ascorbic-acid and Neuroblastoma

Copper-related reactive oxygen species (ROS) formation can lead to neuropathologic degradation associated with Alzheimer's disease (AD) according to amyloid cascade hypothesis. A complexing agent that can selectively chelate with copper ions and capture copper ions from the complex formed by copper ions and amyloid-β (Cu - Aβ complex) may be available in reducing ROS formation. Herein, we described applications of guluronic acid (GA), a natural oligosaccharide complexing agent obtained from enzymatic hydrolysis of brown algae, in reducing copper-related ROS formation. UV-vis absorption spectra demonstrated the coordination between GA and Cu(II). Ascorbic acid consumption and coumarin-3-carboxylic acid fluorescence assays confirmed the viability of GA in reducing ROS formation in solutions containing other metal ions and Aβ. Fluorescence kinetics, DPPH radical clearance and high resolution X - ray photoelectron spectroscopy results revealed the reductivity of GA. Human liver hepatocellular carcinoma (HepG2) cell viability demonstrated the biocompatibility of GA at concentrations lower than 320 μM. Cytotoxic results of human neuroblastoma (SH-SY5Y) cells verified that GA can inhibit copper-related ROS damage in neuronal cells. Our findings, combined with the advantages of marine drugs, make GA a promising candidate in reducing copper-related ROS formation associated with AD therapy.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Ascorbic Acid; Copper; Humans; Neuroblastoma; Reactive Oxygen Species

The ubiquitin-proteasomal pathway regulates the functional expression of many membrane transporters in a variety of cellular systems. Nothing is currently known about the role of ubiquitin E3 ligase, neural precursor cell-expressed developmentally down-regulated gene 4 (Nedd4-1) and the proteasomal degradation pathway in regulating human vitamin C transporter-2 (hSVCT2) in neuronal cells. hSVCT2 mediates the uptake of ascorbic acid (AA) and is the predominantly expressed vitamin C transporter isoform in neuronal systems. Therefore, we addressed this knowledge gap in our study. Analysis of mRNA revealed markedly higher expression of Nedd4-1 in neuronal samples than that of Nedd4-2. Interestingly, Nedd4-1 expression in the hippocampus was higher in patients with Alzheimer's disease (AD) and age-dependently increased in the J20 mouse model of AD. The interaction of Nedd4-1 and hSVCT2 was confirmed by coimmunoprecipitation and colocalization. While the coexpression of Nedd4-1 with hSVCT2 displayed a significant decrease in AA uptake, siRNA-mediated knockdown of Nedd4-1 expression up-regulated the AA uptake. Further, we mutated a classical Nedd4 protein interacting motif ("PPXY") within the hSVCT2 polypeptide and observed markedly decreased AA uptake due to the intracellular localization of the mutated hSVCT2. Also, we determined the role of the proteasomal degradation pathway in hSVCT2 functional expression in SH-SY5Y cells and the results indicated that the proteasomal inhibitor (MG132) significantly up-regulated the AA uptake and hSVCT2 protein expression level. Taken together, our findings show that the regulation of hSVCT2 functional expression is at least partly mediated by the Nedd4-1 dependent ubiquitination and proteasomal pathways.

Topics: Animals; Ascorbic Acid; Endosomal Sorting Complexes Required for Transport; Epithelial Cells; Humans; Mice; Nedd4 Ubiquitin Protein Ligases; Neuroblastoma; Sodium-Coupled Vitamin C Transporters; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination

Neuronal uptake of ascorbic acid (AA) in humans occurs via the human sodium-dependent vitamin C transporter-2 (hSVCT2). Recent studies show that a significantly lower level of vitamin C is present in the blood of epileptic patients. Consequently, focused studies investigating the involved molecular mechanisms for hSVCT2 regulation are vital to enhance vitamin C body homeostasis. Currently, little is known about the role of valproic acid (VPA), a drug utilized to treat epilepsy and a class I histone deacetylase inhibitor (HDACi), on AA uptake in neuronal systems. Thus, this study aims to examine the effect of VPA on hSVCT2 functional expression in neuronal cells. VPA treatment upregulated the AA uptake and this increased AA uptake was associated with a significant increase in hSVCT2 expression and SLC23A2 promoter activity in SH-SY5Y cells. Knockdown of HDAC2, a predominant isoform in neuronal systems, significantly increased hSVCT2 functional expression. VPA treatment in mice displayed increased mouse (m)SVCT2 protein, mRNA and heterogenous nuclear RNA (hnRNA) expression in the brain. In addition, Yin Yang-1 (YY1), a transcription factor that drives the SLC23A2 promoter activity, protein and mRNA expression levels were markedly upregulated in VPA-treated SH-SY5Y cells and mice brain. Together, our findings suggest that VPA upregulates the functional expression of SVCT2 via HDAC2 and transcriptional mechanism(s).

Topics: Animals; Ascorbic Acid; Histone Deacetylase Inhibitors; Humans; Mice; Neuroblastoma; Protein Isoforms; RNA, Heterogeneous Nuclear; RNA, Messenger; Sodium-Coupled Vitamin C Transporters; Transcription Factors; Valproic Acid; Vitamins

The present study demonstrates the efficacies of synthetic 1,8-cineole and an 1,8-cineole-rich supercritical carbon dioxide (SC-CO

Topics: Alzheimer Disease; Amyloid beta-Peptides; Ascorbic Acid; Cell Line, Tumor; Drug Evaluation, Preclinical; Drug Synergism; Elettaria; Eucalyptol; Ferroptosis; Ferrous Compounds; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyl Radical; Neuroblastoma; Peptide Fragments; Phytotherapy; Plant Extracts; Reactive Oxygen Species; Seeds; Spices

Children are at high risk of injuries and wounds. The application of medical grade honey is a promising approach to improving the healing of wounds of various origin and severity. However, the use of medical grade honey in young paediatric patients remains limited. The aim of this study is to show the safety, efficacy and usefulness of medical grade honey in abdominal wounds, of different causes, in paediatric patients.. This was a prospective, observational case series evaluating five young infants with abdominal wounds at the General Hospital in Thessaloniki. All wounds were treated in the same manner with daily medical grade honey applied to the wound area and closely monitored.. All treated wounds rapidly presented granulation tissue formation and underwent re-epithelialisation. Peripheral oedema and inflammation decreased upon initial application. Necrotic tissue was effectively debrided when present. Slough was removed and no signs of infection were detected, irrespective of initial wound presentations. Scar formation was minimal and the full range of motion was preserved in all cases.. Based on this case study, medical grade honey is safe and effective in treating different abdominal wounds, including infected or dehisced wounds as well as burns. The easy application and broad applicability make medical grade honey recommendable as a first-line treatment in paediatric patients.

Topics: Abdominal Injuries; Apitherapy; Appendectomy; Appendicitis; Ascorbic Acid; Bacteroides Infections; Burns; Burns, Chemical; Child; Child, Preschool; Dermatologic Agents; Drug Resistance, Multiple, Bacterial; Edema; Female; Gastrostomy; Greece; Honey; Humans; Infant; Infant, Newborn; Inflammation; Klebsiella Infections; Lanolin; Male; Neuroblastoma; Oils, Volatile; Ointments; Prospective Studies; Re-Epithelialization; Retroperitoneal Neoplasms; Surgical Wound Dehiscence; Surgical Wound Infection; Vitamin E; Vitamins; Zinc Oxide

Scurvy is a disease that is rarely encountered in modern medicine. A condition that was classically associated with sailors, its incidence has decreased dramatically since the discovery of its association with vitamin C deficiency. We present the case of a 2-year-old boy, whose treatment for neuroblastoma was complicated by gastrointestinal disease, which necessitated enteral feeding. While still undergoing treatment, he started to complain about increasing pain in his lower limbs, which appeared to be markedly tender on palpation. Radiographic findings suggested a diagnosis of scurvy, which was subsequently confirmed on serum biochemistry. This was an unexpected finding, as the child had been receiving adequate vitamin C in his enteral feeds. However, his absorption had become severely impaired due to pseudomembranous gastritis and enteritis, leading to his deficient state. He significantly improved after intravenous ascorbic acid replacement and demonstrated a full recovery, both clinically and radiologically. This case highlights the importance of considering scurvy in the differential diagnosis for at-risk patients. Early recognition can facilitate the simple treatment of this potentially serious condition.

Topics: Ascorbic Acid; Diagnosis, Differential; Enteral Nutrition; Enteritis; Gastritis; Humans; Infant; Leg; Male; Neuroblastoma; Scurvy

An ascorbate-mediated production of oxidative stress has been shown to retard tumor growth. Subsequent glycolysis inhibition has been suggested. Here, we further define the mechanisms relevant to this observation. Ascorbate was cytotoxic to human neuroblastoma cells through the production of H

Topics: Animals; Ascorbic Acid; Cell Death; DNA; DNA Damage; DNA Repair; Humans; Hydrogen Peroxide; Mice; Neuroblastoma; Oxidative Stress; Poly(ADP-ribose) Polymerases; Xenograft Model Antitumor Assays

Expression of the sodium and ascorbic acid (AA) cotransporter SVCT2 is induced during the period of cellular arborization and synaptic maturation of early postnatal (P1-P5) rat cerebral neurons. The physiological importance of the transporter for neurons is evidenced by the lethality and delayed neuronal differentiation detected in mice with ablation of SVCT2. The mechanism(s) involved in these defects and the role of SVCT2 in neuronal branching have not been determined yet. To address this, we used lentiviral expression vectors to increase the levels of SVCT2 in N2a cells and analyzed the effects on neurite formation. Expression of a fusion protein containing the human SVCT2wt and EYFP induced an increase in the number of MAP2+ neurites and filopodia in N2a cells. Overexpression of SVCT2 and treatment with AA promoted ERK1/2 phosphorylation. Our data suggest that enhanced expression of the high affinity AA transporter SVCT2, which tightly regulates intracellular AA concentrations, induces neuronal branching that then activates key signaling pathways that are involved in the differentiation and maturation of cortical neurons during postnatal development.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Cell Shape; Dietary Supplements; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; Mice; Neuroblastoma; Phenotype; Phosphorylation; Protein Transport; Sodium-Coupled Vitamin C Transporters

Venezuelan equine encephalitis (VEE) virus causes an acute central nervous system infection in human and animals. Melatonin (MLT), minocycline (MIN) and ascorbic acid (AA) have been shown to have antiviral activities in experimental infections; however, the mechanisms involved are poorly studied. Therefore, the aim of this study was to determine the effects of those compounds on the viral titers, NO production and lipid peroxidation in the brain of mice and neuroblastoma cultures infected by VEE virus. Infected mouse (10 LD50) were treated with MLT (500 μg/kg bw), MIN (50mg/kg bw) or AA (50mg/kg bw). Infected neuroblastoma cultures (MOI: 1); MLT: 0.5, 1, 5mM, MIN: 0.1, 0.2, 2 μM or AA: 25, 50, 75 μM. Brains were obtained at days 1, 3 and 5. In addition, survival rate of infected treated mice was also analyzed. Viral replication was determined by the plaque formation technique. NO and lipid peroxidation were measured by Griess׳ reaction and thiobarbituric acid assay respectively. Increased viral replication, NO production and lipid peroxidation were observed in both, infected brain and neuroblastoma cell cultures compared with uninfected controls. Those effects were diminished by the studied treatments. In addition, increased survival rate (50%) in treated infected animals compared with untreated infected mice (0%) was found. MLT, MIN and AA have an antiviral effect involving their anti-oxidant properties, and suggesting a potential use of these compounds for human VEE virus infection.

Topics: Animals; Antiviral Agents; Ascorbic Acid; Brain; Cell Line, Tumor; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalitis Virus, Venezuelan Equine; Encephalomyelitis, Venezuelan Equine; Lipid Peroxidation; Male; Melatonin; Mice; Minocycline; Neuroblastoma; Neuroprotective Agents; Nitric Oxide; Oxidative Stress; Survival Rate; Treatment Outcome; Viral Load

The increased accumulation of iron in the brain in Alzheimer's disease (AD) is well documented, and excess iron is strongly implicated in the pathogenesis of the disease. The adverse effects of accumulated iron in AD brain may include the oxidative stress, altered amyloid beta-metabolism and the augmented toxicity of metal-bound amyloid beta 42. In this study, we have shown that exogenously added iron in the form of ferric ammonium citrate (FAC) leads to considerable accumulation of amyloid precursor protein (APP) without a corresponding change in the concerned gene expression in cultured SHSY5Y cells during exposure up to 48 h. This phenomenon is also associated with increased β-secretase activity and augmented release of amyloid beta 42 in the medium. Further, the increase in β-secretase activity, in SHSY5Y cells, upon exposure to iron apparently involves reactive oxygen species (ROS) and NF-κB activation. The synthetic flavone negletein (5,6-dihydroxy-7-methoxyflavone), which is a known chelator for iron, can significantly prevent the effects of FAC on APP metabolism in SHSY5Y cells. Further, this compound inhibits the iron-dependent formation of ROS and also blocks the iron-induced oligomerization of amyloid beta 42 in vitro. In concentrations used in this study, negletein alone appears to have only marginal toxic effects on cell viability, but, on the other hand, the drug is capable of ameliorating the iron-induced loss of cell viability considerably. Our results provide the initial evidence of potential therapeutic effects of negletein, which should be explored in suitable animal models of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Ascorbic Acid; Cell Line, Tumor; Ferric Compounds; Flavones; Humans; Hydroxyl Radical; Iron; Iron Chelating Agents; Models, Biological; Neuroblastoma; Neurons; NF-kappa B; Peptide Fragments; Polymerization; Quaternary Ammonium Compounds; Reactive Oxygen Species

Neuroblastoma, a peripheral nervous system cancer that can be highly invasive and metastatic, accounts for 8-10% of all solid childhood tumors in children under the age of 15 years. Despite multiple clinical efforts, prognosis remains poor for this enigmatic disease. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has shown significant antitumor effects. Using the nude mouse xenograft model, we investigated the efficacy of NM. We also tested the effect of NM in vitro, evaluating cell viability, secretion of the matrix metalloproteinases (MMP)-2 and MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 secretion, Matrigel invasion and cellular apoptosis and morphology. Athymic nude mice 5-6 weeks of age were inoculated with 3x10⁶ SK-N-MC neuroblastoma cells subcutaneously and randomly divided into two groups. Group A was fed a regular diet and group B a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. We also tested the effect of NM in vitro. NM inhibited the growth of xenograft tumors by 22% (P=0.04); and, in vitro, NM induced dose-dependent inhibition of cell proliferation with a decrease of 27% (P=0.001) and 36% (P=0.002) at 500 and 1000 µg/ml NM compared to the control, respectively. Zymography revealed MMP-2 secretion in normal cells and PMA (100 ng/ml)‑induced MMP-9 secretion. NM inhibited the secretion of both MMPs with total blockage at a concentration of 100 µg/ml. Reverse zymography demonstrated a dose-dependent increase in TIMP-2 expression by NM. Notable, SK-N-MC human neuroblastoma cells were not invasive through Matrigel. NM induced dose-dependent apoptosis of SK-N-MC cells. The results suggest that NM may have therapeutic potential in treating neuroblastoma.

Topics: Animals; Apoptosis; Ascorbic Acid; Cell Line, Tumor; Cell Survival; Dietary Supplements; Gelatinases; Humans; Lysine; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neuroblastoma; Plant Extracts; Proline; Random Allocation; Tea; Tissue Inhibitor of Metalloproteinase-2; Xenograft Model Antitumor Assays

Ascorbic acid enhances synthesis of norepinephrine from dopamine in adrenal chromaffin cells by serving as a co-factor for chromaffin granule dopamine β-hydroxylase (DβH). However, there is controversy regarding in situ kinetics of the ascorbate effect in chromaffin cells, as well as whether they apply to neuronal cells. In this study we evaluated the stimulation of norepinephrine synthesis from dopamine in cultured SH-SY5Y neuroblastoma cells. These cells contained neither ascorbate nor norepinephrine in culture, but when provided with dopamine, they generated intracellular norepinephrine at rates that were stimulated several-fold by intracellular ascorbate. Ascorbate-induced increases in norepinephrine synthesis in dopamine-treated cells were linear over 60 min, despite saturation of intracellular ascorbate. Norepinephrine accumulation after 60 min of incubation with 100 μM dopamine was half-maximal at intracellular ascorbate concentrations of 0.2-0.5 mM, which fits well with the literature K(m) for ascorbate of DβH using dopamine as a substrate. Moreover, these ascorbate concentrations were generated by initial extracellular ascorbate concentrations of less than 25 μM due to concentrative accumulation by the ascorbate transporter. Treatment with 100 μM dopamine acutely increased cellular superoxide generation, which was prevented by ascorbate loading, but associated with a decrease in intracellular ascorbate when the latter was present at concentrations under 1 mM. These results show that ascorbate promptly enhances norepinephrine synthesis from dopamine by neuronal cells that it does so at physiologic intracellular concentrations in accord with the kinetics of DβH, and that it both protects cells from superoxide and by providing electrons to DβH.

Topics: Analysis of Variance; Antioxidants; Ascorbic Acid; Cell Line, Tumor; Dehydroascorbic Acid; Dopamine; Dopamine beta-Hydroxylase; Dose-Response Relationship, Drug; Extracellular Fluid; Glutathione; Humans; Neuroblastoma; Neurons; Norepinephrine; Superoxides

Ascorbic acid is one of the antioxidant compounds widely used against free radical stress. The present study was undertaken to examine whether ascorbic acid and hydrogen peroxide (H2O2), alone or in combination, could influence cell viability. The murine neuroblastoma cell line, N2a, was used to perform a dose response curve for ascorbic acid. It was observed that ascorbic acid alone at physiological concentrations (0.1-0.4 mM) did not cause any cell death. However, at pharmacological concentrations (1-6 mM), ascorbic acid caused dose-dependent cell death. The lethal concentration at which 50% cells were killed (LC50) was determined to be approximately 3.141 mM ascorbic acid at 24 h. H2O2 up to 300 µM alone did not cause significant cell death. In the combined treatment, when the cells were treated with ascorbic acid at physiological concentrations (0.4 mM) and H2O2 at 400 µM, higher rates of cell death were observed compared to the cell death rates caused by either compound alone. Subsequent experiments revealed that cell death was partly mediated through the loss of total glutathione levels in the cells. These data suggest that the combination of ascorbic acid and H2O2 is disadvantageous for cancer cell survival. Further studies are required to ascertain the physiological significance of these observations.

Topics: Animals; Antineoplastic Agents; Apoptosis; Ascorbic Acid; Cell Line, Tumor; Glutathione; Hydrogen Peroxide; Mice; Neuroblastoma

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

Topics: Acetophenones; Analysis of Variance; Animals; Antidepressive Agents, Tricyclic; Antioxidants; Ascorbic Acid; Brain; Cell Line; Corticosterone; Depressive Disorder; Disease Models, Animal; Drug Administration Schedule; Gene Expression Regulation, Enzymologic; Green Fluorescent Proteins; Hindlimb Suspension; Hippocampus; Humans; Hydrogen Peroxide; Imipramine; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; NADPH Oxidases; Neuroblastoma; Neurons; Phosphoproteins; Reactive Oxygen Species; Restraint, Physical; RNA, Messenger; RNA, Small Interfering; Social Behavior; Superoxides; Swimming; Time Factors

By intravenous (but not oral) application of ascorbate, millimolar serum concentrations can be reached, which are preferentially cytotoxic to cancer cells. Cytotoxicity is mediated by transition metal-dependent generation of H(2)O(2) in the interstitial space. In this study, the sensitivity of neuroblastoma cells (Kelly, SK-N-SH) to ascorbate and H(2)O(2) and their defense mechanisms against H(2)O(2) were investigated. Since aerobic glycolysis (the Warburg effect) is a feature of many tumour cells, their glucose consumption and lactate production were monitored. Furthermore, synthesis and release of ferritin by neuroblastoma cells were analysed in order to examine whether ferritin is possibly an iron source for H(2)O(2) generation. Ascorbate (0.6-5.0 mM) and H(2)O(2) (25-100 muM) were found to be similarly cytotoxic to Kelly and SK-N-SH cells. In each case, cytotoxicity increased if cell concentrations decreased, in accordance with low cell concentrations having lower capacities to detoxify H(2)O(2). Kelly and SK-N-SH cells produced and released remarkable amounts of lactate and ferritin. We propose the selective cytotoxicity of high dose ascorbate to tumour cells to be due to the preferential generation of H(2)O(2) in the acidic and ferritin-rich tumour microenvironment, combined with reduced defense systems against H(2)O(2) as a consequence of aerobic glycolysis.

Topics: Ascorbic Acid; Cell Line, Tumor; Cytotoxins; Ferritins; Humans; Hydrogen Peroxide; Lactic Acid; Neuroblastoma

The small ubiquitin-like modifier (SUMO) proteins have been implicated in the pathology of a number of diseases, including neurodegenerative diseases. The conjugation machinery for SUMOylation consists of a number of proteins which are redox sensitive. Here, under oxidative stress (100 μM hydrogen peroxide), antioxidant (100 μM ascorbate) or control conditions 169 proteins were identified by electrospray ionisation fourier transform ion cyclotron resonance mass spectrometry. The majority of these proteins (70%) were found to contain SUMOylation consensus sequences. From the remaining proteins a small number (12%) were found to contain possible SUMO interacting motifs. The proteins identified included DNA and RNA binding proteins, structural proteins and proteasomal proteins. Several of the proteins identified under oxidative stress conditions had previously been identified as SUMOylated proteins, thus validating the method presented.

Topics: Amino Acid Sequence; Antioxidants; Ascorbic Acid; Cell Line, Tumor; Humans; Hydrogen Peroxide; Mass Spectrometry; Neuroblastoma; Oxidants; Oxidative Stress; Proteins; Sumoylation

Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. As the brain does not synthesize AA, high levels are achieved in this organ by specific uptake mechanisms, which concentrate AA from the bloodstream to the CSF and from the CSF to the intracellular compartment. Two different isoforms of sodium-vitamin C co-transporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na(+)-dependent L-AA transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 was observed in the hippocampus and cortical neurons by in situ hybridization; however, there is no data regarding the expression and distribution of this transporter in the fetal brain. The expression of SVCT2 in embryonal mesencephalic neurons has been shown by RT-PCR suggesting an important role for vitamin C in dopaminergic neuronal differentiation. We analyze SVCT2 expression in human and rat developing brain by RT-PCR. Additionally, we study the normal localization of SVCT2 in rat fetal brain by immunohistochemistry and in situ hybridization demonstrating that SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. SVCT2 expression and function was also confirmed in neurons isolated from brain cortex and cerebellum. The kinetic parameters associated with the transport of AA in cultured neurons and neuroblastoma cell lines were also studied. We demonstrate two different affinity transport components for AA in these cells. Finally, we show the ability of different flavonoids to inhibit AA uptake in normal or immortalized neurons. Our data demonstrates that brain cortex and cerebellar stem cells, neurons and neuroblastoma cells express SVCT2. Dose-dependent inhibition analysis showed that quercetin inhibited AA transport in cortical neurons and Neuro2a cells.

Topics: Animals; Ascorbic Acid; Blotting, Western; Brain Neoplasms; Brain Stem; Cell Line, Tumor; Cerebellum; Cerebral Cortex; DNA, Complementary; Female; Flavonoids; Humans; Immunohistochemistry; In Situ Hybridization; Kinetics; Mice; Neuroblastoma; Neurons; Organic Anion Transporters, Sodium-Dependent; Pregnancy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Sodium; Sodium-Coupled Vitamin C Transporters; Symporters

Dopamine-induced neuronal cytotoxicity has been proposed as a leading pathological mechanism underlying many neuronal degenerative disorders including Parkinson disease. Various hypotheses have been proposed including oxidative stress and dopamine (DA)-induced intracellular signal disorder via DA D1 and D2 receptors. The exact mechanism involved in this process is far from clear. In this study, employing a neuronal blastoma cell line, SH-SY5Y, we tried to elucidate the roles of these different suggested mechanisms in this pathological process. The results showed that DA induced cell toxicity in a dose- and time-dependent way. Selective D1 and D2 DA receptor antagonist could not block the cytotoxic effects, whereas reductive reagent ascorbic acid but not GSH could effectively rescue the cell death, suggesting that DA-induced cell toxicity was caused by an extracellular oxidative stress. This was further supported by the enhancing effects of DA transporter blocker, GBR, which could increase the cell death when pretreated. Finally, ascorbic acid could also protect SY5Y cells from DA-induced cellular apoptotic signal changes including PARP and P53. Our studies suggested that DA exerted its cytotoxic effects via an extracellular metabolism, whereas intracellular transportation could reduce its oxidative stress. Cytotoxicity effects induced by extracellular DA could be protected by reductive agents as ascorbic acid. These results help to broaden our understanding of the mechanisms of DA-induced cell death and may provide potentially therapeutical alternative for the neurodegenerative disorders.

Topics: Apoptosis; Ascorbic Acid; Biological Transport, Active; Cell Death; Cell Line, Tumor; Cytotoxins; Dehydroascorbic Acid; Dopamine; Dopamine Antagonists; Dose-Response Relationship, Drug; Extracellular Space; Glutathione; Humans; Neuroblastoma; Neurons; Oxidative Stress; Signal Transduction; Time Factors

The uptake of ascorbate by neuroblastoma cells using a ruthenium oxide hexacyanoferrate (RuOHCF)-modified carbon fiber disc (CFD) microelectrode (r = 14.5 microm) was investigated. By use of the proposed electrochemical sensor the amperometric determination of ascorbate was performed at 0.0 V in minimum essential medium (MEM, pH = 7.2) with a limit of detection of 25 micromol L(-1). Under the optimum experimental conditions, no interference from MEM constituents and reduced glutathione (used to prevent the oxidation of ascorbate during the experiments) was noticed. The stability of the RuOHCF-modified electrode response was studied by measuring the sensitivity over an extended period of time (120 h), a decrease of around 10% being noticed at the end of the experiment. The rate of ascorbate uptake by control human neuroblastoma SH-SY5Y cells, and cells transfected with wild-type Cu,Zn-superoxide dismutase (SOD WT) or with a mutant typical of familial amyotrophic lateral sclerosis (SOD G93A), was in agreement with the level of oxidative stress in these cells. The usefulness of the RuOHCF-modified microelectrode for in vivo monitoring of ascorbate inside neuroblastoma cells was also demonstrated.

Topics: Ascorbic Acid; Biological Transport; Cell Line, Tumor; Electrochemistry; Ferrocyanides; Humans; Microelectrodes; Neuroblastoma; Oxidation-Reduction; Ruthenium

The mechanisms of catechol-induced cytotoxicity were studied in cultures of neuroblastoma N2a cells. The minimal cytotoxic concentration after 72 h was 20 micromol x l(-1). The EC50 after 72 h was 38 micromol x l(-1). There was not a correlation between the cytotoxicity and the formation of quinones in the medium. Catechol-induced cytotoxicity was increased significantly when superoxide dismutase (SOD) was added. The addition of catalase did not protect cells, but this enzyme reverted the deleterious effect of SOD. The experimental studies showed a detrimental effect of deferoxamine on catechol-induced cytotoxicity suggesting that cells need iron to maintain its metabolism. NF-kappaB inhibitors increased the cytotoxicity, suggesting that this factor is also important for cell viability. L-cysteine and N-acetyl-L-cysteine protected cells significantly in a dose-dependent manner. The use of monochlorobimane showed that catechol induced reduced glutathione (GSH) depletion after 24 h, prior to cell death. The mode of cell death was studied by flow cytometry after double staining with annexin V and propidium iodide. Catechol induced apoptosis after 72 h. Furthermore, catechol also induced nuclear fragmentation. These data showed that catechol-induced cytotoxicity to N2a cell was not directly a consequence of reactive oxygen species production. Rather, it was due to GSH depletion followed by the induction of apoptosis.

Topics: Animals; Antioxidants; Apoptosis; Ascorbic Acid; Catechols; Cell Line, Tumor; Cell Survival; Curcumin; Cysteine; Cytotoxins; Deferoxamine; Glutathione; Mice; Neuroblastoma; NF-kappa B; Reactive Oxygen Species; Sesquiterpenes; Superoxide Dismutase

The replacement of established evidence-based cancer therapy protocols (mainstream therapy) by unevaluated complementary and alternative medicine (CAM) is a challenge in pediatric oncology. We tested the hypothesis that oral application of L-lysine and ascorbic acid (Lysin C Drink) in combination with epigallocatechin-gallate (EGCG) and amino-acids (Epican forte) is effective in a preclinical model of neuroblastoma.. Primary tumors and spontaneous metastases were induced in A/J mice by injection of NXS2 neuroblastoma cells. Mice were treated by daily oral gavage with L-lysine and ascorbic acid (Lysin C Drink) (equivalent to 150 mg ascorbic acid/day/mouse) (treatment A) or with EGCG plus ascorbic- and amino-acids (Epican forte) (9.2 mg/mouse) (treatment B). Treatment A was started in the prophylactic setting (7 days before tumor cell injection) as well as in the therapeutic setting (1 day after tumor cell inoculation). Finally, treatment B was evaluated alone and in combination with treatment A in the therapeutic setting. The effect on primary tumor growth and the development of spontaneous liver metastases was evaluated.. L-lysine and ascorbic acid (Lysin C Drink) and EGCG plus ascorbic- and amino-acids (Epican forte) are ineffective in reduction of primary tumor growth and prevention of spontaneous liver metastases in this model.. Neither a formal clinical development nor the use of these substances can be recommended for neuroblastoma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Catechin; Dietary Supplements; Disease Models, Animal; Female; Liver Neoplasms, Experimental; Lysine; Mice; Mice, Inbred A; Neuroblastoma; Proline

Glutathione (GSH) depletion is widely used to sensitize cells to anticancer treatment inducing the progression of programmed cell death and overcoming chemoresistance. It has been reported that neuroblastoma cells with MYCN amplification are unable to start TRAIL-dependent death and MYCN, in concert with cytotoxic drugs, efficiently induces the mitochondrial pathway of apoptosis through oxidative mechanisms. In this study, we show that GSH loss induced by L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis, leads to overproduction of reactive oxygen species (ROS) and triggers apoptosis of MYCN-amplified neuroblastoma cells. BSO susceptibility of SK-N-BE-2C, a representative example of MYCN-amplified cells, has been attributed to stimulation of total SOD activity in the absence of changes in the level and the activity of catalase. Therefore, the unbalanced intracellular redox milieu has been demonstrated to be critical for the progression of neuroblastoma cell death that was efficiently prevented by antioxidants and rottlerin. These results describe a novel pathway of apoptosis dependent on ROS formation and PKC-delta activation and independent of p53, bcl-2, and bax levels; the selective redox modulation of PKC-delta might be suggested as a potential strategy for sensitizing MYCN-amplified cells to therapeutic approaches.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Ascorbic Acid; Benzopyrans; Biphenyl Compounds; Buthionine Sulfoximine; Calpain; Caspases; Catalase; Cell Line, Tumor; DNA Damage; Glutathione; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Onium Compounds; Protein Kinase C; Reactive Oxygen Species; Superoxide Dismutase; Superoxide Dismutase-1

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Catechin; Cell Growth Processes; Dietary Supplements; Liver Neoplasms, Experimental; Lysine; Mice; Neuroblastoma; Proline; Research Design

The mechanisms of leptin resistance observed in most cases of human obesity are poorly understood. Therefore, we evaluated the effects of nitric oxide (NO) on the leptin-induced activation of Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathways and on the leptin receptor (LEPR) expression using SH-SY5Y cells. Here, we show that the NO donor spermine/NONOate inhibited leptin-induced activation of STAT3 in vitro. The inhibition of leptin-mediated STAT3 phosphorylation caused by excessive NO was partially prevented by a sulfhydryl reducing agent, ascorbic acid. Cellular experiments show that reduced expression of long form leptin receptor (LEPR-b) and STAT3 protein instability induced by NO may be mechanisms of the NO-mediated inhibition of leptin-STAT3 signaling. We also present data showing that the hypothalamic NO content of high-fat (HF)-diet-induced obese mice was higher than that of control mice; this is likely caused by decreased caveolin-1 expression and increased nNOS expression induced by HF diet over 19 weeks. Concurrently with the overproduction of NO, the decrease of hypothalamic LEPR-b in obese mice also supports these in vitro data. Combined results suggest that excess of NO can induce the attenuation of leptin-mediated STAT3 activation through reduced expression of LEPR-b mRNA and instability of STAT3 protein at least in part. Furthermore, our in vivo data indicate that long-term HF diet induces hypothalamic overproduction of NO, which may be related with leptin insensitivity. However, further study is required to warrant direct in vivo evidence of a causal relationship between endogenous excess of hypothalamic NO and central leptin resistance.

Topics: Animals; Ascorbic Acid; Caveolin 1; Cell Line, Tumor; Dietary Fats; Down-Regulation; Drug Antagonism; Gene Expression; Humans; Hypothalamus; Leptin; Male; Mice; Mice, Inbred C57BL; Neuroblastoma; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitrites; Obesity; Phosphorylation; Receptors, Cell Surface; Receptors, Leptin; Spermine; STAT3 Transcription Factor

Among marine sessile organisms, sponges (Porifera) are the major producers of bioactive secondary metabolites that defend them against predators and competitors and are used to interfere with the pathogenesis of many human diseases. Some of these biological active metabolites are able to influence cell survival and death, modifying the activity of several enzymes involved in these cellular processes. These natural compounds show a potential anticancer activity but the mechanism of this action is largely unknown. In this study, we investigated the effects of two Mediterranean sponges, Agelas oroides and Petrosia ficiformis on the viability of human neuroblastoma cells. Upon treatment with the methanolic extract of Petrosia ficiformis, a marked cytotoxic effect was observed at any concentration or time of exposure. In contrast, a time- and dose-dependent effect was monitored for Agelas oroides that induced the development of apoptotic features and ROS production in LAN5 cells. These events were suppressed by calpeptin or zVAD and by vitamin C suggesting that the cell death caused by Agelas oroides was calpain- and caspase-dependent and of oxidative nature. Comet assay showed that this methanolic extract was not able to produce a genotoxic effect. Future studies will be applied to investigate the effect of isolated bioactive compounds from crude extract of this sponge which are potentially useful for cancer therapeutics.

Topics: Agelas; Animals; Ascorbic Acid; Cell Death; Cell Line, Tumor; Cell Survival; Comet Assay; Fluorescein-5-isothiocyanate; Neuroblastoma; Petrosia; Reactive Oxygen Species; Tissue Extracts

Pyrroloquinoline quinone (PQQ), which is an essential nutrient, has been shown to act as an antioxidant. Reactive oxygen species (ROS) are thought to be responsible for neurotoxicity caused by the neurotoxin 6-hydroxydopamine (6-OHDA). In this study, we investigated the ability of PQQ to protect against 6-OHDA-induced neurotoxicity using human neuroblastoma SH-SY5Y. When SH-SY5Y cells were exposed to 6-OHDA in the presence of PQQ, PQQ prevented 6-OHDA-induced cell death and DNA fragmentation. Flow cytometry analysis using the ROS-sensitive fluorescence probe, dihydroethidium, revealed that PQQ reduced elevation of 6-OHDA-induced intracellular ROS. In contrast to PQQ, antioxidant vitamins, ascorbic acid and alpha-tocopherol, had no protective effect. Moreover, we showed that PQQ effectively scavenged superoxide, compared to the antioxidant vitamins. Therefore, our results suggest the protective effect of PQQ on 6-OHDA-induced neurotoxicity is involved, at least in part, in its function as a scavenger of ROS, especially superoxide.

Topics: alpha-Tocopherol; Ascorbic Acid; Cell Line, Tumor; Cell Survival; Flow Cytometry; Free Radical Scavengers; Humans; Neuroblastoma; Neuroprotective Agents; Neurotoxins; Oxidopamine; PQQ Cofactor; Superoxides

Neuroblastoma (NB) is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred.. We could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC), further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells.. Sodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.

Topics: Annexin A5; Antioxidants; Apoptosis; Ascorbic Acid; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Humans; Iron; Membrane Potential, Mitochondrial; Neuroblastoma; Receptors, Transferrin; Trypan Blue

Inhibition of neuronal cyclooxygenase-2 (COX-2) and hence prostaglandin E2 (PGE2) synthesis by non-steroidal anti-inflammatory drugs has been suggested to protect neuronal cells in a variety of pathophysiological situations including Alzheimer's disease and ischemic stroke. Ascorbic acid (vitamin C) has also been shown to protect cerebral tissue in a variety of experimental conditions, which has been attributed to its antioxidant capacity. In the present study, we show that ascorbic acid dose-dependently inhibited interleukin-1beta (IL-1beta)-mediated PGE2 synthesis in the human neuronal cell line, SK-N-SH. Furthermore, in combination with aspirin, ascorbic acid augmented the inhibitory effect of aspirin on PGE2 synthesis. However, ascorbic acid had no synergistic effect along with other COX inhibitors (SC-58125 and indomethacin). The inhibition of IL-1beta-mediated PGE2 synthesis by ascorbic acid was not due to the inhibition of the expression of COX-2 or microsomal prostaglandin E synthase (mPGES-1). Rather, ascorbic acid dose-dependently (0.1-100 microM) produced a significant reduction in IL-1beta-mediated production of 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), a reliable indicator of free radical formation, suggesting that the effects of ascorbic acid on COX-2-mediated PGE2 biosynthesis may be the result of the maintenance of the neuronal redox status since COX activity is known to be enhanced by oxidative stress. Our results provide in vitro evidence that the neuroprotective effects of ascorbic acid may depend, at least in part, on its ability to reduce neuronal COX-2 activity and PGE2 synthesis, owing to its antioxidant properties. Further, these experiments suggest that a combination of aspirin with ascorbic acid constitutes a novel approach to render COX-2 more sensitive to inhibition by aspirin, allowing an anti-inflammatory therapy with lower doses of aspirin, thereby avoiding the side effects of the usually high dose aspirin treatment.

Topics: Antioxidants; Ascorbic Acid; Aspirin; Blotting, Western; Cell Line, Tumor; Cyclooxygenase 2; Dinoprost; Dinoprostone; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression; Humans; Interleukin-1; Neural Inhibition; Neuroblastoma; Neurons

Exit of recycling cholesterol from late endosomes is defective in Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.

Topics: Androstenes; Animals; Antioxidants; Ascorbic Acid; Cell Line; Enzyme Inhibitors; Fibroblasts; Heparan Sulfate Proteoglycans; Humans; Lung; Mice; Models, Biological; Neuroblastoma; Niemann-Pick Diseases; Nitric Oxide; Protein Structure, Secondary; RNA Interference; Suramin

beta-Amyloid causes apoptosis and death in cultured neurons that may be mediated by generation of reactive oxygen species. Since ascorbic acid concentrations are relatively high in brain, we tested whether and how this antioxidant might protect cultured SH-SY5Y neuroblastoma cells from apoptotic cell death. SH-SY5Y cells did not contain ascorbate in culture but readily took it up to achieve intracellular concentrations several-fold those of GSH. Treatment of cells with 2-10 microM beta-amyloid(25-35) decreased both intracellular ascorbate and GSH without affecting rates of ascorbate transport, which suggests that the peptide induces an oxidant stress in the cells. Overnight culture of cells with 10-20 microM beta-amyloid(25-35) induced apoptosis in SH-SY5Y cells when measured as externalization of phosphatidylserine by annexin V binding, as DNA fragmentation in the TUNEL assay, and as caspase-3 activity in cell lysates. Pre-loading cells with ascorbate substantially prevented apoptosis measured by these assays as well as cell death. In addition to preventing apoptosis, ascorbate loading of SH-SY5Y cells also decreased basal rates of generation of endogenous beta-amyloid. Together, these results support the notion that beta-amyloid induces apoptosis and death in neurons due to oxidant stress and suggest that intracellular ascorbate effectively prevents this toxicity.

Topics: Amyloid beta-Peptides; Apoptosis; Ascorbic Acid; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Neuroblastoma; Neuroprotective Agents

Neurons maintain relatively high intracellular concentrations of vitamin C, or ascorbic acid. In this work we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate, as well as how this system responds to oxidant stress induced by glutamate. Cultured SH-SY5Y neuroblastoma cells took up ascorbate, achieving steady-state intracellular concentrations of 6 mM and higher at extracellular concentrations of 200 microM and greater. This gradient was generated by relatively high affinity sodium-dependent ascorbate transport (Km of 113 microM). Ascorbate was also recycled from dehydroascorbate, the reduction of which was dependent on GSH, but not on D-glucose. Glutamate in concentrations up to 2 mM caused an acute concentration-dependent efflux of ascorbate from the cells, which was prevented by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Intracellular ascorbate did not affect radiolabeled glutamate uptake, showing absence of heteroexchange.

Topics: Ascorbic Acid; Biological Transport; Cell Line, Tumor; Glutamic Acid; Glutathione; Humans; Neuroblastoma

Oxidative stress and free radical production have been implicated in Alzheimer's disease, where low levels of the antioxidant vitamin C (ascorbate) have been shown to be associated with the disease. In this study, neuroblastoma SH-SY5Y cells were treated with hydrogen peroxide in the presence of ascorbate in order to elucidate the mechanism(s) of protection against oxidative stress afforded by ascorbate. Protein oxidation, glutathione levels, cell viability and the effects on the proteome and its oxidized counterpart were monitored. SH-SY5Y cells treated with ascorbate prior to co-incubation with peroxide showed increased viability in comparison to cells treated with peroxide alone. This dual treatment also caused an increase in protein carbonyl content and a decrease in glutathione levels within the cells. Proteins, extracted from SH-SY5Y cells that were treated with either ascorbate or peroxide alone or with ascorbate prior to peroxide, were separated by two-dimensional gel electrophoresis and analyzed for oxidation. Co-incubation for 24 hours decreased the number of oxidised proteins (e.g. acyl CoA oxidase 3) and induced brain derived neurotrophic factor (BDNF) expression. Enhanced expression of BDNF may contribute to the protective effects of ascorbate against oxidative stress in neuronal cells.

Topics: Antioxidants; Ascorbic Acid; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Electrophoresis, Gel, Two-Dimensional; Enzyme-Linked Immunosorbent Assay; Glutathione; Humans; Hydrogen Peroxide; Neuroblastoma; Oxidants; Oxidation-Reduction; Oxidative Stress; Proteins; Proteome

Treatment of SH-SY5Y human neuroblastoma cells with copper sulphate (50-300microM) in complete medium for 24h caused an increase in the level of the metal both in whole cells and in isolated mitoplasts. Toxic effects of copper resulted in the impairment of the capability of mitochondrial dehydrogenases to reduce a tetrazolium salt, and, to a lesser extent, in the loss of the integrity of the plasma membrane. The mechanism of toxicity involved the production of reactive oxygen species, amplified by the presence of ascorbate. Decreases in the levels of several mitochondrial proteins (subunits of complex I, complex V, and of the pyruvate dehydrogenase complex) were observed. These findings demonstrate that mitochondria are an early and susceptible target of copper-mediated oxidative stress in neuronal cells and support the hypothesis that mitochondrial damage triggers the neurodegenerative processes associated with copper overload in Wilson's disease.

Topics: Apoptosis; Ascorbic Acid; Cell Line, Tumor; Cell Survival; Copper; Humans; Mitochondria; Mitochondrial Proteins; Neuroblastoma; Reactive Oxygen Species

Interactions of the cell adhesion molecules are known to play important roles in mediating inflammation. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), activates the NF-kappaB signaling pathway, which induces the expression of various genes, such as intercellular adhesion molecule-1 (ICAM-1). In this study, the effect of vitamin C on the ICAM-1 expression induced by TNF-alpha in a human neuroblastoma cell line, SK-N-SH was investigated. Treatment with vitamin C resulted in the downregulation of the TNF-alpha-induced surface expression and ICAM-1 mRNA levels in a concentration-dependent manner. Moreover, a gel shift analysis indicated that vitamin C dose-dependently inhibited the NF-kappaB activation and IkappaBalpha degradation induced by TNF-alpha. Taken together, these results suggest that vitamin C downregulates TNF-alpha-induced ICAM-1 expression via the inhibition of NF-kappaB activation.

Topics: Antioxidants; Ascorbic Acid; Biotransformation; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Nucleus; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Humans; Indicators and Reagents; Intercellular Adhesion Molecule-1; Neuroblastoma; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha

Protein kinases C (PKCs) are a family of isoenzymes sensitive to oxidative modifications and involved in the transduction signal pathways that regulate cell growth. As such, they can act as cellular sensors able to intercept intracellular redox changes and promote the primary adaptive cell response. In this study, we have demonstrated that PKC isoforms are specifically influenced by the amount of intracellular glutathione (GSH). The greatest GSH depletion is associated with a maximal reactive oxygen species (ROS) production and accompanied by an increase in the activity of the delta isoform and a concomitant inactivation of alpha. ROS generation induced early morphological changes in GSH-depleted neuroblastoma cells characterized, at the intracellular level, by the modulation of PKC-delta activity that was involved in the pathway leading to apoptosis. When cells were pretreated with rottlerin, their survival was improved by the ability of this compound to inhibit the activity of PKC-delta and to counteract ROS production. These results define a novel role of PKC-delta in the cell signaling pathway triggered by GSH loss normally associated with many neurodegenerative diseases and clinically employed in the treatment of neuroblastoma.

Topics: Acetophenones; Antioxidants; Apoptosis; Ascorbic Acid; Benzopyrans; Buthionine Sulfoximine; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; Glutathione; Humans; Malondialdehyde; Neuroblastoma; Oxidation-Reduction; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured

We demonstrated that exposure of cells to 50 nM okadaic acid for 2 h induced a reduction in cellular glutathione transferase, glutathione reductase and catalase activity. Likewise, this acid prompted an increase in lipid peroxidation. Treatment of cells with 10(-5) M melatonin or 0.5 microg/ml vitamin C prevented the effects of okadaic acid. These results indicate that okadaic acid induces an oxidative stress imbalance, while melatonin and vitamin C prevent the oxidative stress induced by okadaic acid. Likewise, these data indicate the great importance of oxidative stress in both this experimental model and in the development and course of neurodegenerative disease, especially Alzheimer's disease. They show that melatonin is much more efficient than vitamin C in reducing the extent of oxidative stress. This phenomenon was demonstrated by the smaller dose of melatonin needed to obtain effects similar to those obtained with vitamin C on lipid peroxidation and by the protective effect of melatonin on antioxidant enzyme activity.

Topics: Alzheimer Disease; Animals; Ascorbic Acid; Catalase; Glutathione Transferase; Lipid Peroxidation; Melatonin; Mice; Neuroblastoma; Okadaic Acid; Oxidative Stress; Tumor Cells, Cultured

Dopamine (DA) metabolism and oxidation produce both reactive oxygen species (ROS) and reactive quinones. These chemical species are implicated in dopamine neurotoxicity and neurodegeneration. In the present studies, human neuroblastoma (SK-N-SH) cells were exposed to toxic concentrations of dopamine (333 microM) in order to investigate molecular pathways involved in dopamine toxicity. cDNA hybridization array (microarray) technology demonstrated that GADD45 and GADD153 (growth arrest and DNA-damage inducible) gene expression was increased in dopamine-treated cells (333 microM for 18 h). Subsequent Northern and Western blot analysis confirmed these changes in GADD45 and GADD153 gene expression. The antioxidant, ascorbic acid, significantly reduced the increase in GADD45 gene expression but did not significantly reduce GADD153 gene expression. Currently, the precise function of the GADD gene products is not known. It is known, however, that these genes are upregulated in response to stress to allow cells time to repair macromolecular damage. In the present case, GADD gene expression (manifested as increased mRNA and protein levels) preceded dopamine-induced cytotoxicity. It appears that dopamine, through the formation of reactive oxygen species and quinones, may damage cellular macromolecules to the point of inducing GADD gene expression. Other genes that displayed changes, but that have not been subjected to post-hoc confirmation, include clusterin (increased), ubiquitin (increased), CD27 ligand (increased), CD27BP (increased), and rac-PK-beta (decreased).

Topics: Ascorbic Acid; CCAAT-Enhancer-Binding Proteins; Cell Death; Dopamine; GADD45 Proteins; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Neuroblastoma; Protein Array Analysis; Protein Biosynthesis; Proteins; Transcription Factor CHOP; Transcription Factors; Tumor Cells, Cultured

24-Hydroxycholesterol, the main cholesterol elimination product of the brain is increased in serum of Alzheimer patients. This oxysterol behaves neurotoxic towards the human neuroblastoma cell line, SH-SY5Y. Here we demonstrate, that 24-hydroxycholesterol-induced neurotoxicity in differentiated SH-SY5Y cells was due to apoptosis, as indicated by DNA-fragmentation, caspase-3 activation and a decrease of the mitochondrial membrane potential. Free radicals were generated, resulting in the death of 75% of the cells within 48h; neurotoxicity in differentiated SH-SY5Y cells was partially prevented by physiological concentrations of vitamin E (50-100 microM) in that 75% of the cells survived. Physiological concentrations of estradiol-17beta (1-100nM) elicited a protective effect in differentiated cells, which was not significant; however, in undifferentiated cells a significant protection was noted by this steroid hormone. Vitamin C and melatonin did not prevent 24-hydroxycholesterol-induced neurotoxicity. These in vitro data support the in vivo observed beneficial effects reported as circumstantial evidence of vitamin E and estradiol-17beta treatment in the prevention and therapy of neurodegenerative disease.

Topics: Antioxidants; Ascorbic Acid; Brain; Caspase 3; Caspases; Estradiol; Free Radicals; Humans; Hydroxycholesterols; Melatonin; Membrane Potentials; Mitochondria; Necrosis; Neuroblastoma; Neurons; Oxidative Stress; Tumor Cells, Cultured; Vitamin E

The aim of this study was to investigate the effect of L-DOPA and glia-conditioned medium (GCM) on cell viability, tyrosine hydroxylase (TH) expression, dopamine (DA) metabolism and glutathione (GSH) levels of NB69 cells. L-DOPA (200 microM) induced differentiation of NB69 cells of more than 4 weeks in vitro, as shown by phase-contrast microscopy and TH immunocytochemistry, and decreased replication, as shown by 5-bromodeoxyuridine immunostaining. L-DOPA did not increase the number of necrotic or apoptotic cells, as shown by morphological features, Trypan Blue, lactate dehydrogenase activity, bis-benzimide staining and TUNEL assay. Furthermore, L-DOPA (200 microM) increased Bcl-xL protein expression. Incubation of cells with L-DOPA (50, 100, 200 microM) for 24 h resulted in an increase in TH protein levels (174, 196 and 212% versus control). Neither carbidopa, an inhibitor of L-aromatic amino acid decarboxylase enzyme, nor L-buthionine sulfoximine, which inhibits GSH synthesis, or ascorbic acid, an antioxidant, blocked the L-DOPA-induced effect on TH protein expression. L-DOPA (0, 50, 100 and 200 microM) plus GCM further increased the amount of TH protein (346, 446, 472 and 424%). L-DOPA (200 microM) increased TH protein levels to 132, 191 and 245% of controls after incubation for 24, 48 and 72 h. DA metabolism in NB69 cells was increased in cultures treated with either L-DOPA (200-300 microM) or GCM and these two agents had a synergistic effect on DA metabolism. In addition, L-DOPA (200 microM) or/and GCM-treated cells increased their GSH extracellular levels (223, 257, 300% of controls) after 48 h of treatment. The L-DOPA-induced increase of TH protein expression in NB69 cells was independent of DA production, free radicals and GSH up-regulation.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; bcl-X Protein; Buthionine Sulfoximine; Carbidopa; Cell Differentiation; Culture Media, Conditioned; Culture Media, Serum-Free; Dopamine; Dopamine Agents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutathione; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Levodopa; Neuroblastoma; Neuroglia; Neurons; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Dopamine (DA), while an essential neurotransmitter, is also a known neurotoxin that potentially plays an etiologic role in several neurodegenerative diseases. DA metabolism and oxidation readily produce reactive oxygen species (ROS) and DA can also be oxidized to a reactive quinone via spontaneous, enzyme-catalyzed or metal-enhanced reactions. A number of these reactions are cytotoxic, yet the precise mechanisms by which DA leads to cell death remain unknown. In this study, the neuroblastoma cell line, SK-N-SH, was utilized to examine DA toxicity under varying oxidant states. Cells pretreated with the glutathione (GSH)-depleting compound, L-buthionine sulfoximine (L-BSO), exhibited enhanced sensitivity to DA compared to controls (non-GSH-depleted cells). Furthermore, in cells pretreated with L-BSO, the addition of ascorbate (250 microM) afforded significant protection against DA-induced toxicity, while pyruvate (500 microM) had no protective effect. To further characterize the possibility that DA is associated with oxidative stress, additional studies were carried out with manganese (30 microM) as a pro-oxidant. Manganese and DA (200 microM), although not cytotoxic when individually administered to SK-N-SH cells, had a synergistic action on cytotoxicity. Finally, morphological and molecular markers of programmed cell death (apoptosis) were observed in cells treated with DA and L-BSO. These markers included membrane blebbing and internucleosomal DNA fragmentation. These results suggest that DA toxicity is tightly linked to intracellular oxidant/antioxidant levels, and that environmental factors, such as excessive Mn exposure, may modulate cellular sensitivity to DA.

Topics: Apoptosis; Ascorbic Acid; Biological Assay; Buthionine Sulfoximine; Cell Membrane; Chromatography, High Pressure Liquid; DNA Fragmentation; Dopamine; Drug Synergism; Glutathione; Humans; Manganese; Microscopy, Electron, Scanning; Neuroblastoma; Oxidative Stress; Pyruvic Acid; Tumor Cells, Cultured

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calcium-mobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH-SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild-type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS-transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1-linked FALS.

Topics: Animals; Ascorbic Acid; Calcineurin; Calcium; Dithiothreitol; Hippocampus; Humans; Mice; Mice, Transgenic; Motor Cortex; Motor Neuron Disease; Neuroblastoma; Oxidation-Reduction; Oxidative Stress; Recombinant Proteins; Spinal Cord; Superoxide Dismutase; Transfection; Tumor Cells, Cultured

6-Hydroxydopamine (6-OHDA) was used for ex vivo purging of bone marrow from neuroblastoma cells before autologous transplantation. However, this concept failed because of the rapid autoxidation of 6-OHDA, which leads to the generation of cytotoxic reactive oxygen species (ROS), mainly in the incubation medium before 6-OHDA can be incorporated by neuroblastoma cells.. We based our experiments on the theory that, in contrast, 6-fluorodopamine (6-FDA), which is slowly converted to 6-OHDA at neutral pH, is able to enter neuroblastoma cells via the noradrenaline transporter (NA-T). Therefore, most ROS are generated inside the target cells.. Small amounts of ascorbate prevent the extracellular conversion of 6-FDA to 6-OHDA without affecting its cytotoxicity, leading to an even more selective effect of 6-FDA.. We conclude that 6-FDA is a promising substance for selective destruction of NA-T-positive neuroblastoma cells.

Topics: Ascorbic Acid; Carrier Proteins; Dopamine; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Oxidopamine; Symporters; Tumor Cells, Cultured

Buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis and has been used to sensitize tumor cells to alkylating agents, but has minimal single-agent cytotoxicity for most cell types. We determined the cytotoxicity of BSO for 18 (12 MYCN amplified; 6 MYCN nonamplified) human neuroblastoma cell lines using DIMSCAN, a digital image microscopy cytotoxicity assay. D-L(R:S) BSO was highly cytotoxic (>3 logs of cell kill) for most neuroblastoma cell lines, with 17/18 cell lines having IC90 values (range 2. 1->1000 microM) below equivalent steady state plasma levels of L(R:S) BSO reported in adult human trials. Cell lines with genomic amplification of MYCN were more sensitive to BSO than MYCN nonamplified cell lines (P = 0.04). D-L(R:S) BSO (500 microM for 72 h) induced apoptosis as detected by DNA laddering, nuclear morphology, and TUNEL staining of DNA fragments using flow cytometry. Maximal cell killing occurred within 48 h and was antagonized byic value in neuroblastoma.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Buthionine Sulfoximine; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Gene Amplification; Genes, myc; Glutathione; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Neuroblastoma; Reactive Oxygen Species; Time Factors; Tumor Cells, Cultured; Vitamin E

Hydroxymethyl-glutaryl-CoA-reductase (HMG-CoA-reductase), the key enzyme for cholesterol synthesis and essential for the synthesis of the precursor for p21ras farnesylation, was inhibited in neuroblastoma cells by lovastatin or L-ascorbic acid. Both compounds inhibited clonogenic colony formation of neuroblastoma cells in soft agar. However, while the addition of mevalonate, the product of HMG-CoA-reductase, circumvented the inhibition by lovastatin it had no reversing effect on the inhibition by L-ascorbic acid. The role of reactive oxygen compounds generated by the degradation of catecholamines, and the pro-oxidative effects of L-ascorbic acid are discussed as mechanisms of action of L-ascorbic acid.

Topics: Antineoplastic Agents; Ascorbic Acid; Cell Division; Dose-Response Relationship, Drug; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mevalonic Acid; Neuroblastoma; Tumor Cells, Cultured; Tumor Stem Cell Assay

The kinetics of ascorbate (AscH ) and epinephrine (EP) oxidation in the presence of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ) were studied in 0.05 M phosphate buffer, pH 7.4, at 37 degrees C by using a Clark electrode and ESR techniques. UQ at nanomolar concentrations displayed a pronounced catalytic effect on AscH oxidation which exceeded that of all reported organic catalysts tested in this system. The process was accompanied by the intensive oxygen consumption and increase in the steady-state concentration of the ascorbyl radical Asc.-. The rate of oxygen consumption (R[OX]) was maximal at the moment of reagent mixing ((R[OX]0) and then reduced over a few minutes until a steady-state level ((R[OX])SS) was achieved. (R[OX])0 was found to be proportional to [UQ][AscH-] without regard to the concentrations of the individual reagents; (R[OX])SS was directly related to [UQ] at a given concentration of AscH-. The difference between (R[OX])0 and (R[OX])SS decreased as [AscH-] decreased. The presence of a lipid phase (sodium dodecylsulphate micelles) only moderately decreased UQ activity as a catalyst of AscH- oxidation. Adding micromolar concentrations of UQ induced the acceleration of EP autoxidation. The capability of UQ to catalyze the oxidation of EP exceeded by approximately 25 times that of adrenochrome, a quinoid product of EP oxidation. These catalytic properties of UQ allowed us to predict its pronounced cytotoxicity, especially in the presence of AscH- and to cells of the sympathetic nervous system which are rich in catecholamines. This possibility was confirmed by experiments with human neuroblastoma cells in culture. The capability of UQ to injure neuroblastoma cell line SK-N-SH exceeded that of well-known neurotoxic agents 6-hydroxydopamine and menadione.

Topics: Antineoplastic Agents; Ascorbic Acid; Benzoquinones; Epinephrine; Humans; Kinetics; Neuroblastoma; Oxidation-Reduction; Tumor Cells, Cultured

CuZn superoxide dismutase (SOD) secretion was detected in media of [35S]cysteine-labeled human neuroblastoma SK-N-BE cells precipitated with antihuman CuZn SOD antibodies. The ability of Fe2+/ascorbate oxidative stress to induce CuZn SOD in SK-N-BE cells was evaluated by Western blot analysis. The results showed that, like human hepatocarcinoma cells and human fibroblasts, SK-N-BE cells secrete CuZn SOD. In addition, the CuZn SOD concentration was higher in cells subjected to oxidative stress than in unstressed cells. The secretion of CuZn SOD and the ability of Fe2+/ascorbate to increase its protein content in SK-N-BE cells indicates that this enzyme protects the brain from damage induced by oxidative stress.

Topics: Ascorbic Acid; Ferrous Compounds; Free Radicals; Humans; Kinetics; L-Lactate Dehydrogenase; Malondialdehyde; Neuroblastoma; Oxidative Stress; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Tumor Cells, Cultured

Ascorbic acid is well known to induce noradrenaline synthesis in sympathetic nervous cells. In a series of experiments we found that incubation of the neuroblastoma cell line SK-N-SH with ascorbic acid (100-500 microM) for 2 h results in a significantly enhanced synthesis of 3,4-dihydroxyphenylalanine (DOPA) and dopamine. Additionally, cDNA-polymerase chain reaction (cDNA-PCR) analysis of relative mRNA levels corresponding to the enzymes involved in catecholamine synthesis revealed a 3-fold increase of tyrosine hydroxylase gene expression after 5 days of incubation with ascorbic acid (200 microM), whereas expression of dopamine-beta-hydroxylase was found to be unaltered. In summary the data give evidence that ascorbic acid leads to enhanced DOPA production in SK-N-SH cells by two different mechanisms: at the metabolic level after short-term incubation and by increasing the tyrosine hydroxylase gene expression after long-term incubation. Based on these data we suppose that enhancement of DOPA synthesis by ascorbic acid may be useful in the treatment of early Parkinson's disease.

Topics: Ascorbic Acid; Catecholamines; Chromatography, High Pressure Liquid; Dihydroxyphenylalanine; Dopamine beta-Hydroxylase; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Enhanced oxidative stress has been suggested to be involved in the degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease. The high turnover rate of dopamine and/or unsequestered dopamine may cause an increase of formation of hydrogen peroxide via either oxidative deamination of dopamine by monoamine oxidase or autoxidation. Hydrogen peroxide would be converted to more toxic hydroxyl free radicals. L-beta-3,4-Dihydroxyphenylalanine hydrochloride (L-DOPA), the most useful drug in the symptomatic treatment of Parkinson's disease, has been considered to possess deteriorating degenerative side-effects. The catecholaminergic neuroblastoma SH-SY5Y cells were chosen to investigate the cytotoxic effect of dopamine and L-DOPA. Both dopamine and L-DOPA were found to be cytotoxic towards SH-SY5Y cells. Such toxic effects were accompanied by an increase of oxidative stress in the cell cultures and could be reversed effectively by catalase and to a lesser extent by superoxide dismutase. The non-enzymatic antioxidants L-ascorbic acid, glutathione, N-acetyl-L-cysteine, but not (+)-alpha-tocopherol, also completely protected SH-SY5Y cells against the cytotoxic effects induced by dopamine and L-DOPA. Antioxidative factors, namely free radical scavengers (including N-tert-butyl-alpha-phenylnitrone, salicylic acid, and D-mannitol) and a strong iron chelator, deferoxamine, however, did not protect the SH-SY5Y cells against dopamine and L-DOPA. The generation of reactive oxygen species and the resulting enhanced oxidative stress was clearly involved in the dopamine- and L-DOPA-induced cytotoxic effects. Hydrogen peroxide played the most important role related to cytotoxicity of dopamine and L-DOPA.

Topics: Ascorbic Acid; Catalase; Cell Survival; Deferoxamine; Dopamine; Levodopa; Lipid Peroxidation; Neuroblastoma; Oxidative Stress; Superoxide Dismutase; Tumor Cells, Cultured; Vitamin E

The mobilization of iron from intracellular ferritin by ascorbic acid has been analysed in situ by electron paramagnetic resonance (EPR) spectroscopy. EPR enables a distinction between ferritins and other Fe(3+)-binding cellular components. The ordered iron core of ferritin gives rise to a resonance signal which can be observed only at temperatures above 50 K. In the present study we clearly demonstrate that ascorbic acid is capable of mobilizing iron from ferritin in the cellular system by reduction of the ferric ion core in neuroblastoma SK-N-SH cells. This mechanism may open new ways in the therapy of this hardly curable tumor in stage IV, especially in combination with some cytostatic drugs.

Topics: Ascorbic Acid; Cell Line; Electron Spin Resonance Spectroscopy; Ferritins; Hot Temperature; Humans; Iron; Kinetics; Neuroblastoma; Thermodynamics; Time Factors; Tumor Cells, Cultured

In this paper, we show that human neuroectodermal cells exposed to 1-5 mM hydrogen peroxide or 10 nM-1 mM ascorbate die by programmed cell death induced by oxidative stress. The cell death by peroxide occurs within 4 h and involves approximately 80% of B-mel melanoma cells, while ascorbate causes cell death of approximately 86% of B-mel cells within 24 h. SK-N-BE(2) neuroblastoma cells are more resistant, 32% and 43% cell death for peroxide and ascorbate, respectively. In all cases, cell death causes hypodiploic DNA staining, evaluated by flow cytometry. Both cell lines can efficiently metabolise ascorbate due to significant levels of NADH-dependent semidehydroascorbate reductase and glutathione-dependent dehydroascorbate reductase. The cell death observed suggests a pro-oxidant, rather than anti-oxidant, role for ascorbic acid at physiological concentrations under these experimental conditions.

Topics: Apoptosis; Ascorbic Acid; Dehydroascorbic Acid; DNA, Neoplasm; Humans; Hydrogen Peroxide; Melanoma; Neuroblastoma; Oxidative Stress; Oxidoreductases; Tumor Cells, Cultured

L-3,4-Dihydroxyphenylalanine (L-dopa) is toxic for human neuroblastoma cells NB69 and its toxicity is related to several mechanisms including quinone formation and enhanced production of free radicals related to the metabolism of dopamine via monoamine oxidase type B. We studied the effect of L-DOPA on activities of enzyme complexes in the electron transport chain (ETC) in homogenate preparations from the human neuroblastoma cell line NB69. As a preliminary step we compared the activity of ETC in cellular homogenates with that of purified mitochondria from NB69 cells and rat brain. Specific activities for complex I, complex II-III, and complex IV in NB69 cells were, respectively, 65, 96, and 32% of those in brain mitochondria. Complex I activity was inhibited in a dose-dependent way by 1-methyl-4-phenylpyridinium ion with an EC50 of approximately 150 microM. Treatment with 0.25 mM L-dopa for 5 days reduces complex IV activity to 74% of control values but does not change either complex I or citrate synthase. Ascorbic acid (1 mM), which protects NB69 cells from L-dopa-induced neurotoxicity, increases complex IV activity to 133% of the control and does not change other ETC complexes. Ascorbic acid also reverses L-dopa-induced reduction of complex IV activity in NB69 cells. This observation might indicate that the protection observed with ascorbic acid is related to complex IV activation. In vitro incubation with L-dopa (0.125-4 mM) for 2 min produced a dose-dependent reduction of complex IV without change in complex I and II-III activities.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Ascorbic Acid; Brain; Catecholamines; Electron Transport; Electron Transport Complex IV; Humans; Levodopa; NAD(P)H Dehydrogenase (Quinone); Neuroblastoma; Rats; Tumor Cells, Cultured

Human or mouse epidermal keratinocytes NHEK or Pam212 was less susceptible to ultraviolet (UV)-B irradiation than mouse neuroblastoma NAs1 cells in culture, undergoing apoptosis-like cell death as shown by cell fragmentation and cell membrane integrity disruption. UV susceptibility was appreciably reduced by the reactive oxygen species (ROS)-scavenger L-ascorbic acid-2-phosphate (Asc2P) endowed with long-lasting functions but not by L-ascorbic acid (Asc) for each cell type. DehydroAsc reduced UV susceptibility of Pam212 or NAs1 established cell lines but not of normal diploid NHEK cells destined to be thereafter submitted to cellular senescence. The susceptibility reduction may not be ascribed to extracellular Asc2P or DehAsc, which was removed by aspirating and/or rinsing upon irradiation after the intracellular channelyzer analysis and dead cell-specific DNA-intercalator ethidium homodimer/fluorometry, respectively. Thus, the three cell types differed in UV susceptibility partly because of their different ROS-scavenging abilities, which may be potently promoted by Asc2P or dehydroAsc but not Asc.

Topics: Animals; Ascorbic Acid; Cell Membrane; Dehydroascorbic Acid; Free Radical Scavengers; Humans; Keratinocytes; Mice; Neuroblastoma; Tumor Cells, Cultured; Ultraviolet Rays

Ascorbic acid at pharmacologically attainable concentrations effectively inhibited the growth of the catecholamine-positive neuroblastoma cell line SK-N-SH; it inhibited LS cells to a smaller extent and catecholamine-negative SK-N-LO cell growth least effectively. In all three cell lines high concentrations of H2O2 were found. Since ascorbic acid was shown to release iron from ferritin in vitro and to keep it in the reduced state, we suggested that it acted as a pro-oxidant in ferritin-rich neuroblastoma cells in the presence of H2O2 and Fe2+ (Fenton reaction), implying iron release from cellular ferritin. We show here that iron could be mobilized from cellular ferritin by 1 mM ascorbic acid in iron-59-preloaded SK-N-SH and LS cells, but not in SK-N-LO cells. In agreement with these results, DNA strand break formation by ascorbate was only observed in SK-N-SH and LS cells. In SK-N-LO cells, DNA strand breaks could be induced by a combination of 1 mM ascorbic acid and 100 microM H2O2. Since cell-damaging effects caused by chemotherapy further facilitate iron release from ferritin, we conclude that ascorbate could be a powerful enhancer of some cytostatic drugs in neuroblastoma therapy.

Topics: Ascorbic Acid; Catecholamines; Drug Synergism; Ferritins; Humans; Hydrogen Peroxide; Iron; Neuroblastoma; Tumor Cells, Cultured

Analytical capillary isotachophoresis was used to determine ascorbic acid (AA) in different matrices (cell-free system, neuroblastoma cell extracts and urine). The system for purging bone marrow of neuroblastoma cells, including 6-hydroxydopamine (6-OHDA) and AA, was analysed with regard to the interaction of AA with 6-OHDA and its autoxidation product, hydrogen peroxide. Furthermore, analyses concerning the uptake of AA into neuroblastoma cells as well as its excretion in urine after uptake of large amounts were carried out.

Topics: Ascorbic Acid; Cell Line; Chromatography, High Pressure Liquid; Electrochemistry; Electrophoresis; Humans; Hydrogen Peroxide; Neuroblastoma; Oxidopamine; Oxygen Consumption; Tumor Cells, Cultured

Levodopa, at concentrations of 0.25 x 10(-4) M or larger, is toxic for the human neuroblastoma cell NB69. Toxicity is associated with high levels of quinones, increased activity of complex II-III, and lack of changes of complex I of the mitochondrial respiratory chain. Deprenyl, which does not alter the production of quinones, has a partial protective effect. Tocopherol, 23 or 115 x 10(-6) M, lacks significant preventive effect on levodopa toxicity, but ascorbic acid, 10(-3) M, prevents levodopa toxicity and quinone formation. Deprenyl, 10(-4) M, provides additional protection in cultures treated with levodopa and ascorbic acid. Our results indicate that ascorbic acid and deprenyl prevent levodopa neurotoxicity by unrelated mechanisms. Both compounds should be considered as complementary drugs to test for slowing the progression of Parkinson's disease.

Topics: Ascorbic Acid; Brain; Brain Neoplasms; Catecholamines; Cell Line; Drug Interactions; Female; Humans; Levodopa; Male; Neuroblastoma; Parkinson Disease; Quinones; Selegiline

A commercially available enzyme immunoassay was used to determine ferritin content and subsequently the loading and release of iron from ferritin in neuroblastoma cells. LS cells were incubated with 59Fe for 24 h, lysed, and the cytoplasmic ferritin was bound to monoclonal antibodies coupled to globules. After determination of the ferritin content the same globules with bound radioactive ferritin were measured in a gamma-counter. To illustrate the applicability of this test system, increased iron loading of cellular ferritin could be demonstrated in cycloheximide-treated cells; furthermore, release of iron was documented after incubation of LS cells with a combination of 6-hydroxydopamine and ascorbate. The assay turned out to be a simple method for determination of changes in 59Fe content of ferritin in neuroblastoma cells.

Topics: Ascorbic Acid; Cycloheximide; Ferritins; Ferrous Compounds; Humans; Immunoenzyme Techniques; Iron; Iron Radioisotopes; Neuroblastoma; Oxidopamine; Tumor Cells, Cultured

Long-term results are presented of 28 patients who were diagnosed with neuroblastoma at more than 12 months of age and who received melphalan 180 mg/m2 (n = 6) or 240 mg/m2 (n = 22) to consolidate remissions of Stage IV disease or to control refractory disease. Twenty-four patients also received dianhydrogalactitol 180 to 240 mg/m2, and 11 received total body irradiation 450 to 600 cGy. Autologous bone marrow transplantation (ABMT) was performed with marrow that was unpurged (n = 2) or purged ex vivo (n = 26) with 6-hydroxydopamine (6-OHDA) 20 micrograms/ml plus ascorbate 200 micrograms/ml. The median time to an absolute neutrophil count of 500/microliters was 21 days and to self-sustaining platelet counts more than 20,000/microliters, 28 days. One patient required infusion of unpurged reserve marrow. Two groups of patients underwent ABMT: (1) 17 patients (Group I) who were in first remission a median of 7 months after diagnosis; and (2) 11 patients (Group II) who had refractory disease or were in second remission. For Group I, event-free survival was 29% at 12 months and 6% at 24 months post-ABMT. All Group II patients died of disease or ABMT-related toxicity. Overall, of the 28 patients, one is a long-term relapse-free survivor; five died of ABMT-related toxicity; ten patients with tumors present at ABMT had progressive disease within 6 months of ABMT; and 12 patients with no measurable disease at ABMT relapsed 4 to 32 months (median, 12) post-ABMT. Among the latter, six relapses involved the primary site, and six were restricted to distant sites. These results--in accord with the long-term outcome in other series--suggest that for neuroblastoma high-dose melphalan cannot be relied on to ablate residual disease or to salvage patients with refractory tumors. In addition, the pattern of relapse in several patients could be explained by infusion of incompletely purged autografts; this would support recent laboratory evidence that 6-OHDA/ascorbate is a suboptimal purging method.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Bone Marrow Transplantation; Child; Child, Preschool; Combined Modality Therapy; Cyclophosphamide; Cytarabine; Dianhydrogalactitol; Doxorubicin; Female; Fluorouracil; Humans; Hydroxydopamines; Hydroxyurea; Infant; Male; Melphalan; Neoplasm Staging; Neuroblastoma; Oxidopamine; Prognosis; Radiotherapy Dosage; Remission Induction; Survival Rate; Vincristine; Whole-Body Irradiation

Neuroblastoma cells accumulate ascorbic acid and iron. It was hypothesized that these features could be exploited for sensitizing neuroblastoma cells for therapy in combination with reactive oxygen intermediates. In the present study the effects of 6-hydroxydopamine (6-OHDA) and H2O2 on metabolic parameters critical for cell survival were investigated in cells with low and high ferritin content in the presence and absence of ascorbate. Human neuroblastoma SK-N-SH cells were pretreated with 100 microM FeSO4 and 10 microM desferrioxamine, respectively, for 24 h yielding cells with different ferritin contents. The effects of 6-OHDA and H2O2 (25 microM-250 microM) in the absence and presence of 1 mM ascorbic acid on DNA strand break formation, activation of poly(ADP-ribose) polymerase, and finally decrease in NAD+ and ATP concentration were investigated. All these parameters were influenced by 6-OHDA and H2O2 in a concentration-dependent manner in a similar way. The effects were most pronounced in ferritin-rich cells and in the presence of ascorbic acid. Using isolated CCC PM2 DNA, 6-OHDA and ascorbic acid caused strand breaks that were prevented in the presence of mannitol or desferrithiocine. H2O2-mediated strand breaks were observed only in the presence of ascorbic acid. Based on these data and data published by others a model explaining the deleterious effects of ascorbic acid on neuroblastoma cells is presented. It is suggested that continuous application of a high dosage of ascorbic acid might be a useful approach in neuroblastoma therapy.

Topics: Adenosine Triphosphate; Ascorbic Acid; DNA; DNA Damage; Humans; Hydrogen Peroxide; Iron; NAD; Neuroblastoma; Oxidopamine; Poly(ADP-ribose) Polymerases; Tumor Cells, Cultured

To develop an effective neuroblastoma (NB) purging condition, we have compared in vitro cytotoxicity of 6-hydroxydopamine (6-OHDA) and ascorbic acid, with 4-hydroperoxycyclophosphamide (4-HC) on three NB cell lines (SK-N-BE2, SMS-SAN, and LA-N-1) and also upon human hematopoietic stem cells. Our study included mixing NB cells with 20-fold excess of irradiated bone marrow buffy coat cells to simulate the borderline remission marrow. When NB cells were treated without marrow cells, all three NB cell lines were very sensitive to 6-OHDA; complete inhibition of SK-N-BE2 and SMS-SAN cells was achieved at 10 micrograms/ml, and greater than 4 log inhibition of LA-N-1 was observed at 100 micrograms/ml of 6-OHDA. Addition of marrow cells caused marked reduction of the 6-OHDA-induced cytotoxicity of NB cells, and under similar conditions, colony-forming units-granulocyte-macrophage (CFU-GM) growth was not inhibited significantly. In the absence of normal marrow cells, 60 minutes of treatment with 100 microM of 4-HC produced complete inhibition (greater than 4.5 log) of SK-N-BE2 and SMS-SAN cells, greater than 4 log inhibition of LA-N-1 cells, and 97% of CFU-GM. Addition of marrow cells reduced the cytotoxicity of 4-HC, and 100 microM of 4-HC produced 99.8% inhibition of LA-N-1 colony growth. Shortening incubation duration to 30 minutes resulted in further reduction of 4-HC cytotoxicity; 100 microM of 4-HC caused 98.3%, 45%, and 33% inhibition of LA-N-1 cells, marrow CFU-GM, and burst-forming units-erythrocytes (BFU-E), respectively. At 200 microM, complete inhibition (greater than 4 log) of LA-N-1 colony growth was noted, and 9.9% of CFU-GM and 9.3% of BFU-E growth was observed. These data favor the use of 4-HC for purging marrow of NB, cells in the clinical autologous marrow transplantation.

Topics: Antibodies, Monoclonal; Ascorbic Acid; Cyclophosphamide; Hematopoietic Stem Cells; Humans; Hydroxydopamines; Neuroblastoma; Oxidopamine; Tumor Cells, Cultured

Topics: Animals; Ascorbic Acid; Cyclic AMP; Drug Tolerance; Endorphins; Etorphine; Glioma; GTP Phosphohydrolases; Hybrid Cells; Mice; Morphine Dependence; Neuroblastoma; Tumor Cells, Cultured

The patterns of the cytolytic effects of 6-hydroxydopamine (6-OHDA), with/without ascorbate, on C-1300 and three other cloned mouse neuroblastoma cell lines (N1E-115, NS-20, N-18) were studied in vitro. The sensitivity to 6-OHDA differed and the three cloned cell lines were more sensitive than the wild type C-1300 cell line. Ascorbate synergistically potentiated the cytolytic effect of 6-OHDA to all four cell lines. The 6-OHDA cytotoxicity was eliminated by the addition of exogenous catalase but not by addition of other oxygen free radical scavengers, thereby suggesting that the hydrogen peroxide formed might influence the cells, extracellularly. In addition, the critical time for tumor cell lysis was the first 60 min of the reaction. The cytotoxicity induced by the unmasked cyclophosphamide, 4-hydroperoxycyclophosphamide, was synergistically enhanced in the presence of a nontoxic concentration of 6-OHDA and ascorbate. These data suggest that reactive oxygen intermediates may prove to be a good tool for destroying neuroblastoma cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Cell Line; Cisplatin; Clone Cells; Doxorubicin; Drug Evaluation, Preclinical; Drug Synergism; Female; Free Radicals; Hydrogen Peroxide; Hydroxydopamines; Mice; Mice, Inbred A; Neuroblastoma; Oxidopamine; Spleen; Time Factors; Vincristine

L-Ascorbic acid inhibits the growth of mouse neuroblastoma and human endometrial carcinoma cells at concentrations greater than 100 microM. Under the same concentrations used in cell culture study, normal human lung fibroblasts show less sensitivity to the antiproliferative effect of ascorbate than tumor cell lines. The antitumor activity of ascorbate can be greatly potentiated by the combination with copper ions or copper chelates. The exposure of normal and tumor cells to the mixtures of ascorbate and copper chelates, especially Cu2+-o-phenanthroline and Cu2+-2,9-dimethyl-o-phenanthroline complexes, resulted in the killing of a large proportion of cell populations whereas the organic ligand portion of metal complexes was much less toxic. These copper chelates in combination with ascorbate showed different degrees of DNA-scission activities which could not be correlated with their cytotoxicities in the cell culture study. It is suggested that the primary targets of these antiproliferative agents may be on the biological sites such as cell membrane other than DNA in the nucleus which has been commonly assumed as the critical target for most free radical-generating antitumor drugs.

Topics: Animals; Ascorbic Acid; Bacteriophage lambda; Cell Division; Cell Line; Chelating Agents; Copper; DNA, Viral; Female; Humans; Kinetics; Lung; Mice; Neuroblastoma; Uterine Neoplasms

6-Hydroxydopamine(6-OHDA), a specific neurotoxin against sympathetic nerve cells, is a drug already used for purging of bone marrow from neuroblastoma cells before autologous bone marrow transplantation. However, we could not detect significant differences in the toxicity of 6-OHDA against neuroblastoma and other tumor cells under the purging conditions clinically used. In contrast, bone marrow stem cells were much more resistant. The unspecific toxic effect of 6-OHDA is caused by H2O2 or H2O2-derived products which are generated by auto-oxidation in the incubation medium before a significant amount of 6-OHDA is taken up by the cells. Withdrawal of oxygen during the incubation period and subsequent incubation with an oxygen containing medium led to a more specific destruction of neuroblastoma cells which can take up 6-OHDA selectively.

Topics: Ascorbic Acid; Bone Marrow; Catalase; Cell Survival; Cells, Cultured; Dithionite; Humans; Hydrogen Peroxide; Hydroxydopamines; Neuroblastoma; Oxygen; Superoxide Dismutase; Superoxides

The effect of high concentrations of L-ascorbic acid on the in vivo and in vitro growth of human neuroblastoma has been investigated. Directly implemented into cell culture it decreased the DNA, RNA and protein synthesis, and mitosis of neuroblastoma cells, without affecting normal neuronal cells. In vivo treatment of young nude mice bearing human neuroblastoma with 500 mg/kg L-ascorbic acid for the first 10 days markedly inhibited the growth of tumor mass. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in tumor cells treated with 1 X 10(-4) M L-ascorbic acid for 2 h. DNA-DNA and DNA-protein cross links in cells treated with L-ascorbic acid were revealed by the proteinase potassium assay. The results indicated that L-ascorbic acid can be a very effective and selective agent against human neuroblastoma.

Topics: Ascorbic Acid; Cells, Cultured; DNA, Neoplasm; DNA, Single-Stranded; Humans; Neuroblastoma

Topics: Adenylyl Cyclases; Adrenergic alpha-Agonists; Animals; Ascorbic Acid; Brain; Cations; Cell Line; Cell Membrane; Cricetinae; Cricetulus; Enkephalins; Hybrid Cells; Mice; Narcotics; Neuroblastoma; Prostaglandins; Receptors, Serotonin

In NG108-15 hybrid cells ascorbate suppresses the delayed etorphine-induced compensatory increase in the levels of cyclic AMP. It has, however, no effect on the early response of the cells to etorphine, as manifested in a transient decrease in the levels of cyclic AMP.

Topics: Ascorbic Acid; Cyclic AMP; Etorphine; Glioma; Hybrid Cells; Morphinans; Neuroblastoma; Receptors, Opioid

6-Hydroxydopamine (6-OHDA) is a neurotoxin for catecholaminergic neurons and neuroblasts. Since frequent marrow involvement in neuroblastoma restricts the exploitation of stored autologous bone marrow for rescue postchemotherapy, the potential for tumor-specific in vitro specificity of 6-OHDA was studied. The cytotoxic effect of 6-OHDA on 12 human neuroblastoma cell lines was compared to the effect on nonneuroblastoma cell lines. Most neuroblastoma cell lines were very sensitive to 6-OHDA (average concentration killing 50% of cells, 22 microgram/ml; range, 2.8 to 65.4). Cells derived from catecholamine-producing tumors were more sensitive to 6-OHDA than were those from non-catecholamine producers. By contrast, human fibroblasts, lymphoblastoid cell lines, and normal marrow were relatively insensitive to 6-OHDA; the concentration needed to kill 50% of cells for most of these cells exceeded 100 microgram/ml. Leukemia cell lines and a rhabdomyosarcoma cell line were intermediate in sensitivity. Ascorbate and 6-OHDA were synergistic in toxicity for human neuroblastoma cells. Thus, in vitro addition of 6-OHDA and ascorbate was rapidly lethal for human neuroblastoma cells at concentrations which were minimally toxic for hematopoietic cells. This differential toxicity provides a possible means for selective destruction of neuroblastoma cells in bone marrow harvested for autologous transplantation.

Topics: Ascorbic Acid; Bone Marrow; Bone Marrow Transplantation; Catecholamines; Cell Line; Humans; Hydroxydopamines; Neuroblastoma; Transplantation, Autologous

Ascorbic acid causes concentration-dependent and time-dependent effects on [3H]-serotonin (3H-5HT) uptake into differentiated neuroblastoma N-2a cells. Preincubation of cells with ascorbic acid inhibits both passive diffusion and active transport of 3H-5HT (0.1 microM). The kinetic characteristics of the active uptake process change with ascorbic acid treatment, resulting in an increase in the Km from 0.27 microM to 3.0 microM and in the Vmax from 453 to 2369 fmol/min/10(6) cells. This inhibitory effect of ascorbic acid appears to be due to its reducing properties.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; Kinetics; Neuroblastoma; Serotonin; Temperature; Time Factors

Determination of LD50 values showed 3 human neuroblastoma cell lines to be 2-8-fold more sensitive to 6-hydroxydopamine (6-OHDA) than a mouse sarcoma cell line. Treatment of the cells with 6-OHDA and ascorbic acid decreased the cytotoxicity of 6-OHDA for the sarcoma cells and increased cytotoxicity for the 3 neuroblastoma cell lines.

Topics: Animals; Ascorbic Acid; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Drug Synergism; Humans; Hydroxydopamines; Mice; Neoplasms, Experimental; Neuroblastoma; Oxidopamine; Sarcoma

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Cell Transformation, Neoplastic; Cells, Cultured; Drug Synergism; Drug Therapy, Combination; Glioma; Mice; Neoplasms, Experimental; Neuroblastoma; Vincristine; Vitamin E

Topics: Animals; Ascorbic Acid; Cell Survival; Cells, Cultured; Glioma; Methylmercury Compounds; Mice; Neuroblastoma; Neurons; Vitamin E

Mouse neuroblastoma (NB) cells in culture were more sensitive to sodium L-ascorbate than were rat glioma cells by the criterion of growth inhibition (due to cell death and reduction in cell division). Sodium L-ascorbate at nonlethal concentrations potentiated the effect of 5-fluorouracil (FUra), x-irradiation, bleomycin, RO20-1724, prostaglandin E1, and sodium butyrate on NB cells but did not produce such an effect on glioma cells. Sodium L-ascorbate did not enhance the effect of vincristine, 6-thioguanine, or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) except at higher drug doses and it reduced the cytotoxic effect of methotrexate and 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) on NB cells. Sodium D-ascorbate produced effects similar to those produced by sodium L-ascorbate on NB cells. L-Ascorbic acid-2-sulfate (barium salt) affected neither the growth rate nor the effect of 5-FUra on NB cells. Glutathione, a reducing agent, was more toxic to NB cells in comparison to D- OR L-ascorbate; however, at a similar concentration it failed to potentiate the effect of 5-FUra on NB cells.

Topics: Animals; Antibiotics, Antineoplastic; Ascorbic Acid; Catalase; Cell Division; Cell Survival; Cells, Cultured; Drug Synergism; Fluorouracil; Glioma; Glutathione; L Cells; Mice; Neuroblastoma; Rats; X-Rays