Cultured Neuroblastoma Cells Research Articles

Forty Years of Sodium Channels: Structure, Function, Pharmacology, and Epilepsy.

Voltage-gated sodium channels initiate action potentials in brain neurons. In the 1970s, much was known about the function of sodium channels from measurements of ionic currents using the voltage clamp method, but there was no information about the sodium channel molecules themselves. As a postdoctoral fellow and staff scientist at the National Institutes of Health, I developed neurotoxins as molecular probes of sodium channels in cultured neuroblastoma cells. During those years, Bruce Ransom and I crossed paths as members of the laboratories of Marshall Nirenberg and Philip Nelson and shared insights about sodium channels in neuroblastoma cells from my work and electrical excitability and synaptic transmission in cultured spinal cord neurons from Bruce's pioneering electrophysiological studies. When I established my laboratory at the University of Washington in 1977, my colleagues and I used those neurotoxins to identify the protein subunits of sodium channels, purify them, and reconstitute their ion conductance activity in pure form. Subsequent studies identified the molecular basis for the main functions of sodium channels-voltage-dependent activation, rapid and selective ion conductance, and fast inactivation. Bruce Ransom and I re-connected in the 1990s, as ski buddies at the Winter Conference on Brain Research and as faculty colleagues at the University of Washington when Bruce became our founding Chair of Neurology and provided visionary leadership of that department. In the past decade my work on sodium channels has evolved into structural biology. Molecular modeling and X-ray crystallographic studies have given new views of sodium channel function at atomic resolution. Sodium channels are also the molecular targets for genetic diseases, including Dravet Syndrome, an intractable pediatric epilepsy disorder with major co-morbidities of cognitive deficit, autistic-like behaviors, and premature death that is caused by loss-of-function mutations in the brain sodium channel NaV1.1. Our work on a mouse genetic model of this disease has shown that its multi-faceted pathophysiology and co-morbidities derive from selective loss of electrical excitability and action potential firing in GABAergic inhibitory neurons, which disinhibits neural circuits throughout the brain and leads directly to the epilepsy, premature death and complex co-morbidities of this disease. It has been rewarding for me to use our developing knowledge of sodium channels to help understand the pathophysiology and to suggest potential therapeutic approaches for this devastating childhood disease.

Ginkgolide K attenuates neuronal injury after ischemic stroke by inhibiting mitochondrial fission and GSK-3β-dependent increases in mitochondrial membrane permeability.

Ginkgolide K (GK) belongs to the ginkgolide family of natural compounds found in Ginkgo biloba leaves, which have been used for centuries to treat cerebrovascular and cardiovascular diseases. We evaluated the protective effects of GK against neuronal apoptosis by assessing its ability to sustain mitochondrial integrity and function. Co-immunoprecipitation showed that Drp1 binding to GSK-3β was increased after an oxygen-glucose deprivation/reperfusion (OGD/R) insult in cultured neuroblastoma cells. This induced Drp1 and GSK-3β translocation to mitochondria and mitochondrial dysfunction, which was attenuated by GK. GK also reduced mitochondrial fission by increasing Drp1 phosphorylation at Ser637 and inhibiting mitochondrial Drp1 recruitment. In addition, GK exposure induced GSK-3β phosphorylation at Ser9 and enhanced the interaction between adenine nucleotide translocator (ANT) and p-GSK-3β. This interaction suppressed the interaction between ANT and cyclophilin D (CypD), which inhibited mitochondrial permeability transition pore (mPTP) opening. Similarly, suppression of mitochondrial fission by Mdivi-1 also inhibited GSK-3β-induced mPTP opening. Treating mice with GK prevented GSK-3β and Drp1 translocation to mitochondria and attenuated mitochondrial dysfunction after middle cerebral artery occlusion. We therefore propose that by inhibiting mitochondrial fission and attenuating mPTP opening, GK exerts neuroprotective effects that mitigate or prevent neuronal damage secondary to ischemic stroke.

Neuroprotective effects of phloretin and its glycosylated derivative on rotenone-induced toxicity in human SH-SY5Y neuronal-like cells.

Phloretin and phlorizin are the two strong natural antioxidants whose biological and pharmacological applications are rapidly growing in different human pathological conditions. The neuroprotective activity of the two flavonoids has been analyzed on cell culture of neuroblastoma cells. The neuroprotective activity of the two flavonoids has been analyzed on cell culture of neuroblastoma cells and evaluated by testing cell vitality, mitochondrial transmembrane potential and ROS production, antioxidant enzymes detection, activation of caspase 3, DNA damage, protein carbonylation, lipid peroxidation, and superoxide anion scavenging activity. Incubation of cells with rotenone caused cell death and significant increase in intracellular reactive oxygen species, activation of caspase 3, and variation in mitochondrial transmembrane potential. Although, rotenone exposure caused a significant increase of antioxidant enzymes, high levels of lipid peroxidation were also observed. Phloretin or phlorizin, at micromolar concentration, reduced rotenone-induced cell death by scavenging ability against superoxide anion radical, one of the main effectors of rotenone toxicity at level of mitochondrial respiratory chain complex I. Under our experimental conditions, a reduction of the intracellular ROS levels with consequent normalization of the aforementioned antioxidant enzymes occurred. Concomitantly, we observed the inhibition of caspase 3 activity and DNA damage. This study shows the promising neuroprotective ability of the two dihydrochalcones able to protect human differentiated neuroblastoma cells (commonly used as model of Parkinson's disease) from injury induced by rotenone, actively scavenging ROS, normalizing mitochondrial transmembrane potential and consequently avoiding energy depletion. © 2017 BioFactors, 43(4):549-557, 2017.

Didymin: an orally active citrus flavonoid for targeting neuroblastoma.

Neuroblastoma, a rapidly growing yet treatment responsive cancer, is the third most common cancer of children and the most common solid tumor in infants. Unfortunately, neuroblastoma that has lost p53 function often has a highly treatment-resistant phenotype leading to tragic outcomes. In the context of neuroblastoma, the functions of p53 and MYCN (which is amplified in ~25% of neuroblastomas) are integrally linked because they are mutually transcriptionally regulated, and because they together regulate the catalytic activity of RNA polymerases. Didymin is a citrus-derived natural compound that kills p53 wild-type as well as drug-resistant p53-mutant neuroblastoma cells in culture. In addition, orally administered didymin causes regression of neuroblastoma xenografts in mouse models, without toxicity to non-malignant cells, neural tissues, or neural stem cells. RKIP is a Raf-inhibitory protein that regulates MYCN activation, is transcriptionally upregulated by didymin, and appears to play a key role in the anti-neuroblastoma actions of didymin. In this review, we discuss how didymin overcomes drug-resistance in p53-mutant neuroblastoma through RKIP-mediated inhibition of MYCN and its effects on GRK2, PKCs, Let-7 micro-RNA, and clathrin-dependent endocytosis by Raf-dependent and -independent mechanisms. In addition, we will discuss studies supporting potential clinical impact and translation of didymin as a low cost, safe, and effective oral agent that could change the current treatment paradigm for refractory neuroblastoma.

Thiamine and lipophilic thiamine precursors protect against oxidative damage in cultured neuroblastoma cells

Thiamine and lipophilic thiamine precursors protect against oxidative damage in cultured neuroblastoma cells

Downregulation of bone morphogenetic protein receptor 2 promotes the development of neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In this study, we examined the expression of bone morphogenetic protein receptor 2 (BMPR2) in primary NB and adjacent non-tumor samples (adrenal gland). BMPR2 expression was significantly downregulated in NB tissues, particularly in high-grade NB, and was inversely related to the expression of the NB differentiation markers ferritin and enolase. The significance of the downregulation was further explored in cultured NB cells. While enforced expression of BMPR2 decreased cell proliferation and colony-forming activity, shRNA-mediated knockdown of BMPR2 led to increased cell growth and clonogenicity. In mice, NB cells harboring BMPR2 shRNA showed significantly increased tumorigenicity compared with control cells. We also performed a retrospective analysis of NB patients and identified a significant positive correlation between tumor BMPR2 expression and overall survival. These findings suggest that BMPR2 may play an important role in the development of NB.

Active Control of Dye Release for Neuronal Tracing Using PEDOT-PSS Coated Electrodes.

Penetrating neural probes comprising arrays of microelectrodes are commonly used to monitor local field potentials and multi-unit activity in animal brain over time frames of weeks. To correlate these recorded signals to specific tissue areas, histological analysis is performed after the experimental endpoint. Even if the lesion of the penetrating probe shaft can be observed, a precise reconstruction of the exact electrode positions is still challenging. To overcome these experimental difficulties, we developed a new concept, whereupon recording electrodes are coated with a poly (3, 4-ethylenedioxythiophene/ polystyrenesulfonate) (PEDOT/PSS)-based film. The conducting polymer acts as dye reservoir over several weeks and afterwards provides controlled delivery of neurotracers. This paper presents a recording electrode based on a PEDOT/PSS bilayer optimized for dye delivery and with reduced impedance. Controlled exchange of neurotracer dye is successfully demonstrated in vitro using spectrofluorometry and in neuroblastoma cell cultures. A second PEDOT/PSS capping layer on top of the dye reservoir lowers the passive leakage of dye by a factor of 6.4 and prevents a direct contact of the dye filled layer with the cells. Stability tests over four weeks demonstrate the electrochemical stability of the PEDOT coating, as well as retained functionality of the dye delivery system.

Copper brain protein protection against free radical-induced neuronal death: Survival ratio in SH-SY5Y neuroblastoma cell cultures

In Creutzfeldt Jakob, Alzheimer and Parkinson diseases, copper metalloproteins such as prion, amyloid protein precursor and α-synuclein are able to protect against free radicals by reduction from cupric Cu+2 to cupreous Cu+. In these pathologies, a regional copper (Cu) brain decrease correlated with an iron, zinc or manganese (Mn) increase has previously been observed, leading to local neuronal death and abnormal deposition of these metalloproteins in β-sheet structures. In this study we demonstrate the protective effect of Cu metalloproteins against deleterious free-radical effects. With neuroblastoma SH-SY5Y cell cultures, we show that bovine brain prion protein in Cu but not Mn form prevents free radical-induced neuronal death. The survival ratio of SH-SY5Y cells has been measured after UV irradiation (free radical production), when the incubating medium is supplemented with bovine brain homogenate in native, Cu or Mn forms. This ratio, about 28% without any addition or with bovine brain protein added in Mn form, increases by as much as 54.73% with addition to the culture medium of native bovine brain protein and by as much as 95.95% if the addition is carried out in cupric form. This protective effect of brain copper protein against free radical-induced neuronal death has been confirmed with Inductively Coupled Plasma Mass Spectrometry Mn and Cu measurement in bovine brain homogenates: respectively lower than detection limit and 9.01μg/g dry weight for native form; lower than detection limit and 825.85μg/g dry weight for Cu-supplemented form and 1.75 and 68.1μg/g dry weight in Mn-supplemented brain homogenate.

Abstract 581: Bone marrow-derived B-cell hybridomas from neuroblastoma patients generate antibodies that bind to patients’ own tumors

Abstract Today antibody therapy is considered to be one of the most important and successful strategies to treat a variety of cancers. For example, the addition of antibodies such as Herceptin and Avastin to a chemotherapy regimen has shown improved survival in the treatment of breast cancer and colorectal cancer, respectively. Main problems with this kind of therapy are that many patients are not candidates because their tumors do not overexpress the drug target and that patient develop resistance to the targeted drug. A method to rapidly develop different sets of therapeutic antibodies would greatly contribute to the field of targeted anticancer therapy. This work evaluated the feasibility of using residual clinical material from pediatric neuroblastoma patients to generate antibodies to autologous tumor. Neuroblastoma is the most common extracranial solid tumor in children, accounting for 8-10% of all childhood cancers. Most patients with neuroblastoma are young and commonly present with metastatic disease. Bone marrow aspirate from neuroblatoma patients was the source material for the mononuclear cells and the tumor cells used in present study. Tumor cells were cultured and xenograft tumors were produced in mice. Hybridomas were generated by electrofusion of stimulated bone marrow mononuclear cells with plasmacytoma P3×63.Ag8.653 under hypo-osmolar condition using Eppendorf Multiporator/Helix chamber. Following hypoxanthine-aminopterin-thymidine (HAT) selection and monoclonal distribution, the culture supernatants were assayed for immunoglobulin secretion by ELISA. The supernatants from the positive clones were evaluated by immunofluorescence microscopy for binding to cultured neuroblastoma cells and neuroblastoma xenograft tissue sections derived from the same patient from which the hybridomas were generated. The results demonstrated that multiple hybridomas of bone marrow mononuclear cells secreted monoclonal antibodies that bound autologous neuroblastoma cells. Further evaluation of the tumor-binding antibodies on a panel of normal human tissues showed no binding to most of the tissues in the panel. Successful outcome of these experiments demonstrate the feasibility of generating human monoclonal antibodies from residual marrow specimens that bind autologous neuroblastoma cells. However, it remains to be determined whether these antibodies are bioactive and whether this approach will be generally applicable in more patients with neuroblastoma. It may be concluded that the strategy described here, which exploits the cancer patient's own immune repertoire, has a great potential for neuroblastoma target discovery and developing antibodies with possible therapeutic and/or diagnostic utility in cancer. Citation Format: Girja S. Shukla, Giselle S. Sholler, Yujing Sun, Stephanie C. Pero, Chelsea L. Carman, Ping Zhao, David N. Krag. Bone marrow-derived B-cell hybridomas from neuroblastoma patients generate antibodies that bind to patients’ own tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 581.

Protective Effects of Thiotriazolinum and Mildronate Against Mercury Chloride Toxicity in Neuroblastoma Cell Culture

We studied the toxic action of mercury (II) chloride (10.9 μM) on cultured IMR-32 neuroblastoma cells and possible protective effects of the antioxidant thiotriazolinum and cardioprotector mildronate (concentrations of the former agent in the medium 0.01-10.0 mg/ml). Isolated addition of thiotriazolinum and a combination of thiotriazolinum + mildronate to the medium did not cause dramatic negative effects (number of dead cells at all concentrations used did not exceed 7–10% of the population under control conditions). Thiotriazolinum in concentrations of 0.1 and 0.01 mg/ml demonstrated a significant neuroprotective activity against the toxic action of mercury (II) chloride: the mean number of living cells under such conditions of culturing was, on average, about 83.0% vs 54.7% in the case where isolated action of HgCl2 was used. The mean number of living (morphologically unaffected) cells cultured in the medium containing 10.9 μM HgCl2 with the addition of mildronate + thiotriazolinum reached 87.0% of the control value taken as 100%. Therefore, under the toxic action of HgCl2 on cultured IMR-32 neuroblastoma cells, the combination of mildronate and thiotriazolinum demonstrated a significant protective effect, which was more intense than that of thiotriazolinum at its isolated application. Thiotriazolinum in concentrations of 0.001 to 0.1 mg/ml exerts rather moderate negative effects on the studied culture of neuroblastoma cells.

CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression.

Previous studies have indicated the important roles of MYCN in tumorigenesis and progression of neuroblastoma (NB), the most common extracranial solid tumor derived from neural crest in childhood. However, the regulatory mechanisms of MYCN expression in NB still remain largely unknown. In this study, through mining public microarray databases and analyzing the cis-regulatory elements and chromatin immunoprecipitation data sets, we identified CCCTC-binding factor (CTCF) as a crucial transcription factor facilitating the MYCN expression in NB. RNA immunoprecipitation, RNA electrophoretic mobility shift assay, RNA pull down and in vitro binding assay indicated the physical interaction between CTCF and MYCN opposite strand (MYCNOS), a natural noncoding RNA surrounding the MYNC promoter. Gain- and loss-of-function studies revealed that MYCNOS facilitated the recruitment of CTCF to its binding sites within the MYCN promoter to induce chromatin remodeling, resulting in enhanced MYCN levels and altered downstream gene expression, in cultured NB cell lines. CTCF cooperated with MYCNOS to suppress the differentiation and promote the growth, invasion and metastasis of NB cells in vitro and in vivo. In clinical NB tissues and cell lines, CTCF and MYCNOS were upregulated and positively correlated with MYCN expression. CTCF was an independent prognostic factor for unfavorable outcome of NB, and patients with high MYCNOS expression had lower survival probability. Taken together, these results demonstrate that CTCF cooperates with noncoding RNA MYCNOS to exhibit oncogenic activity that affects the aggressiveness and progression of NB through transcriptional upregulation of MYCN.

Autoimmune autonomic ganglionopathy: Bench to Bedside

Autoimmune autonomic ganglionopathy (AAG) presents as severe generalized sympathetic and parasympathetic autonomic failure. This disorder is also known as acute pandysautonomia or idiopathic subacute autonomic neuropathy. Themost disablingmanifestations are orthostatic hypotension (OH) and gastrointestinal dysmotility. An autoimmune pathogenesis was suggested by the subacute onset, frequent associationwith antecedent viral illness, occasional association with cancer or other autoimmune disorders, and spontaneous recovery in some patients. Many cases have a rapid onset, but others have a chronic progressive course. Clinical and experimental observations demonstrate that AAG is an antibody-mediated disorder. (1) AAG patients have high levels of serum antibodies specific for the neuronal AChR in autonomic ganglia. This ganglionic AChR mediates fast synaptic transmission in all autonomic ganglia. Ganglionic AChR antibodies found in AAG patients are distinct from muscle AChR antibodies found in patients with MG. (2) Ganglionic AChR antibody levels correlate with severity of autonomic deficits. Patients with high antibody levels have a combination of OH, dry eyes and dry mouth, Adies pupil, gastroparesis, and neurogenic bladder. (3) Induction of ganglionic AChR antibodies in rabbits by immunization leads to experimental AAG which reproduces many of the cardinal features of the human disease. (4) Passive transfer of ganglionic AChR antibodies tomice causes transient autonomic failure. In electrophysiological studies, ganglionic AChR antibodies produce a reduction in ganglionic AChR current in cultured neuroblastoma cells and inhibit synaptic transmission in isolated autonomic ganglia. AAG is an antibody-mediated channelopathy that produces a severe, but treatable, form of autonomic failure.

Oleuropein aglycone induces autophagy via the AMPK/mTOR signalling pathway: a mechanistic insight.

The healthy effects of plant polyphenols, some of which characterize the so-called Mediterranean diet, have been shown to arise from epigenetic and biological modifications resulting, among others, in autophagy stimulation. Our previous work highlighted the beneficial effects of oleuropein aglycone (OLE), the main polyphenol found in the extra virgin olive oil, against neurodegeneration both in cultured cells and in model organisms, focusing, in particular, autophagy activation. In this study we investigated more in depth the molecular and cellular mechanisms of autophagy induction by OLE using cultured neuroblastoma cells and an OLE-fed mouse model of amylod beta (Aβ) deposition. We found that OLE triggers autophagy in cultured cells through the Ca2+-CAMKKβ-AMPK axis. In particular, in these cells OLE induces a rapid release of Ca2+ from the SR stores which, in turn, activates CAMKKβ, with subsequent phosphorylation and activation of AMPK. The link between AMPK activation and mTOR inhibition was shown in the OLE-fed animal model in which we found that decreased phospho-mTOR immunoreactivity and phosphorylated mTOR substrate p70 S6K levels match enhanced phospho-AMPK levels, supporting the idea that autophagy activation by OLE proceeds through mTOR inhibition. Our results agree with those reported for other plant polyphenols, suggesting a shared molecular mechanism underlying the healthy effects of these substances against ageing, neurodegeneration, cancer, diabetes and other diseases implying autophagy dysfunction.

Association of Neuroprotective Effect of Di-O-Demethylcurcumin on Aβ25-35-Induced Neurotoxicity with Suppression of NF-κB and Activation of Nrf2.

Amyloid-β peptides (Aβ), a major component of senile plaques, play an important role in the development and progression of Alzheimer's disease. Several lines of evidence have demonstrated that Aβ-induced neuronal death is mediated by oxidative stress. The present study aimed to evaluate the potential involvement of di-O-demethylcurcumin, an analog of curcuminoid, on Aβ-induced neurotoxicity in culture neuroblastoma cells (SK-N-SH cells) through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and the suppression of nuclear factor-κB (NF-κB) signaling pathway and their downstream targets. The results showed that pretreatment with di-O-demethylcurcumin elevated cell viability and decreased the level of reactive oxygen species. Moreover, treatment with di-O-demethylcurcumin promoted the translocation of Nrf2 protein from the cytoplasm to the nucleus, increased the expression of Nrf2-ARE pathway-related downstream proteins including heme oxygenase (HO-1), NAD(P)H:quinone oxidoreductase 1 and glutamate-cysteine ligase catalytic subunit, and increased the activity of superoxide dismutase enzymes. On the other hand, di-O-demethylcurcumin suppressed the degradation of IκBα, translocation of the p65 subunit of NF-κB from cytoplasm to nucleus and thereby, attenuated the expression of inducible nitric oxide synthase protein and nitric oxide production. Taken together, these results suggest that neuroinflammatory effect of di-O-demethylcurcumin might potentially be due to inhibit NF-κB and activate Nrf2 signaling pathways induced by Aβ25-35.

Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles.

An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases.

Melatonin decreases brain apoptosis, oxidative stress, and CD200 expression and increased survival rate in mice infected by Venezuelan equine encephalitis virus.

Pro-inflammatory and oxidative events during brain Venezuelan equine encephalitis virus infection could lead to apoptosis and induce anti-inflammatory responses (increased expression of CD200). The aim of this study was to determine the effect of melatonin on brain apoptosis, oxidative stress, and CD200 molecule in mice and neuroblastoma cultures infected by Venezuelan equine encephalitis virus. Mice were infected with 10 median lethal doses (LD50) of Venezuelan equine encephalitis virus, treated with melatonin (500 µg/kg bw; three days before infection and during all experimental time) and sacrificed on days 1, 3, and 5 postinfection. Brain samples were obtained at those periods of time. In addition, infected neuroblastoma cell cultures (multiplicity of infection [MOI]: 1) were treated with 0, 0.1, 0.5, and 1 mM of melatonin and analyzed at 2, 4, and 6 h. CD200 and apoptosis expressions were analyzed by immunohistochemistry and TUNEL assay, respectively. Nitrites and malondialdehyde were determined by appropriate biochemical methods. Increased brain expression of apoptosis, nitrite, and malondialdehyde productions and CD200 of infected mice were found. Melatonin diminished those expressions. Similarly, high apoptosis expression and nitrite and malondialdehyde productions on infected neuroblastoma cultures were diminished by melatonin. Melatonin increased the survival rate (25%) in Venezuelan equine encephalitis virus-infected animals compared with untreated infected mice (0%). Neurological damage during brain Venezuelan equine encephalitis virus infection could be mediated by apoptosis and oxidative stress and CD200 molecule could be an important anti-inflammatory response. Melatonin could be beneficial reducing apoptosis and oxidative stress.

Melatonin, minocycline and ascorbic acid reduce oxidative stress and viral titers and increase survival rate in experimental Venezuelan equine encephalitis

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 (500μg/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, 2μM or AA: 25, 50, 75μM. 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.

Effect of sulfasalazine on human neuroblastoma: analysis of sepiapterin reductase (SPR) as a new therapeutic target.

BackgroundNeuroblastoma (NB) is an aggressive childhood malignancy in children up to 5 years of age. High-stage tumors frequently relapse even after aggressive multimodal treatment, and then show therapy resistance, typically resulting in patient death. New molecular-targeted compounds that effectively suppress tumor growth and prevent relapse with more efficacy are urgently needed. We and others previously showed that polyamines (PA) like spermidine and spermine are essential for NB tumorigenesis and that DFMO, an inhibitor of the key PA synthesis gene product ODC, is effective both in vitro and in vivo, securing its evaluation in NB clinical trials. To find additional compounds interfering with PA biosynthesis, we tested sulfasalazine (SSZ), an FDA-approved salicylate-based anti-inflammatory and immune-modulatory drug, recently identified to inhibit sepiapterin reductase (SPR). We earlier presented evidence for a physical interaction between ODC and SPR and we showed that RNAi-mediated knockdown of SPR expression significantly reduced native ODC enzyme activity and impeded NB cell proliferation.MethodsHuman NB mRNA expression datasets in the public domain were analyzed using the R2 platform. Cell viability, isobologram, and combination index analyses as a result of SSZ treatment with our without DFMO were carried out in NB cell cultures. Molecular protein-ligand docking was achieved using the GRAMM algorithm. Statistical analyses were performed with the Kruskal-Wallis test, 2log Pearson test, and Student’s t test.ResultsIn this study, we show the clinical relevance of SPR in human NB tumors. We found that high SPR expression is significantly correlated to unfavorable NB characteristics like high age at diagnosis, MYCN amplification, and high INSS stage. SSZ inhibits the growth of NB cells in vitro, presumably due to the inhibition of SPR as predicted by computational docking of SSZ into SPR. Importantly, the combination of SSZ with DFMO produces synergistic antiproliferative effects in vitro.ConclusionsThe results suggest the use of SSZ in combination with DFMO for further experiments, and possible prioritization as a novel therapy for the treatment of NB patients.

MiRNA-337-3p suppresses neuroblastoma progression by repressing the transcription of matrix metalloproteinase 14.

Recent evidence shows the emerging roles of endogenous microRNAs (miRNAs) in repressing gene transcription. However, the miRNAs inhibiting the transcription of matrix metalloproteinase 14 (MMP-14), a membrane-anchored MMP crucial for the tumorigenesis and aggressiveness, still remain largely unknown. In this study, through mining computational algorithm program and genome-wide Argonaute profiling dataset, we identified one binding site of miRNA-337-3p (miR-337-3p) within the MMP-14 promoter. We demonstrated that miR-337-3p was under-expressed and inversely correlated with MMP-14 expression in clinical specimens and cell lines of neuroblastoma (NB), the most common extracranial solid tumor in childhood. Patients with high miR-337-3p expression had greater survival probability. miR-337-3p suppressed the promoter activity, nascent transcription, and expression of MMP-14, resulting in decreased levels of vascular endothelial growth factor, in cultured NB cell lines. Mechanistically, miR-337-3p recognized its binding site and recruited Argonaute 2 to facilitate the enrichment of repressive epigenetic markers and decrease the binding of RNA polymerase II and specificity protein 1 on the MMP-14 promoter. Gain- and loss-of-function studies demonstrated that miR-337-3p suppressed the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. In addition, restoration of MMP-14 expression rescued the NB cells from changes in these biological features. Taken together, these data indicate that miR-337-3p directly binds the MMP-14 promoter to repress its transcription, thus suppressing the progression of NB.

MiRNA-584-5p exerts tumor suppressive functions in human neuroblastoma through repressing transcription of matrix metalloproteinase 14

Matrix metalloproteinase 14 (MMP-14) is a membrane-anchored MMP crucial for tumorigenesis and aggressiveness, and is highly expressed in neuroblastoma (NB), the most common extracranial solid tumor in childhood. Recent evidence shows the emerging roles of endogenous promoter-targeting microRNAs (miRNAs) in regulating gene transcription. However, the roles of miRNAs in the transcription of MMP-14 still remain largely unknown. In this study, through mining computational algorithm program and Argonaute-chromosome interaction dataset, we identified one binding site of miRNA-584-5p (miR-584-5p) within the MMP-14 promoter. In NB tissues, miR-584-5p was under-expressed and inversely correlated with MMP-14 expression, and was an independent prognostic factor for favorable outcome of patients. miR-584-5p precursor attenuated the expression of MMP-14 in a Dicer-dependent manner, resulting in decreased levels of vascular endothelial growth factor, in cultured NB cell lines. In addition, miR-584-5p suppressed the promoter activity of MMP-14, and mutation of miR-584-5p binding site abolished these effects. Mechanistically, miR-584-5p recruited Argonaute 2 to facilitate the enrichment of enhancer of zeste homolog 2, histone H3 lysine 27 trimethylation, and histone H3 lysine 9 dimethylation on MMP-14 promoter in NB cells, which was abolished by repressing the miR-584-5p-promoter interaction. Gain- and loss-of-function studies demonstrated that miR-584-5p suppressed the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Moreover, restoration of MMP-14 expression rescued the NB cells from changes in these biological features. Taken together, these results indicate that promoter-targeting miR-584-5p exerts tumor suppressive functions in NB through repressing the transcription of MMP-14.