Augmentation Of Reactive Oxygen Species Research Articles

Protective Assessment of Novel (Bncs Formulation) against Brain Tumor.

Oxidative stress refers to non-homeostatic elevation within intracellular reactive oxygen species (ROS) levels and is associated with several neuro-related pathological conditions. Diclofenac is a commonly prescribed non-steroidal anti-inflammatory drug (NSAID) for treating aches and pain by reducing inflammation. Diclofenac is also associated with the induction of apoptotic cell death by altering the homeostatic balance within mitochondria. In the present report, the neuroprotective effects of BNC formulation constituted by Bacopa monnieri leaves, Nigella sativa and Curcuma longa rhizome seeds were investigated. The synthesized formulation was characterized using FT-IR and LC-MS along with organoleptic evaluation. Thereafter neuroprotective efficacy of BNC formulation was subsequently investigated against Diclofenac-induced oxidative stress in SH-SY5Y cells. The cells were pretreated with synthesized formulation and subsequently evaluated for amelioration in Diclofenac-induced cytotoxicity, and ROS augmentation. The neuroprotective effect of synthesized formulation was further explored by evaluating the changes in nuclear morphology, and apoptosis alleviation with concomitant regulatory effects on caspase-3 and -9 activation. Diclofenac was found to be considerably cytotoxic against human neuroblastoma SHSY5Y cells. Intriguingly, Diclofenac-mediated toxicity was reduced significantly in SH-SY5Y cells pretreated with BNC formulation. Augmented ROS levels within Diclofenac-treated SHSY5Y cells were also reduced in the BNC formulation pretreated SH-SY5Y cells. Furthermore, BNC formulation pretreated SH-SY5Y cells also exhibited reduced dissipation of mitochondrial membrane potential, caspase-3 and -9, along with apoptosis after Diclofenac treatment. These findings indicated that, indeed, Diclofenac induces considerable ROSmediated apoptosis in SH-SY5Y cells, which further intriguingly ameliorated Diclofenacmediated cytotoxic effects on SH-SY5Y cells. This manuscript further collected information about available National and International patents published or granted in preparation of and thereof applications against motor and non-motor brain dysfunctions.

Cadmium toxicity: its' uptake and retaliation by plant defence system and ja signaling.

Cadmium (Cd+2) renders multifarious environmental stresses and highly toxic to nearly all living organisms including plants. Cd causes toxicity by unnecessary augmentation of ROS that targets essential molecules and fundamental processes in plants. In response, plants outfitted a repertory of mechanisms to offset Cd toxicity. The main elements of these are Cd chelation, sequestration into vacuoles, and adjustment of Cd uptake by transporters and escalation of antioxidative mechanism. Signal molecules like phytohormones and reactive oxygen species (ROS) activate the MAPK cascade, the activation of the antioxidant system andsynergistic crosstalk between different signal molecules in order to regulate plant responses to Cd toxicity. Transcription factors like WRKY, MYB, bHLH, bZIP, ERF, NAC etc., located downstream of MAPK, and are key factors in regulating Cd toxicity responses in plants. Apart from this, MAPK and Ca2+signaling also have a salient involvement in rectifying Cd stress in plants. This review highlighted the mechanism of Cd uptake, translocation, detoxification and the key role of defense system, MAPKs, Ca2+ signals and jasmonic acid in retaliating Cd toxicity via synchronous management of various other regulators and signaling components involved under stress condition.

Elabela blunts doxorubicin-induced oxidative stress and ferroptosis in rat aortic adventitial fibroblasts by activating the KLF15/GPX4 signaling.

Doxorubicin (DOX) is a chemotherapeutic drug for a variety of malignancies, while its application is restricted by the cardiovascular toxic effects characterized by oxidative stress. Ferroptosis is a novel iron-dependent regulated cell death driven by lipid peroxidation. Our study aimed to investigate the role of Elabela (ELA) in DOX-induced oxidative stress and ferroptosis. In cultured rat aortic adventitial fibroblasts (AFs), stimulation with DOX dramatically induced cytotoxicity with reduced cell viability and migration ability, and enhanced lactate dehydrogenase (LDH) activity. Importantly, ELA and ferrostatin-1 (Fer-1) mitigated DOX-mediated augmentation of reactive oxygen species (ROS) in rat aortic AFs, accompanied by upregulated levels of Nrf2, SLC7A11, GPX4, and GSH. In addition, ELA reversed DOX-induced dysregulation of apoptosis- and inflammation-related factors including Bax, Bcl2, interleukin (IL)-1β, IL6, IL-10, and CXCL1. Intriguingly, knockdown of Krüppel-like factor 15 (KLF15) by siRNA abolished ELA-mediated alleviation of ROS production and inflammatory responses. More importanly, KLF15 siRNA impeded the beneficial roles of ELA in DOX-pretreated rat aortic AFs by suppressing the Nrf2/SLC7A11/GPX4 signaling. In conclusion, ELA prevents DOX-triggered promotion of cytotoxicity, and exerts anti-oxidative and anti-ferroptotic effects in rat aortic AFs via activation of the KLF15/GPX4 signaling, indicating a promising therapeutic value of ELA in antagonizing DOX-mediated cardiovascular abnormality and disorders.

Oxidative stress-induced premature senescence and aggravated denervated skeletal muscular atrophy by regulating progerin–p53 interaction

BackgroundProgerin elevates atrophic gene expression and helps modify the nuclear membrane to cause severe muscle pathology, which is similar to muscle weakness in the elderly, to alter the development and function of the skeletal muscles. Stress-induced premature senescence (SIPS), a state of cell growth arrest owing to such stimuli as oxidation, can be caused by progerin. However, evidence for whether SIPS-induced progerin accumulation is connected to denervation-induced muscle atrophy is not sufficient.MethodsFlow cytometry and a reactive oxygen species (ROS) as well as inducible nitric oxide synthase (iNOS) inhibitors were used to assess the effect of oxidation on protein (p53), progerin, and nuclear progerin–p53 interaction in the denervated muscles of models of mice suffering from sciatic injury. Loss-of-function approach with the targeted deletion of p53 was used to assess connection among SIPS, denervated muscle atrophy, and fibrogenesis.ResultsThe augmentation of ROS and iNOS-derived NO in the denervated muscles of models of mice suffering from sciatic injury upregulates p53 and progerin. The abnormal accumulation of progerin in the nuclear membrane as well as the activation of nuclear progerin–p53 interaction triggered premature senescence in the denervated muscle cells of mice. The p53-dependent SIPS in denervated muscles contributes to their atrophy and fibrogenesis.ConclusionOxidative stress-triggered premature senescence via nuclear progerin–p53 interaction that promotes denervated skeletal muscular atrophy and fibrogenesis.

Hybrid Carbon Dot Assembly as a Reactive Oxygen Species Nanogenerator for Ultrasound-Assisted Tumor Ablation.

The efficacy of reactive oxygen species (ROS)-based therapy is substantially constrained by the limited ROS generation, stern activation conditions, and lack of a straightforward reaction paradigm. Carbon dots (CDs) have been highly sought after for therapeutic applications for their biocompatibility and intrinsic fluorescence imaging capabilities, making them suitable for ROS generation. Herein, we synthesized a CD-based ultrasmall hybrid nanostructure possessing active sites of Mo, Cu, and IR-780 dye. After cooperative self-assembly with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol), the obtained assembly (CMIR-CDa) exhibits near-infrared fluorescence imaging and photoacoustic tomography. Interestingly, CMIR-CDa can generate singlet oxygen (1O2), hydroxyl radical (·OH), and superoxide radical anion (O2• –) upon ultrasound stimulus owing to its sonosensitizing and enzyme-mimicking properties, showing an enhanced efficacy for tumor ablation in vivo. The collective in vitro and in vivo results indicate that CMIR-CDa has a high potency as an ROS nanogenerator under US irradiation, even at a low concentration. The present study offers an approach for engineering hybrid CDs in a bioinspired way for intratumoral ROS augmentation in response to deep tissue penetrable external stimuli.

Compensatory expression of NRF2-dependent antioxidant genes is required to overcome the lethal effects of Kv11.1 activation in breast cancer cells and PDOs

Potassium channels are important regulators of cellular homeostasis and targeting these proteins pharmacologically is unveiling important mechanisms in cancer cell biology. Here we demonstrate that pharmacological stimulation of the Kv11.1 potassium channel activity results in mitochondrial reactive oxygen species (ROS) production and fragmentation in breast cancer cell lines and patient-derived organoids independent of breast cancer subtype. mRNA expression profiling revealed that Kv11.1 activity significantly altered expression of genes controlling the production of ROS and endoplasmic-reticulum (ER) stress. Characterization of the transcriptional signature of breast cancer cells treated with Kv11.1 potassium channel activators strikingly revealed an adaptive response to the potentially lethal augmentation of ROS by increasing Nrf2-dependent transcription of antioxidant genes. Nrf2 in this context was shown to promote survival in breast cancer, whereas knockdown of Nrf2 lead to Kv11.1-induced cell death. In conclusion, we found that the Kv11.1 channel activity promotes oxidative stress in breast cancer cells and that suppression of the Nrf2-mediated anti-oxidant survival mechanism strongly sensitized breast cancer cells to a lethal effect of pharmacological activation of Kv11.1.

Raising the 'Good' Oxidants for Immune Protection.

Redox medicine is a new therapeutic concept targeting reactive oxygen species (ROS) and secondary reaction products for health benefit. The concomitant function of ROS as intracellular second messengers and extracellular mediators governing physiological redox signaling, and as damaging radicals instigating or perpetuating various pathophysiological conditions will require selective strategies for therapeutic intervention. In addition, the reactivity and quantity of the oxidant species generated, its source and cellular location in a defined disease context need to be considered to achieve the desired outcome. In inflammatory diseases associated with oxidative damage and tissue injury, ROS source specific inhibitors may provide more benefit than generalized removal of ROS. Contemporary approaches in immunity will also include the preservation or even elevation of certain oxygen metabolites to restore or improve ROS driven physiological functions including more effective redox signaling and cell-microenvironment communication, and to induce mucosal barrier integrity, eubiosis and repair processes. Increasing oxidants by host-directed immunomodulation or by exogenous supplementation seems especially promising for improving host defense. Here, we summarize examples of beneficial ROS in immune homeostasis, infection, and acute inflammatory disease, and address emerging therapeutic strategies for ROS augmentation to induce and strengthen protective host immunity.

Inhibition of microRNA-375 ameliorated ketamine-induced neurotoxicity in human embryonic stem cell derived neurons

Excessive exposure to commonly used anesthetic agents, such as ketamine, may induce permanent damage to immature human brains. In this work, we used a human embryonic stem cell (hESC)-derived neuron model to assess the expression and function of human microRNA 735 (hsa-miR-375) in regulating ketamine-induced neural cell death and neural toxicity in vitro. In the in vitro culture, hESC-derived neurons were incubated with ketamine for 72 h. After that, cell viability, reactive oxygen species activity, neural apoptosis, neurite degeneration, and hsa-miR-375 gene expression were assessed, respectively. We found ketamine induced neural death, reactive oxygen species augmentation, neural apoptosis, neurite degeneration and hsa-miR-375 upregulation, in hESC-derived neurons. In addition, we discovered that, lentivirus-mediated mR-375 downregulation protected ketamine-induced neural cell death and neural toxicity. Also, human brain derived neurotrophic factor (BDNF) was found to be directly and reversely regulated by hsa-miR-375. Moreover, BDNF downregulation was shown to functionally reverse the protective effect of miR-375 downregulation on ketamine-induced neural cell death and neural toxicity. Overall, this work provided strong evidence showing hsa-miR-375 is an active regulator in anesthesia-induced neural cell death and neural toxicity, possibly via inverse regulation on BDNF gene.

Novel Colchicine Analogues Target Mitochondrial PT Pores Using Free Tubulins and Induce ROS-Mediated Apoptosis in Cancerous Lymphocytes.

B-acute lymphoblastic leukemia (B-ALL) is the frequent pediatric malignity. Chemotherapy is the most practical approaches to deal with such malignancies. Microtubule-targeted agents are one of the most strategic drugs which formerly used in chemotherapy. Although colchicine-binding anti-tubulin agents exhibited promising effects in clinical trials, their exact mechanism of action is not fully understood. In this study, the effects of two newly synthesized of colchicine derivatives were investigated on cell viability of cancerous and normal lymphocytes. The viability test was carried out by MTT assay. Apoptosis vs. necrosis was measured by double staining with annexin V/PI, and caspase-3 as the ultimate mediator of apoptotic measured through the colorimetric assay. Parameters of mitochondrial damage (ROS formation, MMP (Mitochondrial Membrane Potential) decline, mitochondrial swelling, and cytochrome c release following treatment by colchicine derivatives. By focusing on mitochondrial parameters, we showed that following treatment by two newly synthesized colchicine derivatives, apoptosis is triggered in cancerous B-lymphocytes. We demonstrated these compounds could activate apoptosis in cancerous lymphocytes by augmentation of reactive oxygen species (ROS), a decline in mitochondrial membrane potential (MMP), mitochondrial swelling, release of cytochrome c, and also caspase-3 activation. Considering the obtained evidence, these inhibitors could be the new therapeutic strategies in ALL treatment.

MYCN drives glutaminolysis in neuroblastoma and confers sensitivity to an ROS augmenting agent

Heightened aerobic glycolysis and glutaminolysis are characteristic metabolic phenotypes in cancer cells. Neuroblastoma (NBL), a devastating pediatric cancer, is featured by frequent genomic amplification of MYCN, a member of the Myc oncogene family that is primarily expressed in the early stage of embryonic development and required for neural crest development. Here we report that an enriched glutaminolysis gene signature is associated with MYCN amplification in children with NBL. The partial knockdown of MYCN suppresses glutaminolysis in NBL cells. Conversely, forced overexpression of MYCN in neural crest progenitor cells enhances glutaminolysis. Importantly, glutaminolysis induces oxidative stress by producing reactive oxygen species (ROS), rendering NBL cells sensitive to ROS augmentation. Through a small-scale metabolic-modulator screening, we have found that dimethyl fumarate (DMF), a Food and Drug Administration-approved drug for multiple sclerosis, suppresses NBL cell proliferation in vitro and tumor growth in vivo. DMF suppresses NBL cell proliferation through inducing ROS and subsequently suppressing MYCN expression, which is rescued by an ROS scavenger. Our findings suggest that the metabolic modulation and ROS augmentation could be used as novel strategies in treating NBL and other MYC-driven cancers.

Proteolytic cleavage of vascular adhesion protein-1 induced by vascular endothelial growth factor in retinal capillary endothelial cells.

To investigate the mechanism of soluble vascular adhesion protein-1 (sVAP-1) accumulation induced by vascular endothelial growth factor (VEGF) in the vitreous of patients with diabetic retinopathy (DR). Experimental. Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with proliferative diabetic retinopathy (PDR) with or without intravitreal bevacizumab (IVB) injection were determined by ELISA. The effect of VEGF on both mRNA expression of Vap-1 and secretion of sVAP-1 in rat retinal capillary endothelial cells (TR-iBRB2) was analyzed by real-time PCR and western blotting, respectively. In addition, the impact of VEGF on production and activation ratios of matrix metalloproteinase (MMP)-2 and MMP-9 was examined by gelatin zymography. Hydrogen peroxide production and reactive oxygen species (ROS) levels were assessed in the supernatants of TR-iBRB2 cells treated with VEGF. IVB injection decreased vitreous levels of sVAP-1 and HEL in patients with PDR. VEGF stimulation released sVAP-1 protein from TR-iBRB2 cells as a consequence of membrane-anchored VAP-1 shedding by MMP-2 and MMP-9. In addition, VEGF increased hydrogen peroxide generation and ROS augmentation through spermine oxidation by sVAP-1 as semicarbazide-sensitive amine oxidase (SSAO) in the supernatant of cultured endothelial cells. The current data demonstrate that proangiogenic factor VEGF induces sVAP-1 release from retinal capillary endothelial cells and facilitates hydrogen peroxide generation via enzymatic property of sVAP-1, followed by the increase of oxidative stress, one of the crucial factors in the pathogenesis of DR.

Cold atmospheric plasma-modulated phorbol 12-myristate 13-acetate-induced differentiation of U937 cells to macrophage-like cells

Monocytes are recruited to injured tissue sites and differentiate into tissue macrophages or dendritic cells to protect against pathogens and repair the damaged tissues. Phorbol-12-myristate-13-acetate (PMA) is a well-known stimulus commonly used for differentiation of monocytes into macrophage-like cells (MϕLC). Here, we report the effect of Cold Atmospheric Plasma (CAP) on PMA-induced U937 differentiation into MϕLC. Treatment of U937 cells with PMA for 3 days and resting for 4 days increased the size of cytoplasm as compared with nucleus, and exposure to CAP before addition of PMA led to further increase in cytoplasm indicating the ability of CAP to modulate the differentiation of monocytes. Exposure of U937 cells to CAP or PMA increased cellular reactive oxygen species (ROS) level and the combination led to further augmentation of ROS. Treatment of U937 cells with PMA displayed a biphasic activation of proinflammatory transcription factor NF-κB, which plays an important role in differentiation and pretreatment with CAP further increased PMA induced NF-κB-DNA-binding activity. CAP also increased lipopolysaccharide induced secretion of TNF-α and IL-6 in MϕLC. Further investigation revealed that MϕLC or CAP-treated MϕLC were more resistant to anticancer drugs such as doxorubicin and 5-fluorouracil (5-FU) than U937 cells. Our present studies suggest an alternate protocol to modulate the differentiation of U937 cells into MϕLC by combining CAP and PMA.

Potent In Vitro Synergism of Fluconazole and Osthole against Fluconazole-Resistant Candida albicans.

Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole.

Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

Emodin mitigates the oxidative stress induced by cisplatin in osteosarcoma MG63 cells.

Previously, the application of cisplatin in chemotherapy was limited due to the significant side effects on normal cell growth. In the present study, the concomitant application of emodin with cisplatin was demonstrated to ameliorate cisplatin-induced oxidative stress and markedly suppress tumor cell proliferation for the first time. Human osteosarcoma MG-63 cells were treated with cisplatin alone or in combination with emodin. The cell viability was determined by MTS assays and the augmentation of reactive oxygen species were determined by fluorogenic probes; in addition, a stable MG-63 subline bearing antioxidant response element (ARE)-driven luciferase expression was developed to monitor the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-ARE signaling pathway. The results indicated that cisplatin or emodin may inhibit MG-63 cell proliferation in a time- or dose-dependent manner, respectively. Concomitant treatment with cisplatin and emodin demonstrated synergic anti-tumor effects. Cisplatin augmented reactive oxygen species in the MG-63 cells, followed by the translocation of Nrf2 from the cytoplasm into the nucleus, which triggered ARE-driven luciferase expression. The addition of emodin diminished the previously described phenomenon, resulting in decreased ROS augmentation, translocation of Nrf2 and ARE-driven luciferase activity. In conclusion, emodin could ameliorate cisplatin-induced oxidative stress and protect the cells from oxidative stress-induced damage. The findings of the present study provide a novel strategy for the treatment of osteosarcoma using emodin and cisplatin.

Hydroxylated Dimeric Naphthoquinones Increase the Generation of Reactive Oxygen Species, Induce Apoptosis of Acute Myeloid Leukemia Cells and Are Not Substrates of the Multidrug Resistance Proteins ABCB1 and ABCG2.

Selective targeting of the oxidative state, which is a tightly balanced fundamental cellular property, is an attractive strategy for developing novel anti-leukemic chemotherapeutics with potential applications in the treatment of acute myeloid leukemia (AML), a molecularly heterogeneous disease. Dimeric naphthoquinones (BiQs) with the ability to undergo redox cycling and to generate reactive oxygen species (ROS) in cancer cells are a novel class of compounds with unique characteristics that make them excellent candidates to be tested against AML cells. We evaluated the effect of two BiQ analogues and one monomeric naphthoquinone in AML cell lines and primary cells from patients. All compounds possess one halogen and one hydroxyl group on the quinone cores. Dimeric, but not monomeric, naphthoquinones demonstrated significant anti-AML activity in the cell lines and primary cells from patients with favorable therapeutic index compared to normal hematopoietic cells. BiQ-1 effectively inhibited clonogenicity and induced apoptosis as measured by Western blotting and Annexin V staining and mitochondrial membrane depolarization by flow cytometry. BiQ-1 significantly enhances intracellular ROS levels in AML cells and upregulates expression of key anti-oxidant protein, Nrf2. Notably, systemic exposure to BiQ-1 was well tolerated in mice. In conclusion, we propose that BiQ-induced therapeutic augmentation of ROS in AML cells with dysregulation of antioxidants kill leukemic cells while normal cells remain relatively intact. Further studies are warranted to better understand this class of potential chemotherapeutics.

Increased Energy Demand during Adrenergic Receptor Stimulation Contributes to Ca2+ Wave Generation

While β-adrenergic receptor (β-AR) stimulation ensures adequate cardiac output during stress, it can also trigger life-threatening cardiac arrhythmias. We have previously shown that proarrhythmic Ca2+ waves during β-AR stimulation temporally coincide with augmentation of reactive oxygen species (ROS) production. In this study, we tested the hypothesis that increased energy demand during β-AR stimulation plays an important role in mitochondrial ROS production and Ca2+-wave generation in rabbit ventricular myocytes. We found that β-AR stimulation with isoproterenol (0.1 μM) decreased the mitochondrial redox potential and the ratio of reduced to oxidated glutathione. As a result, β-AR stimulation increased mitochondrial ROS production. These metabolic changes induced by isoproterenol were associated with increased sarcoplasmic reticulum (SR) Ca2+ leak and frequent diastolic Ca2+ waves. Inhibition of cell contraction with the myosin ATPase inhibitor blebbistatin attenuated oxidative stress as well as spontaneous SR Ca2+ release events during β-AR stimulation. Furthermore, we found that oxidative stress induced by β-AR stimulation caused the formation of disulfide bonds between two ryanodine receptor (RyR) subunits, referred to as intersubunit cross-linking. Preventing RyR cross-linking with N-ethylmaleimide decreased the propensity of Ca2+ waves induced by β-AR stimulation. These data suggest that increased energy demand during sustained β-AR stimulation weakens mitochondrial antioxidant defense, causing ROS release into the cytosol. By inducing RyR intersubunit cross-linking, ROS can increase SR Ca2+ leak to the critical level that can trigger proarrhythmic Ca2+ waves.

Heavy metal stress and some mechanisms of plant defense response.

Unprecedented bioaccumulation and biomagnification of heavy metals (HMs) in the environment have become a dilemma for all living organisms including plants. HMs at toxic levels have the capability to interact with several vital cellular biomolecules such as nuclear proteins and DNA, leading to excessive augmentation of reactive oxygen species (ROS). This would inflict serious morphological, metabolic, and physiological anomalies in plants ranging from chlorosis of shoot to lipid peroxidation and protein degradation. In response, plants are equipped with a repertoire of mechanisms to counteract heavy metal (HM) toxicity. The key elements of these are chelating metals by forming phytochelatins (PCs) or metallothioneins (MTs) metal complex at the intra- and intercellular level, which is followed by the removal of HM ions from sensitive sites or vacuolar sequestration of ligand-metal complex. Nonenzymatically synthesized compounds such as proline (Pro) are able to strengthen metal-detoxification capacity of intracellular antioxidant enzymes. Another important additive component of plant defense system is symbiotic association with arbuscular mycorrhizal (AM) fungi. AM can effectively immobilize HMs and reduce their uptake by host plants via binding metal ions to hyphal cell wall and excreting several extracellular biomolecules. Additionally, AM fungi can enhance activities of antioxidant defense machinery of plants.

Is antioxidant therapy effective for advanced hypertension and renal injury?

nappropriate augmentation of reactive oxygen species(ROS) production reduces the bioavailability of anti-oxidant defenses [1,2] and plays a pivotal role in thepathophysiology of hypertension and renal injury [3–5].Experimental studies have indicated that enhanced ROSproduction is involved in vascular remodeling and endo-thelium dysfunction during the development of hyperten-sion [6]. Earlier clinical studies have also suggested a strongassociation between lower antioxidant levels and cardio-vascular risks [7].To date, much effort has been directed toward thebenefits of antioxidant therapy. Clinical studies, includingthe Cambridge Heart Antioxidant Study [8] and the Anti-oxidant Supplementation in Atherosclerosis PreventionStudy [9], have shown the beneficial effects of antioxidanttherapy for cardiovascular events. Large epidemiologicalstudies have also reported that dietary intake of anti-oxidants inverselycorrelateswithhypertension[9,10].Simi-larly, treatment with ascorbic acid significantly improvedSBP and DBP in mild-to-moderate hypertensive patients[11]. In experimental models of hypertension, vitamin Calone or in combination with vitamin E increased thesynthesis of nitric oxide and reduced blood pressure[12,13]. Furthermore, an antioxidant rich diet was shownto relieve hypertension and reduces renal immune cellinfiltration [14]. By contrast, many clinical trials failed todemonstrate any beneficial effects of antioxidant therapyon blood pressure [15–17] and cardiovascular events[18–20]. For example, the Heart Protection Study Collabo-rative Group studied high-risk individuals and showed thatalthoughantioxidantvitaminsupplementationsubstantiallyincreased blood vitamin concentrations, it did not produceany significant reductions in the incidence of any type ofcardiovascular disease [19].In this issue of Journal of Hypertension, the article‘Superoxide dismutase mimetic, tempol, aggravates renalinjury in advanced-stage stroke-prone spontaneouslyhypertensive rats’ by Sugama et al. [21] shows that anti-oxidanttherapywithasuperoxidemimetic,tempol,didnotreduce blood pressure in salt-loaded old stroke pronespontaneously hypertensive rats (SHRSP). The authors alsoshowed that although tempol effectively reduced oxidativestress in the kidney, it actually exacerbated the progressionof proteinuria and histological damage, such as glomerulo-sclerosis and interstitial fibrosis. These data suggestthat alternative pathways that are ROS-independent areinvolved in the pathogenesis of the advanced stage ofhypertension and renal injury in SHRSP. It is also possiblethat oxidative stress is merely a consequence of the acti-vation of these alternative pathways. However, Park et al.[22] had performed similar experiments in SHRSP andshown that long-term treatment with tempol from earlystages of hypertension did attenuate the progression ofhypertension through the prevention of vascular remodel-ing, and that this was associated with decreased vascularsuperoxide production and increased plasma antioxidantlevels.Thus, thequestion arisesastowhyantioxidanttherapiesshow such conflicting results in clinical and experimentalstudies [23]. Importantly, it is clear from the clinical studiesthatdosedifferencesdonotseemtoberesponsibleforsuchdiscrepant results [23]. One possible explanation for thediscrepancy in these studies is the different stage of hyper-tension and renal injury. In both clinical and experimentalstudies,beneficialeffectsofantioxidantsweremostlyfoundwhen interventions were started at an early stage. Forexample, Park et al. [22] administered tempol from an earlyage and showed an attenuation in the development ofhypertension in SHRSP, whereas Sugama et al. [21] failedto observe a blood pressure-lowering effect of tempolif treatment started at an advanced hypertensive stage.Inatomi et al. [24] have also shown that in patients withmild cardioembolic stroke, treatment with an antioxidant,edaravone, resulted in early functional improvement, butthese effects were not observed in late stages.Thefindingthatantioxidanttherapieshavenobeneficialeffects on advanced hypertension, as well as cardiovas-cular and renal diseases, suggest a potentialinvolvement of

Dimeric Naphthoquinones: Novel Anti-Leukemic Agents Modulating Cellular Redox Status

BackgroundThe oxidative state is significantly perturbed in leukemia cells, compared to normal cells, due to generation of increased reactive oxygen species (ROS) and/or dysregulated antioxidant mechanisms. In a molecularly heterogeneous disease such as acute myeloid leukemia (AML), targeting the oxidative state, a fundamental physiological property, is an attractive strategy for developing novel anti-leukemic agents. We hypothesized that regardless of oncogenic mutations, therapeutic augmentation of ROS in AML cells with dysregulated antioxidants would kill leukemia cells and leukemia stem cells, while normal stem cells would remain relatively intact. Quinones can initiate and propagate free radical reactions. The electron-accepting nature of quinones could in principle be tuned to yield selective cytotoxicity for cells with a particular redox signature. Dimeric naphthoquinones (BiQs), with known ability to generate ROS in cancer cells, are a novel class of compounds with unique characteristics that make them excellent candidates to be tested against leukemia cells. The primary objective of this study was to determine the potency of BiQs in leukemia cells and to assess the therapeutic index of BiQs in AML cells, in relation to normal hematopoietic stem cells. The secondary objective was to determine whether BiQs induce apoptosis, increase ROS, target mitochondrial membrane potential, and/or affect antioxidant proteins. MethodsWe tested two BiQs (E6a and B1a) and one mononaphthoquinone in two AML cell lines, MOLM-14 (FLT3-ITD) and THP-1 (FLT3-WT), two normal karyotype primary AML samples (AML15, AML16), both with FLT3-WT, and fresh normal bone marrow cells. IC50 values were determined by exposing cell lines and bone marrow cells to BiQs for 72 h and 48 h, respectively. Each experiment was terminated with Alamar Blue (Sigma). Cell viability assays were carried out similarly except that the endpoint was trypan blue exclusion. For clonogenic assays, cells were plated in methylcellulose and exposed to BiQs either prior to plating or both prior to plating and during culture. Colonies were counted using an automated colony counter (Microbiology Intl.). Apoptosis was measured using the FITC Annexin V Kit (BD Pharmingen), and mitochondrial potential was assessed using the MitoRed Kit (Millipore); cells were analyzed by FACScan (BD Biosciences) and Flow Jo Software (Tree Flo). Standard Western blot techniques were used for measurement of caspase 3 and Nrf2 protein. Numbers are mean ± SD. ResultsThe IC50 values (µM) of E6a for MOLM-14, THP1, AML15 and AML16 were 5.5 ± 0.8, 4.2 ± 1.9, 5.1, and 0.4, respectively. The IC50 values (µM) of B1a for MOLM-14, THP1, AML15 and AML16 were 6.2 ± 0.7, 5.6 ± 0.1, 5.4, and 3.0, respectively. The IC50 value of E6a for normal bone marrow cells was 14.5 µM. The mononaphthoquinone did not show anti-leukemic activity. Viability tests showed a 52 ± 8% and 33 ± 9% increase in MOLM-14 cell kill after exposure to 5 µM E6a and 5 µM B1a, compared to vehicle, versus 69 ± 12% and 59 ± 2% in THP-1. Treatment with E6a, compared to vehicle, resulted in a concentration-dependent decrease in colony formation in MOLM-14 (52 ± 17 [10 µM] vs 228 ± 170 [5 µM] vs 338 ± 72 colonies). Interestingly, when MOLM-14 cells were treated with E6a for 24 h then placed in methylcellulose in continuous presence of E6a for 9 days, colony formation was completely inhibited with 5 µM E6a compared to vehicle (2.5 ± 1 vs 294 ± 43 colonies). Treatment of AML16 with 5 µM E6a for 24 h resulted in a 1.5-fold increase in apoptosis, and a 1.6-fold increase in loss of mitochondrial membrane potential compared to vehicle. After 6 h exposure of MOLM-14 and AML16 to 5 µM E6a, cleavage of caspase 3 was detected. After 2 h exposure of AML16 to 5 µM E6a and 5 µM B1a, Nrf2 proteins was significantly increased in response to oxidative stress. ConclusionsWe have demonstrated that BiQs possess anti-leukemia activity with a reasonable therapeutic index in relation to normal bone marrow cells. Measurements of effects on intracellular ROS and on proteins involved in oxidative stress are ongoing, and drug transport by multidrug resistance-associated ATP-binding cassette proteins including P-glycoprotein will be studied. The more active BiQ will be studied further in an AML primagraft model. Disclosures:No relevant conflicts of interest to declare.