclic voltammetry recordings confirmed that presynaptic inhibition of dopamine release by the KOR agonist U69,593 was not blocked by in vivo pretreatment with norBNI under conditions that prevented KOR-mediated aversion and analgesia. We employed a novel in vivo proxy sensor of KOR activation, adenovirus associated double floxed inverted-HyPerRed, and demonstrated that KOR activation stimulates cJun kinase-dependent reactive oxygen species (ROS) production in somatic regions of ventral tegmental area dopamine neurons, but did not activate ROS production in dopamine terminals. The compartment selective action helps explain how dopamine somatic, but not terminally expressed, KORs are inactivated by norBNI. These results further elucidate molecular signaling mechanisms mediating receptor-inactivating KOR antagonist action and advance medication development for this novel class of stress-resilience medications. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) antagonists are being developed as novel proresilience therapeutics for the treatment of mood and substance use disorders. This study showed that the long-acting KOR antagonists are affected by both the sex of the animal and the subcellular compartment in which the receptor is expressed.

Cytotoxicity of a naturally occuring spirostanol saponin, progenin III, towards a broad range of cancer cell lines by induction of apoptosis, autophagy and necroptosis

This study was aimed to investigate the cytotoxic potential of a natural compound, progenin III on a broad range of cancer cell lines, including various sensitive and drug-resistant phenotypes. The cytotoxicity, progenin III-induced autophagic, ferroptotic and necroptotic cell death were evaluated by the resazurin reduction assay (RRA). Spectrophotometric analysis of caspases activity was performed using caspase-Glo assay. Flow cytometry was applied for cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H2DCFH-DA). Progenin III and the reference molecule, doxorubicin exerted cytotoxic effects towards the 18 cancer cell lines tested including animal and human cell lines. The IC50 values obtained ranged from 1.59 μM (towards CCRF-CEM leukemia cells) to 31.61 μM (against the BRAF-V600E homozygous mutant SKMel-28 melanoma cells) for progenin III. Normal sensitivity was achieved with CEM/ADR5000 cells and HCT116p53−/− adenocarcinoma cells respectively compared to their sensitive congeners CCRF-CEM cells and HCT116 p53+/+ cells. Progenin III induced apoptosis in CCRF-CEM cells mediated by caspases 3/7 activation, MMP alteration and increase ROS production, and otherwise autophagy and necroptosis. Progenin III is a potential anticancer molecule that deserves further investigations to develop a novel drug to combat malignant diseases including refractory cancers.

The Role of Water in the Effect of Weak Combined Magnetic Fields on Production of Reactive Oxygen Species (ROS) by Neutrophils

Various models have been used to demonstrate the pronounced effects of the microwave frequency range electromagnetic fields, as well as weak and very weak combined collinear magnetic fields (CMF) with static and variable components in the order of micro- and nano-tesla. One of such models, previously shown to be sensitive to variations in the parameters of applied magnetic fields, is the neutrophil respiratory burst. Using luminol-enhanced cell chemiluminescence assay, we studied the effects of the CMF exerted directly on neutrophil suspensions and, indirectly, through aqueous solutions. To experimentally create a uniform standard weak CMF with 60 µT static and 100 nT alternating magnetic field components, we engineered a shielded magnetic field induction device. CMF applied directly to neutrophils enhanced reactive oxygen species (ROS) production by more than 36%. The pronounced stimulating effect was observed only when using the signals that included the frequency of 12.6 Hz that corresponds to the ion cyclotron resonance (ICR) frequency of the hydrated hydronium ion. Similarly, to direct exposure, CMF pre-treatment of a water sample subsequently added to the neutrophil suspension increased ROS production by 66%. The effect of CMF pre-treatment was retained after a series of dilutions and mechanical treatment but disappeared in “magnetic vacuum” or without mechanical influence. Therefore, weak and super weak magnetic fields may indirectly, via water, activate ROS production by neutrophils, provided that modulation of super weak component of collinear field corresponds to the ICR frequency of the hydrated hydronium ion.

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis.

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.

Morphological, biochemical and functional studies to evaluate bovine oocyte vitrification

The aim of the present study was to evaluate the effect of vitrification on morphological, biochemical and functional parameters of matured bovine oocytes at different recovery times. To this end, matured bovine oocytes were vitrified using the Cryotech® kit (a minimum-volume system) and then incubated in maturation medium for different post-warming durations (0 h, 3 h or 21 h). Morphology, viability and biochemical parameters were assessed at each time point mentioned above and the recovery of the metaphase plate was analyzed at 2 h, 3 h and 4 h post-warming. The vitrification-warming process did not affect the viability or morphology of oocytes at any time point. However, the recovery of the metaphase plate occurred mostly between 3 and 4 h rather than at 2 h after warming (P < 0.05). Both control and vitrified-warmed oocytes showed changes in cytosolic oxidative activity, quantification of active mitochondria, reactive oxygen species (ROS) levels and redox status at the different time points studied (P < 0.05). However, differences between control and vitrified-warmed oocytes were found only in the quantification of active mitochondria and ROS production (P < 0.05). Finally, in vitro fertilization and embryo culture were carried out as functional studies to establish whether vitrification-warming affected oocyte competence, and a significant decrease was found both in the cleavage rate and embryo development (P < 0.05). We concluded that major improvements in oocyte vitrification, at list with Cryotech® kit, are still needed to avoid variations in oocyte metabolism which could contribute to the reduction in the developmental competence of bovine oocytes.

Cytotoxicity of the crude extract and constituents of the bark of Fagara tessmannii towards multi-factorial drug resistant cancer cells

Ethnopharmacological relevanceFagara tessmannii Engl. is an African medicinal plant used in Cameroonian traditional medicine to treat various types of cancers. Aim of the studyThis work was designed to determine the cytotoxicity of the crude extract (FTB), fractions (FTBa-d) and compounds isolated from the bark of Fagara tessmannii, namely lupeol (1), fagaramide (2), zanthoxyline (3), hesperidin (4), nitidine chloride (5), fagaridine chloride (6), and β-sitosterol-3-O-β-D-glucopyranoside (7). The study was extended to the mode of induction of apoptosis by FTB, compounds 5 and 6. Materials and methodsThe resazurin reduction assay was used to evaluate the cytotoxicity of samples. The cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were measured by flow cytometry. Column chromatography was used for the purification of FTB. Meanwhile, nuclear magnetic resonance (NMR) spectroscopic analysis was applied for structural elucidation. ResultsThe crude extract, fractions FTBa, FTBc, FTBd as well as compounds 5 and 6 revealed cytotoxicity towards the 9 tested cancer cell lines. The IC50 values ranged from 17.34 µg/mL (towards U87MG.ΔEGFR glioblastoma cells) to 40.68 µg/mL (against CCRF-CEM leukemia cells) for FTB, from 16.78 µg/mL (towards U87. MGΔEGFR cells) to 37.42 µg/mL (against CEM/ADR5000 leukemia cells) for FTBa, from 19.47 µg/mL (towards U87. MG glioblastoma cells) to 41.62 µg/mL (against CCRF-CEM cells) for FTBc, from 14.17 µg/mL (against HCT116p53-/- colon adenocarcinoma cells) to 22.28 µg/mL (towards CEM-ADR5000 cells) for FTBd, from 1.75 µM (against CCRF-CEM cells) to 23.52 µM (against U87. MGΔEGFR cells) for compound 5, from 1.69 µM (against CCRF-CEM cells) to 22.06 µM (against HepG2 hepatocarcinoma cells) for compound 6 and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin. FTB induced apoptosis in CCRF-CEM cells mediated by enhanced ROS production. Compound 5 induced apoptosis through caspases activation and increase ROS production. Meanwhile, 6 induced apoptosis mediated by caspases activation, MMP alteration and enhanced ROS production. ConclusionFagara tessmannii as well as its constituents 5 and 6 revealed considerable cytotoxicity and may be suitable candidates deserving to be further explored to develop new anticancer drugs to combat sensitive and resistant phenotypes.

Cytotoxicity of ungeremine towards multi-factorial drug resistant cancer cells and induction of apoptosis, ferroptosis, necroptosis and autophagy

BackgroundSuccessful cancer chemotherapy is hampered by resistance of cancer cells to established anticancer drugs. Numerous natural products reveal cytotoxicity towards tumor cells. PurposeThe present study was aimed to determine the cytotoxicity of a betaine-type alkaloid, ungeremine, towards 9 cancer cell lines including various sensitive and drug-resistant phenotypes. The mode of action of this compound was further investigated. MethodsThe cytotoxicity, ferroptotic and necroptotic cell death were determined by the resazurin reduction assay. Caspase activation was evaluated using the caspase-Glo assay. Flow cytometry was applied for the analysis of cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H2DCFH-DA). Apoptotic, necroptotic and autophagic markers were determined by Western blotting. CCRF-CEM leukemia cells were used for all mechanistic studies. ResultsUngeremine displayed cytotoxic activity towards the 9 cancer cell lines tested, including drug-sensitive and MDR phenotypes. The IC50values obtained varied from 3.67 µM (in MDA-MB-231-BCRP breast carcinoma cells) to 75.24 µM (against in CEM/ADR5000 leukemia cells) for ungeremine and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin (control drug). Ungeremine induced ferroptosis, necroptosis, autophagy as well as apoptosis mediated by caspase activation, MMP alteration and increase ROS production. ConclusionThe present investigation showed that ungeremine is a promising cytotoxic compoundthat could be further explored in the future to develop new anticancer drugs to fight sensitive and resistant phenotypes.

The oxidoreductase p66Shc acts as tumor suppressor in BRAFV600E-transformed cells.

Metabolic reprogramming, as exemplified by the shift from oxidative phosphorylation to glycolysis, is a common feature of transformed cells. In many tumors, altered metabolism is also reflected in increased reactive oxygen species (ROS) levels, which contribute to proliferation and survival signaling. However, despite high ROS levels, cancer cells can be efficiently killed by further increasing ROS production. We have shown previously that both wild‐type and oncogenic CRAF and BRAF prevent excessive mitochondrial ROS production. Subsequently, it has been demonstrated that raising ROS levels in BRAFV600E‐transformed melanoma cells by inhibiting BRAF or MEK rendered them susceptible to cell death induction. To understand how oncogenic BRAF affects mitochondrial ROS production in melanoma, we studied the mitochondrial ROS‐producing oxidoreductase p66Shc, which is frequently overexpressed in tumors. Using NIH 3T3 BRAFV600E fibroblasts and the melanoma cell lines A375 and M238 carrying the same BRAF mutation, we show that under treatment with the ROS‐inducing agent phenethyl isothiocyanate (PEITC), oncogenic BRAF renders cells refractory to p66ShcS36 phosphorylation, which is essential for p66Shc activation and mitochondrial ROS production. Consistent with this, the activation of JNK1/2, which phosphorylate S36, was blunted, while other mitogen‐activated protein kinases were not affected. Inhibition of JNK1/2 efficiently prevented ROS production, while BRAF and MEK inhibitors increased ROS levels. Vemurafenib‐resistant M238R melanoma cells were impaired in S36 phosphorylation and ROS production following PEITC treatment. Moreover, they failed to increase ROS levels after MEK/BRAF inhibition. Finally, shRNA‐mediated knockdown of p66Shc led to increased growth of BRAFV600E‐transformed NIH 3T3 cells in soft agar assay. Taken together, these data suggest that phosphorylation‐activated p66Shc functions as a tumor suppressor in melanoma cells.

Cytotoxicity of 18 Cameroonian medicinal plants against drug sensitive and multi-factorial drug resistant cancer cells

Ethnopharmacological relevanceRecommendations have been made stating that ethnopharmacological usages such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases should be taken into account if selecting plants for anticancer screening, since these reflect disease states bearing relevance to cancer or cancer-like symptoms. Cameroonian medicinal plants investigated in this work are traditionally used to treat cancer or ailments with relevance to cancer or cancer-like symptoms. Aim of the studyIn this study, 21 methanol extracts from 18 Cameroonian medicinal plants were tested in leukemia CCRF-CEM cells, and the best extracts were further tested on a panel of human cancer cell lines, including various multi-drug-resistant (MDR) phenotypes. Mechanistic studies were performed with the three best extracts. Materials and methodsResazurin reduction assay was used to evaluate cytotoxicity and ferroptotic effects of methanol extracts from different plants. Flow cytometry was used to analyze cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) of extracts from Curcuma longa rhizomes (CLR), Lycopersicon esculentum leaves (LEL), and Psidium guajava bark (PGB). ResultsIn a pre-screening of all extracts, 13 out of 21 (61.9%) had IC50 values below 80 µg/mL. Six of these active extracts displayed IC50 values below 30 µg/mL: Cola pachycarpa leaves (CPL), Curcuma longa rhizomes (CLR), Lycopersicon esculentum leaves, Persea americana bark (PAB), Physalis peruviana twigs (PPT) and Psidium guajava bark (PGB). The best extracts displayed IC50 values from 6.25 µg/mL (against HCT116 p53-/-) to 10.29 µg/mL (towards breast adenocarcinoma MDA-MB-231-BCRP cells) for CLR, from 9.64 µg/mL (against breast adenocarcinoma MDA-MB-231 cells) to 57.74 µg/mL (against HepG2 cells) for LEL and from 1.29 µg/mL (towards CEM/ADR5000 cells) to 62.64 µg/mL (towards MDA-MB-231 cells) for PGB. CLR and PGB induced apoptosis in CCRF-CEM cells via caspases activation, MMP depletion and increase ROS production whilst LEL induced apoptosis mediated by caspases activation and increase ROS production. ConclusionThe best botanicals tested were CLR and LEL, which are worth to be explored in more detail to fight cancers including MDR phenotypes.

Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca2+) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium accumulation is a critical mediator in MAM-induced necroptosis through sustained JNK activation and mitochondrial ROS production. Our study also provided new insights into the molecular regulation of necroptosis in human colon cancer cells.

Cu(II) enhances the effect of Alzheimer's amyloid-β peptide on microglial activation.

BackgroundAggregated forms of amyloid-β (Aβ) peptides are important triggers for microglial activation, which is an important pathological component in the brains of Alzheimer’s patients. Cu(II) ions are reported to be coordinated to monomeric Aβ, drive Aβ aggregation, and potentiate Aβ neurotoxicity. Here we investigated whether Cu(II) binding modulates the effect of Aβ on microglial activation and the subsequent neurotoxicity.MethodsAβ peptides were incubated with Cu(II) at an equimolar ratio to obtain the Cu(II)-Aβ complex. Primary and BV-2 microglial cells were treated with Cu(II)-Aβ, Aβ, or Cu(II). The tumor necrosis factor-α (TNF-α) and nitric oxide levels in the media were determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was detected by MitoSOX oxidation.ResultsIncubation of Cu(II) with Aβ confers different chemical properties on the resulting complex. At the subneurotoxic concentrations, Cu(II)-Aβ (but not Aβ or Cu(II) alone) treatment induced an activating morphological phenotype of microglia and induced the microglial release of TNF-α and nitric oxide as well as microglia-mediated neuronal damage. Cu(II)-Aβ-triggered microglial activation was blocked by nuclear factor (NF)-κB inhibitors and was accompanied with NF-κB activation. Moreover, Cu(II)-Aβ induced hydrogen peroxide release, which was not affected by NADPH oxidase inhibitors. Mitochondrial superoxide production was increased after Cu(II)-Aβ stimulation. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), inhibited Cu(II)-Aβ-elicited microglial release of TNF-α and nitric oxide as well as the microglia-mediated neurotoxic effect.ConclusionOur observations suggest that Cu(II) enhances the effect of Aβ on microglial activation and the subsequent neurotoxicity. The Cu(II)-Aβ-triggered microglial activation involves NF-κB activation and mitochondrial ROS production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0343-3) contains supplementary material, which is available to authorized users.

Comparison of Reactive Oxygen Species Production Activity and Binding Ability of Porphyrins in Cell Membrane Models

Photo dynamic therapy (PDT) is a widespread medical treatment based on the light-triggered generation of reactive oxygen species (ROS) by porphyrin derivatives. ROS may cause oxidative damage to membranes as well as to DNA and, in consequence, ultimately kill cells. Hence, the binding ability, the location within liposomes as simple cellular membrane models, and the ROS production ability of porphyrins are of outstanding interest. Earlier we determined the location of mesoporphyrin IX dimethyl ester (MPE) and its non-esterified form, mesoporphyrin IX dihydrochloride (MPCl) in small unilamellar vesicles (SUV) with fluorescence line narrowing spectroscopy (FLN). Here we investigated the production of ROS by the photosensitizers in the aqueous medium of the vesicles and in the lipid bilayer environment. The monocomponent vesicles were formed of various saturated phosphatidylcholines. The amount of generated oxygen radicals in the aqueous media was measured on the basis of the produced tri-iodide (I3-) from potassium iodide (KI) in the presence of molybdenum (MoO4) catalyst, which was followed by absorption spectrophotometry. The ROS in the lipophilic membranes and in near-membrane regions was measured with a dihydrorhodamine derivative by fluorescence spectroscopy. We observed in general that the binding ability of MPE is considerably higher than that of MPCl. In aqueous media (without liposomes) MPCl was highly effective in ROS formation whereas in case of MPE no similar effect was observed. Liposome-incorporated MPCl produced ROS in much higher amounts than the MPE in the aqueous medium of the liposomes. In near-membrane regions MPE produced ROS in the same amount as MPCl.

OP1-2 - Akt mediated ROS-dependent selective targeting of mutant KRAS tumors

Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers.

NADPH oxidase and the cardiovascular toxicity associated with smoking.

Smoking is one of the most serious but preventable causes of cardiovascular disease (CVD). Key aspects of pathological process associated with smoking include endothelial dysfunction, a prothrombotic state, inflammation, altered lipid metabolism, and hypoxia. Multiple molecular events are involved in smokinginduced CVD. However, the dysregulations of reactive oxygen species (ROS) generation and metabolism mainly contribute to the development of diverse CVDs, and NADPH oxidase (NOX) has been established as a source of ROS responsible for the pathogenesis of CVD. NOX activation and resultant ROS production by cigarette smoke (CS) treatment have been widely observed in isolated blood vessels and cultured vascular cells, including endothelial and smooth muscle cells. NOX-mediated oxidative stress has also been demonstrated in animal studies. Of the various NOX isoforms, NOX2 has been reported to mediate ROS generation by CS, but other isoforms were not tested thoroughly. Of the many CS constituents, nicotine, methyl vinyl ketone, and α,β-unsaturated aldehydes, such as, acrolein and crotonaldehyde, appear to be primarily responsible for NOX-mediated cytotoxicity, but additional validation will be needed. Human epidemiological studies have reported relationships between polymorphisms in the CYBA gene encoding p22phox, a catalytic subunit of NOX and susceptibility to smoking-related CVDs. In particular, G allele carriers of A640G and -930A/G polymorphisms were found to be vulnerable to smoking-induced cardiovascular toxicity, but results for C242T studies are conflicting. On the whole, evidence implicates the etiological role of NOX in smoking-induced CVD, but the clinical relevance of NOX activation by smoking and its contribution to CVD require further validation in human studies. A detailed understanding of the role of NOX would be helpful to assess the risk of smoking to human health, to define high-risk subgroups, and to develop strategies to prevent or treat smoking-induced CVD.

β-Amyloid-evoked Apoptotic Cell Death is Mediated Through MKK6–p66shc Pathway

We have previously shown the involvement of p66shc in mediating apoptosis. Here, we demonstrate the novel mechanism of β-Amyloid-induced toxicity in the mammalian cells. β-Amyloid leads to the phosphorylation of p66shc at the serine 36 residue and activates MKK6, by mediating the phosphorylation at serine 207 residue. Treatment of cells with antioxidants blocks β-Amyloid-induced serine phosphorylation of MKK6, reactive oxygen species (ROS) generation, and hence protected cells against β-Amyloid-induced cell death. Our results indicate that serine phosphorylation of p66shc is carried out by active MKK6. MKK6 knock-down resulted in decreased serine 36 phosphorylation of p66shc. Co-immunoprecipitation results demonstrate a direct physical association between p66shc and WT MKK6, but not with its mutants. Increase in β-Amyloid-induced ROS production was observed in the presence of MKK6 and p66shc, when compared to triple mutant of MKK6 (inactive) and S36 mutant of p66shc. ROS scavengers and knock-down against p66shc, and MKK6 significantly decreased the endogenous level of active p66shc, ROS production, and cell death. Finally, we show that the MKK6-p66shc complex mediates β-Amyloid-evoked apoptotic cell death.

Mitochondrial Dysfunction, Oxidative Stress and Diabetic Cardiovascular Disorders

The prevalence of diabetes has been rapidly increasing in world-wide countries. The dominant cause of mortality in diabetic patients is cardiovascular complications. Mechanism for the susceptibility of diabetic patients to cardiovascular disorders remains unclear. Elevated oxidative stress was detected in diabetic patients or in animal models. Mitochondria are one of major sources of reactive oxygen species (ROS) in cells. Mitochondrial dysfunction and DNA mutations have been detected in diabetic patients. Diabetes-associated metabolic disorders, including hyperglycemia, hypertriglyceridemia, hypercholesterolemia, hypoalphalipoproteinemia, and increased levels of advanced glycation end products, glycated and oxidized lipoproteins, are associated with oxidative stress. Glycated or oxidized low density lipoproteins (LDL) impair the activities of mitochondrial respiratory chain complex enzymes in vascular endothelial cells (EC). Dysfunction in mitochondrial respiration may increase the formation of ROS in mitochondria. NADPH oxidase (NOX) generates ROS in cytosol. Increased NOX activity was detected in diabetic patients. Glycated and oxidized LDL increase the expression of NOX and ROS production in EC. Diabetes-associated metabolic disorders may lead to mitochondrial dysfunction, NOX activation and excess ROS production, which results in oxidative stress and promotes cardiovascular disorders in diabetic patients. Statins, metformin and anthocyanidins may help to attenuate oxidative stress in vasculature induced by diabetes-associated metabolic disorders.

Calcium and the damage pathways in muscular dystrophyThis article is one of a selection of papers published in this special issue on Calcium Signaling.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. Experiments on the mdx mouse, a model of DMD, have shown that mdx muscles are particularly susceptible to stretch-induced damage. In this review, we discuss evidence showing that a series of stretched contractions of mdx muscle fibres causes a prolonged increase in resting intracellular calcium concentration ([Ca2+]i). The rise in [Ca2+]i is caused by Ca2+ entry through a class of stretch-activated channels (SACNSC) for which one candidate gene is TRPC1. We review the evidence for activation of SACNSC in muscle by reactive oxygen species (ROS) and suggest that stretch-induced ROS production is part of the pathway that triggers increased channel activity. When the TRPC1 gene was transfected into C2 myoblasts, expression occurred throughout the cell. Only when the TRPC1 gene was coexpressed with caveolin-3 did the TRPC1 protein express in the membrane. When TRPC1 was expressed in the membrane, it could be activated by ROS to produce Ca2+ entry and this entry was inhibited by PP2, an inhibitor of src kinase. These results suggest that stretched contractions activate ROS production, which activates src kinase. Activity of this kinase causes opening of SACNSC and allows Ca2+ entry. This pathway appears to be a significant cause of muscle damage in DMD.