Murine Neuroblastoma Neuro-2a Cells Research Articles

The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

Soloxolone Methyl Reduces the Stimulatory Effect of Leptin on the Aggressive Phenotype of Murine Neuro2a Neuroblastoma Cells via the MAPK/ERK1/2 Pathway.

Despite the proven tumorigenic effect of leptin on epithelial-derived cancers, its impact on the aggressiveness of neural crest-derived cancers, notably neuroblastoma, remains largely unexplored. In our study, for the first time, transcriptome analysis of neuroblastoma tissue demonstrated that the level of leptin is elevated in neuroblastoma patients along with the severity of the disease and is inversely correlated with patient survival. The treatment of murine Neuro2a neuroblastoma cells with leptin significantly stimulated their proliferation and motility and reduced cell adhesion, thus rendering the phenotype of neuroblastoma cells more aggressive. Given the proven efficacy of cyanoenone-bearing semisynthetic triterpenoids in inhibiting the growth of neuroblastoma and preventing obesity in vivo, the effect of soloxolone methyl (SM) on leptin-stimulated Neuro2a cells was further investigated. We found that SM effectively abolished leptin-induced proliferation of Neuro2a cells by inducing G1/S cell cycle arrest and restored their adhesiveness to extracellular matrix (ECM) proteins to near control levels through the upregulation of vimentin, zonula occludens protein 1 (ZO-1), cell adhesion molecule L1 (L1cam), and neural cell adhesion molecule 1 (Ncam1). Moreover, SM significantly suppressed the leptin-associated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and ribosomal protein S6 kinase A1 (p90RSK), which are key kinases that ensure the survival and proliferation of cancer cells. Further molecular modeling studies demonstrated that the inhibitory effect of SM on the mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathway can be mediated by its direct interaction with ERK2 and its upstream regulators, son of sevenless homolog 1 (SOS) and mitogen-activated protein kinase kinase 1 (MEK1). Taken together, our findings in murine Neuro2a cells provide novel evidence of the stimulatory effect of leptin on the aggressiveness of neuroblastoma, which requires further detailed studies in human neuroblastoma cells and relevant animal models. The obtained results indicate that SM can be considered a promising drug candidate capable of reducing the impact of adipokines on tumor progression.

Effects of Ethanol on Expression of Coding and Noncoding RNAs in Murine Neuroblastoma Neuro2a Cells.

Excessive use of alcohol can induce neurobiological and neuropathological alterations in the brain, including the hippocampus and forebrain, through changes in neurotransmitter systems, hormonal systems, and neuroimmune processes. We aimed to investigate the effects of ethanol on the expression of coding and noncoding RNAs in a brain-derived cell line exposed to ethanol. After exposing Neuro2a cells, a neuroblastoma cell line, to ethanol for 24 and 72 h, we observed cell proliferation and analyzed up- and downregulated mRNAs and long noncoding RNAs (lncRNAs) using total RNA-Seq technology. We validated the differential expression of some mRNAs and lncRNAs by RT-qPCR and analyzed the expression of Cebpd and Rnu3a through knock-down of Cebpd. Cell proliferation was significantly reduced in cells exposed to 100 mM ethanol for 72 h, with 1773 transcripts up- or downregulated by greater than three-fold in ethanol-treated cells compared to controls. Of these, 514 were identified as lncRNAs. Differentially expressed mRNAs and lncRNAs were mainly observed in cells exposed to ethanol for 72 h, in which Atm and Cnr1 decreased, but Trib3, Cebpd, and Spdef increased. On the other hand, lncRNAs Kcnq1ot1, Tug1, and Xist were changed by ethanol, and Rnu3a in particular was greatly increased by chronic ethanol treatment through inhibition of Cebpd. Our results increase the understanding of cellular and molecular mechanisms related to coding and noncoding RNAs in an in vitro model of acute and chronic exposure to ethanol.

CRISPR-Mediated Activation of αV Integrin Subtypes Promotes Neuronal Differentiation of Neuroblastoma Neuro2a Cells.

Neuronal differentiation is a complex process whose dysfunction can lead to brain disorders. The development of new tools to target specific steps in the neuronal differentiation process is of paramount importance for a better understanding of the molecular mechanisms involved, and ultimately for developing effective therapeutic strategies for neurodevelopmental disorders. Through their interactions with extracellular matrix proteins, the cell adhesion molecules of the integrin family play essential roles in the formation of functional neuronal circuits by regulating cell migration, neurite outgrowth, dendritic spine formation and synaptic plasticity. However, how different integrin receptors contribute to the successive phases of neuronal differentiation remains to be elucidated. Here, we implemented a CRISPR activation system to enhance the endogenous expression of specific integrin subunits in an in vitro model of neuronal differentiation, the murine neuroblastoma Neuro2a cell line. By combining CRISPR activation with morphological and RT-qPCR analyses, we show that integrins of the αV family are powerful inducers of neuronal differentiation. Further, we identify a subtype-specific role for αV integrins in controlling neurite outgrowth. While αVβ3 integrin initiates neuronal differentiation of Neuro2a cells under proliferative conditions, αVβ5 integrin appears responsible for promoting a complex arborization in cells already committed to differentiation. Interestingly, primary neurons exhibit a complementary expression pattern for β3 and β5 integrin subunits during development. Our findings reveal the existence of a developmental switch between αV integrin subtypes during differentiation and suggest that a timely controlled modulation of the expression of αV integrins by CRISPRa provides a means to promote neuronal differentiation.

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

Pinhole Closure Improves Spatial Resolution in Confocal Scanning Microscopy.

Confocal microscopy is a simple, super-resolution technique, which does not produce a marked increase in resolution compared to other advanced techniques, such as super-resolution nanoscopy. Here, we present a simple protocol to acquire "slightly, but easily resolved" images by pinhole closure (<1 airy unit) in a conventional confocal scanning microscope equipped with an avalanche photodiode, a detector with high sensitivity. We use murine neuroblastoma Neuro2a cells to demonstrate the image resolution obtained via this protocol without the use of any special software to enhance image quality.

Biologically Active Metabolites from the Marine Sediment-Derived Fungus Aspergillus flocculosus.

Four new compounds were isolated from the Vietnamese marine sediment-derived fungus Aspergillus flocculosus, one aspyrone-related polyketide aspilactonol G (2), one meroterpenoid 12-epi-aspertetranone D (4), two drimane derivatives (7,9), together with five known metabolites (1,3,5,6,8,10). The structures of compounds 1–10 were established by NMR and MS techniques. The absolute stereoconfigurations of compounds 1 and 2 were determined by a modified Mosher’s method. The absolute configurations of compounds 4 and 7 were established by a combination of analysis of ROESY data and coupling constants as well as biogenetic considerations. Compounds 7 and 8 exhibited cytotoxic activity toward human prostate cancer 22Rv1, human breast cancer MCF-7, and murine neuroblastoma Neuro-2a cells.

The Neuroprotective Role of the GM1 Oligosaccharide, II3Neu5Ac-Gg4, in Neuroblastoma Cells.

Recently, we demonstrated that the GM1 oligosaccharide, II3Neu5Ac-Gg4 (OligoGM1), administered to cultured murine Neuro2a neuroblastoma cells interacts with the NGF receptor TrkA, leading to the activation of the ERK1/2 downstream pathway and to cell differentiation. To understand how the activation of the TrkA pathway is able to trigger key biochemical signaling, we performed a proteomic analysis on Neuro2a cells treated with 50μM OligoGM1 for 24h. Over 3000 proteins were identified. Among these, 324 proteins were exclusively expressed in OligoGM1-treated cells. Interestingly, several proteins expressed only in OligoGM1-treated cells are involved in biochemical mechanisms with a neuroprotective potential, reflecting the GM1 neuroprotective effect. In addition, we found that the exogenous administration of OligoGM1 reduced the cellular oxidative stress in Neuro2a cells and conferred protection against MPTP neurotoxicity. These results confirm and reinforce the idea that the molecular mechanisms underlying the GM1 neurotrophic and neuroprotective effects depend on its oligosaccharide chain, suggesting the activation of a positive signaling starting at plasma membrane level.

Enhancement of biocompatibility on aligned electrospun poly(3‐hydroxybutyrate) scaffold immobilized with laminin towards murine neuroblastoma Neuro2a cell line and rat brain‐derived neural stem cells (mNSCs)

Electrospinning has been extensively used to construct tissue‐engineered scaffolds because of its ability to provide the fibrous scaffold with structurally analogous to the naturally occurring protein in the extracellular matrix of native tissues. In addition, the modification of scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation, and differentiation. In the present contribution, we prepared and investigated the potential used of aligned electrospun poly(3‐hydroxybutyrate) (PHB) scaffold immobilized with bioactive molecule to serve as nervous scaffold. Laminin was successfully immobilized on the surface using covalent binding between functional groups of modified scaffolds and protein. The ability to use for neural regeneration was evaluated in vitro towards murine neuroblastoma Neuro2a cell line and mouse brain‐derived neural stem cells. The surface modification with laminin immobilized on the PHB fibrous scaffolds supported the attachment and promoted the proliferation of Neuro2a very wells. Despite the good attachment and proliferation of Neuro2a and mouse brain‐derived neural stem cells were not able to proliferate on the neat PHB, hydrolyzed PHB and laminin immobilized on hydrolyzed PHB fibrous scaffold.

U6 snRNA expression prevents toxicity in TDP-43-knockdown cells.

Depletion of amyotrophic lateral sclerosis (ALS)-associated transactivation response (TAR) RNA/DNA-binding protein 43 kDa (TDP-43) alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs) as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function.

Tricholides A and B and Unnarmicin D: New Hybrid PKS-NRPS Macrocycles Isolated from an Environmental Collection of Trichodesmium thiebautii.

Bioassay-guided isolation of the lipophilic extract of Trichodesmium thiebautii bloom material led to the purification and structure characterization of two new hybrid polyketide-non-ribosomal peptide (PKS-NRPS) macrocyclic compounds, tricholides A and B (1 and 2). A third macrocyclic compound, unnarmicin D (3), was identified as a new depsipeptide in the unnarmicin family, given its structural similarity to the existing compounds in this group. The planar structures of 1–3 were determined using 1D and 2D NMR spectra and complementary spectroscopic and spectrometric procedures. The absolute configurations of the amino acid components of 1–3 were determined via acid hydrolysis, derivitization with Marfey’s reagent and HPLC-UV comparison to authentic amino acid standards. The absolute configuration of the 3-hydroxydodecanoic acid moiety in 3 was determined using a modified Mosher’s esterification procedure on a linear derivative of tricharmicin (4) and additionally by a comparison of 13C NMR shifts of 3 to known depsipeptides with β-hydroxy acid subunits. Tricholide B (2) showed moderate cytotoxicity to Neuro-2A murine neuroblastoma cells (EC50: 14.5 ± 6.2 μM).

Effect of the surface topography and chemistry of poly(3-hydroxybutyrate) substrates on cellular behavior of the murine neuroblastoma Neuro2a cell line

Interactions between cells and substrates play an important role in tissue development during the process of tissue regeneration. Substrates that mimic the surface topography and chemical composition of the extracellular matrix (ECM) lead to enhanced cellular interactions. Electrospinning can easily produce aligned fibrous substrates with an architecture that structurally resembles tissue ECM and can provide contact guidance during tissue regeneration. However, the sole use of substrate materials may not be sufficient for the treatment of damaged tissue due to a lack of biochemical guidance, which helps to promote cell adhesion and proliferation. In the present contribution, we evaluated the effect of the surface properties of various surface-modified electrospun fibrous and solution-cast film PHB substrates in vitro on the murine neuroblastoma Neuro2a cell line. A neat electrospun fibrous and a solution-cast PHB scaffolds were used as the internal control. The results from cell studies suggest that the laminin–PHB fibrous substrate provided better support for the attachment and proliferation of Neuro2a cells than the other substrates. The cellular viability increased from 116% for 4 h of cell seeding to 187% for 3 days of cell seeding. These results suggest that the surface topography and chemistry significantly impact the Neuro2a cell line. The introduction of contact guidance, such as that provided by the fiber diameter and alignment, and biochemical guidance, such as that achieved by the immobilization of adhesive proteins, enhanced cell attachment and proliferation. These results emphasize the importance of surface properties with respect to cellular behavior.

Lichen-derived compounds show potential for central nervous system therapeutics

BackgroundNatural products from lichens are widely investigated for their biological properties, yet their potential as central nervous system (CNS) therapeutic agents is less explored. PurposeThe present study investigated the neuroactive properties of selected lichen compounds (atranorin, perlatolic acid, physodic acid and usnic acid), for their neurotrophic, neurogenic and acetylcholine esterase (AChE) activities. MethodsNeurotrophic activity (neurite outgrowth) was determined using murine neuroblastoma Neuro2A cells. A MTT assay was performed to assess the cytotoxicity of compounds at optimum neurotrophic activity. Neuro2A cells treated with neurotrophic lichen compounds were used for RT-PCR to evaluate the induction of genes that code for the neurotrophic markers BDNF and NGF. Immunoblotting was used to assess acetyl H3 and H4 levels, the epigenetic markers associated with neurotrophic and/or neurogenic activity. The neurogenic property of the compounds was determined using murine hippocampal primary cultures. AChE inhibition activity was performed using a modified Ellman's esterase method. ResultsLichen compounds atranorin, perlatolic acid, physodic acid and (+)-usnic acid showed neurotrophic activity in a preliminary cell-based screening based on Neuro2A neurite outgrowth. Except for usnic acid, no cytotoxic effects were observed for the two depsides (atranorin and perlatolic acid) and the alkyl depsidone (physodic acid). Perlatolic acid appears to be promising, as it also exhibited AChE inhibition activity and potent proneurogenic activity. The neurotrophic lichen compounds (atranorin, perlatolic acid, physodic acid) modulated the gene expression of BDNF and NGF. In addition, perlatolic acid showed increased protein levels of acetyl H3 and H4 in Neuro2A cells. ConclusionThese lichen depsides and depsidones showed neuroactive properties in vitro (Neuro2A cells) and ex vivo (primary neural stem or progenitor cells), suggesting their potential to treat CNS disorders.

Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain)

Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1K98E,E145A,L169F with three substitutions in the VP1 protein—K98E, E145A and L169F—productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection.Emerging Microbes and Infections (2016) 5, e60; doi:10.1038/emi.2016.56; published online 22 June 2016

Ex vivo induction of antitumor DEC-205+ CD11c+ cells in a murine neuroblastoma model by co-stimulation with doxorubicin, lipopolysaccharide and interleukin-4.

The antigen-presenting capacity of specific cells and tumor immunogenicity involved in innate cellular immunity are important for initiating an antitumor response to advanced neuroblastoma. The present study was performed to establish a method of producing antigen-presenting cells that induced an immune response to murine neuroblastoma cells through culture with neuroblastoma cells that had undergone immunogenic cell death. Immunogenic death of neuro-2a murine neuroblastoma cells was induced by exposure to doxorubicin. Mouse bone marrow cells were cultured in medium containing granulocyte-macrophage colony-stimulating factor, followed by the addition of doxorubicin-treated neuro-2a cells to the culture with or without lipopolysaccharide (LPS) and/or interleukin-4. Subsequently, cluster of differentiation (CD) 8α+ lymphocytes were co-cultured with neuro-2a cells and the adherent bone marrow cells obtained by the above procedure to evaluate CD8α+ lymphocyte proliferation and interferon-γ production. Furthermore, the surface antigen profile of adherent bone marrow cells was analyzed by flow cytometry. When adherent bone marrow cells were treated with LPS and/or interleukin-4, followed by co-culture with CD8α+ lymphocytes and neuro-2a cells, interferon-γ production by the CD8α+ cells increased in response to anti-CD3/CD28 antibody stimulation. CD11c major histocompatibility complex II (MHC II) double-positive cells were increased among adherent cells derived from cultured bone marrow cells. These cells were positive for DEC-205, but not CD8α. These findings suggest that co-culture of bone marrow-derived cells with tumor cells (that have undergone immunogenic death by exposure to doxorubicin) plus stimulation by LPS and interleukin-4 induces antigen-presenting cells that can evoke an immune response to neuroblastoma. Bone marrow-derived DEC-205+ CD11c+ MHC II+ dendritic cells are key antigen-presenting cells in the induction of an immune response following phagocytosis of doxorubicin-treated neuroblastoma cells.

Systemic TNFα Gene Therapy Synergizes With Liposomal Doxorubicine in the Treatment of Metastatic Cancer

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

Modified polyethyleneimine with histidine–lysine short peptides as gene carrier

There are several strategies that can be utilized to improve transfection efficiency while reducing the cytotoxicity of polyethyleneimine (PEI) as a promising non-viral gene delivery vector. In this study, we evaluated the potential use of lysine-histidine (KH) peptides in modifying the PEI 10 kDa structure and enhancing its efficiency while maintaining low toxicity of PEI. PEI 10 kDa was modified with 6-bromohexanoic acid (alkyl) to increase its lipophilicity. Then, ethylenediamine (EDA) was attached to the carboxylic groups of PEI-hexanoate to restore the primary amines of PEI. Subsequently, six different KH short peptides were conjugated to PEIs and evaluated for the effect of the KH sequence on vector transfection efficiency and cytotoxicity. The transfection efficiency of PEI-peptides complexed with a luciferase reporter gene (pRLCMV) in Neuro-2A murine neuroblastoma cells showed that the PEI conjugated to KHHHKKHHHK peptide had a significantly higher rate of gene transfection efficiency in comparison with other KH peptides. This peptide was conjugated to PEI-alkyl and PEI-alkyl-EDA and significant improvement in efficiency with minimal cytotoxicity was observed. The results obtained suggest that the sequence and content of KH peptides will have a significant impact on the transfection efficiency of modified PEI 10 kDa.

A gastrin‐releasing peptide receptor antagonist stimulates Neuro2a neuroblastoma cell growth: Prevention by a histone deacetylase inhibitor

Gastrin-releasing peptide (GRP) acts as an autocrine growth factor for neuroblastoma and other types of cancer, and its cell-surface receptor, GRPR, is overexpressed in advanced-stage human neuroblastoma. GRPR knockdown and GRPR antagonism inhibit the growth of experimental neuroblastoma. Here we show that a GRPR antagonist promotes rather than inhibits the growth of neuroblastoma cells. The GRPR antagonist, RC-3095, at 0.1 nM inhibited, whereas at 100 nM stimulated proliferation of Neuro2a murine neuroblastoma cells in vitro. The stimulatory effects were prevented by the histone deacetylase inhibitor (HDACi), sodium butyrate (NaB). Expression of GRPR mRNA in Neuro2a cells was analyzed by RT-PCR. These findings provide evidence that a GRPR antagonist can stimulate the growth of cancer cells, and suggest that GRPR might interact with epigenetic mechanisms in regulating neuroblastoma cell growth.

F‐spondin plays a critical role in murine neuroblastoma survival by maintaining IL‐6 expression

F-spondin is associated with the regulation of axonal growth and the development of the nervous system. Its mechanism of action, however, is not clearly understood. In this study, we found that murine neuroblastoma Neuro-2a cells expressed a significant level of IL-6, but only trace amounts of IL-12, tumor necrosis factor alpha and nitric oxide. Knock-down of F-spondin mRNA in murine neuroblastoma NB41A3 and Neuro-2a cells using small interfering RNAs led to decreased IL-6 levels along with lower resistance to serum starvation and cytotoxic amyloid beta(1-42) (Abeta(1-42)) peptide. Restoring decline of F-spondin or IL-6 induced by F-spondin knock-down through adding exogenous F-spondin, IL-6 or over-expressing F-spondin reversed the cell death induced by Abeta(1-42) peptide or serum starvation. The decrease of IL-6 level was positively correlated with decrease of NF-kappaB and inhibition of p38 mitogen-activated protein kinase (MAPK). Over-expressing MEKK, a kinase activator of the p38 MAPK pathway, increased IL-6 production, restored the decrease of p38 induced by F-spondin knock-down, and rescued the cells from death caused by Abeta(1-42) peptide. Taken together, these results suggest that F-spondin may play a critical role in murine neuroblastoma survival under adverse conditions by maintaining IL-6 level via a MEKK/p38 MAPK/NF-kappaB-dependent pathway.

Induction of Apoptosis in Murine Neuroblastoma by Systemic Delivery of Transferrin-Shielded siRNA Polyplexes for Downregulation of Ran

The polymer, OEI-HD, based on beta-propionamide-cross-linked oligoethylenimine and its chemical transferrin conjugate were evaluated for siRNA delivery into murine Neuro2A neuroblastoma cells in vitro and in vivo. An 80% silencing of luciferase expression in neuroblastoma cells, stably transfected with a luciferase gene, was obtained using standard OEI-HD polyplexes or transferrin-conjugated shielded OEI-HD polyplexes. The Ras-related nuclear protein Ran was selected as a therapeutically relevant target protein. Systemic delivery of transferrin-conjugated OEI-HD/RAN siRNA formulations (three intravenous applications at 3 days interval) resulted in >80% reduced Ran protein expression, apoptosis, and a reduced tumor growth in Neuro2A tumors of treated mice. The treatment was not associated with signs of acute toxicity or significant changes in weight, hematology parameters, or liver enzymes (AST, ALT, or AP) of mice. All our results demonstrate that OEI-HD/siRNA formulations can knockdown genes in tumor cells in vitro and in vivo in mice in the absence of unspecific toxicity.