Neuroblastoma Cell Line SH-SY5Y Research Articles

The Effects of Urolithin B and Auraptene on Quinolinic Acid-induced Toxicity in the SH-SY5Y Neuroblastoma Cell Line

The pathological accumulation of quinolinic acid (QA) is often associated with neuritis and neuronal cell death in several neurodegenerative diseases, through the overproduction of free radicals. Urolithin B and auraptene have been reported to exert potent antioxidant effects - however, little is known about the protective effects of these compounds against QA-induced neurotoxicity. Therefore, this study aimed to explore the in vitro protective effects of urolithin B and auraptene against QA-induced neurotoxicity in the SH-SY5Y neuroblastoma cell line. The MTT assay was used to evaluate cell viability, and flow cytometry was carried out to evaluate effects on the cell cycle and apoptosis. The intracellular levels of reactive oxygen species (ROS) were also determined. Our findings showed that auraptene at non-toxic concentrations had no protective effect on QA-induced toxicity. However, urolithin B at concentrations of 0.6 μM and 2.5 μM enhanced the viability of cells treated with QA. Moreover, while the percentage of apoptotic cells (i.e. in the sub-G1 phase) was shown to significantly increase after QA treatment, pre-treatment with urolithin B reduced the number of these apoptotic cells. Furthermore, urolithin B, as an antioxidant, also significantly reduced QA-induced ROS production. Our findings suggest that urolithin B may possess potent antioxidant and neuroprotective effects against QA-induced neurotoxicity that merit further investigation.

Artemisinin Alleviates Cerebral Ischemia/Reperfusion-Induced Oxidative Damage via Regulating PHB2-Mediated Autophagy in the Human Neuroblastoma SH-SY5Y Cell Line.

Oxidative stress plays a key role in cerebral ischemia/reperfusion injury. Artemisinin (ART) has antioxidative stress activity in addition to its powerful antimalarial effects. In this article, we investigated the effect of ART on OGD/R-induced oxidative stress injury and its underlying mechanisms. We used oxygen-glucose deprivation/reoxygenation (OGD/R) to establish an in vitro model of cerebral ischemia/reperfusion (I/R) injury. CCK-8 and lactate dehydrogenase (LDH) release were used to assess cellular damage. Measurement of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and mitochondrial membrane potential (MMP) estimates oxidative stress-induced damage and protection from ART effect. OGD/R treatment aggravated oxidative stress damage, whereas ART reversed the effects of OGD/R. Autophagy is closely related to oxidative stress; in order to confirm whether the antioxidative stress effect of ART is related to PHB2-mediated autophagy, we examined the protein expression of prohibitin 2 (PHB2), TOMM20, p62, and the conversion of microtubule-associated protein light chain 3I (LC3I) to LC3II and found that the protein expression of PHB2, TOMM20, p62, and LC3II/LC3I was significantly correlated with OGD/R treatment. The colocalization of PHB2 and LC3, TOMM20, and LC3 was reduced after OGD/R treatment, and ART reversed this change. After silencing PHB2, the protective effect of ART against OGD/R-induced oxidative stress injury was reduced, the protein expressions of PHB2, TOMM20 and LC3II/LC3I and the colocalization of PHB2 and LC3, TOMM20, and LC3 were decreased. We used chloroquine to block the lysosomal pathway and found that ART increased the conversion of LC3I to LC3II, silencing PHB2 which inhibited the conversion of LC3I to LC3II, and impaired mitophagy. Our findings showed that ART attenuated OGD/R-induced oxidative stress damage through PHB2-mediated mitophagy. To the current knowledge, our study is the first to demonstrate that ART attenuates OGD/R-induced oxidative stress injury through PHB2-mediated autophagy in the human neuroblastoma SH-SY5Y cell line, which provided new insights into the treatment of OGD/R injury.

Evaluation of the Cytotoxic Efficacy of Thymoquinone and Capsaicin in the SH-SY5Y Neuroblastoma Cell Line

In our study, it was aimed to examine the effects of thymoquinone, the active ingredient of Nigella sativa, which has known anticancer activities, and capsaicin, which is an important part of the endocannabinoid system, on the neuroblastoma cell line SH-SY5Y cells. SH-SY5Y cells were grown in culture in conventional culture flasks in DMEM medium at 37 °C and 5% CO2. When the cells were 70-80% confluent, morphological changes were examined under an inverted microscope. The cells were passaged into 96 microplates, and after passage, different concentrations of thymoquinone (2.5; 5; 10; 25; 50; 100; 200; 300 µM/ml) and capsaicin (0.675; 1.25; 2.5; 5; 10; 20; 50; 100 µM/ml) were applied to the cells. After administration, cytotoxic effect and proliferation rates/cell proliferation were analyzed by the MTT method. When compared to the control group, cultured cells treated with 200 and 300 μM thymoquinone and 5 and 100 μM capsaicin had reduced cell proliferation at statistically significant levels (p

Salsolinol Protects SH-SY5Y Cells Against MPP+ Damage and Increases Enteric S100-Immunoreactivity in Wistar Rats

A dopamine derivative, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, known as salsolinol (SAL), has increasingly gained attention since its first detection in the urine of Parkinson’s disease patients treated with levodopa, and has been proposed as a possible neurotoxic contributor to the disease. Yet, so far, the neurobiological role of SAL remains unclear. Thus, the main aims of our study were to compare the neurotoxic potential of SAL with MPP+ (1-methyl-4-phenylpyridinium ion) in vitro, and to examine intestinal and metabolic alterations following intraperitoneal SAL administration in vivo. In vitro, SH-SY5Y neuroblastoma cell line was monitored following MPP+ and SAL treatment. In vivo, Wistar rats were subjected to SAL administration by either osmotic intraperitoneal mini-pumps or a single intraperitoneal injection, and after two weeks, biochemical and morphological parameters were assessed. SH-SY5Y cells treated with MPP+ (1000 μM) and SAL (50 µM) showed increase in cell viability and fluorescence intensity in comparison with the cells treated with MPP+ alone. In vivo, we predominantly observed decreased collagen content in the submucosal layer, decreased neuronal density with comparable ganglionic area in the jejunal myenteric plexus, and increased glial S100 expression in both enteric plexuses, yet with no obvious signs of inflammation. Besides, glucose and triglycerides levels were lower after single SAL-treatment (200 mg/kg), and low- to high-density lipoprotein (LDL/HDL) ratio and aspartate to alanine aminotransferases (AST/ALT) ratio levels were higher after continuous SAL-treatment (200 mg/kg in total over 2 weeks). Low doses of SAL were non-toxic and exhibited pronounced neuroprotective properties against MPP+ in SH-SY5Y cell line, which supports the use of SAL as a reference compound for in vitro studies. In vivo results give insight into our understanding of gastrointestinal remodeling following intraperitoneal SAL administration, and might represent morphological correlates of a microglial-related enteric neurodegeneration and dopaminergic dysregulation.

Neuroprotective Effects of Neuropeptide Y on Human Neuroblastoma SH-SY5Y Cells in Glutamate Excitotoxicity and ER Stress Conditions.

Neuropeptide Y (NPY), a sympathetic neurotransmitter, is involved in various physiological functions, and its dysregulation is implicated in several neurodegenerative diseases. Glutamate excitotoxicity, endoplasmic reticulum (ER) stress, and oxidative stress are the common mechanisms associated with numerous neurodegenerative illnesses. The present study aimed to elucidate the protective effects of NPY against glutamate toxicity and tunicamycin-induced ER stress in the human neuroblastoma SH-SY5Y cell line. We exposed the SH-SY5Y cells to glutamate and tunicamycin for two different time points and analyzed the protective effects of NPY at different concentrations. The protective effects of NPY treatments were assessed by cell viability assay, and the signalling pathway changes were evaluated by biochemical techniques such as Western blotting and immunofluorescence assays. Our results showed that treatment of SH-SY5Y cells with NPY significantly increased the viability of the cells in both glutamate toxicity and ER stress conditions. NPY treatments significantly attenuated the glutamate-induced pro-apoptotic activation of ERK1/2 and JNK/BAD pathways. The protective effects of NPY were further evident against tunicamycin-induced ER stress. NPY treatments significantly suppressed the ER stress activation by downregulating BiP, phospho-eIF2α, and CHOP expression. In addition, NPY alleviated the Akt/FoxO3a pathway in acute oxidative conditions caused by glutamate and tunicamycin in SH-SY5Y cells. Our results demonstrated that NPY is neuroprotective against glutamate-induced cell toxicity and tunicamycin-induced ER stress through anti-apoptotic actions.

Transcriptome Analysis Reveals the Anti-Tumor Mechanism of Eucalyptol Treatment on Neuroblastoma Cell Line SH-SY5Y

Eucalyptol (1.8-cineole), an active component in traditional Chinese medicine Artemisia argyi for moxibustion. Previous studies have shown that eucalyptol has anti-tumor effects on leukemia and colon cancer. Nonetheless, the effect and mechanism of eucalyptol on neuroblastoma remains unclear. In the present study, we intended to reveal the effect and mechanism of eucalyptol treatment on the neuroblastoma cell line SH-SY5Y through transcriptome analysis. In the group treated with eucalyptol, 566 brain genes were up-regulated, while 757 genes were down-regulated. GO function analysis showed that positive regulation of cell cycle was down-regulated in biological processes. Meanwhile, cancer-related pathways were identified in KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis, including pathways in cancer, PI3K-Akt signaling pathway, cAMP signaling pathway, TGF-beta signaling pathway, Hippo signaling pathway, p53 signaling pathway, and additional pathways. Furthermore, we found a key gene, such as MYC, by constructing a network of cancer related pathways with differentially expressed genes and transcription factor analysis. In conclusion, our research indicates that MYC might play a central role in the anit-tumor mechanisms of eucalyptol.

Neuroprotective Effects of Lycium Barbarum Fruit Extract on Pink1B9Drosophila Melanogaster Genetic Model of Parkinson's Disease.

Lycium barbarum (LB) is a famous traditional Chinese medicinal plant as well as food supplement possessing various pharmacological functions such as anti-aging and antioxidant effects. The Parkinson's disease (PD)-related kinase Pink1 plays vital role in maintaining the neuron cell homeostasis, having been recognized as a potential target for the development of anti-PD drugs. In this work, the neuroprotective effects of methanol extract of LB fruit (LBFE) were investigated using a Drosophila PD model (PINK1B9) and a human neuroblastoma SH-SY5Y cell line. We found that when LBFE was supplied to the PINK1B9 flies at 6, 12, and 18 days of age, it raised the ATP and dopamine levels at all ages, extended life span, improved motor behavior, and rescued olfactory deficits of the PINK1B9 flies. In addition, histopathological examinations indicated that muscle atrophy in thoraces of the mutant flies was significantly repaired. Finally, LBFE was able to rescue the SH-SY5Y cells against MPP+-induced neurotoxicity. This work reports for the first time the anti-PD potential of L. barbarum fruit extract in PINK1 mutant fruit flies, presenting a new viewpoint for studing the mechanism of action of LBFE.

Phenotype and Neuronal Cytotoxic Function of Glioblastoma Extracellular Vesicles.

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. Extracellular vesicles (EVs) released by tumor cells play a critical role in cellular communication in the tumor microenvironment promoting tumor progression and invasion. We hypothesized that GBM EVs possess unique characteristics which exert effects on endogenous CNS cells including neurons, producing dose-dependent neuronal cytotoxicity. We purified EVs from the plasma of 20 GBM patients, 20 meningioma patients, and 21 healthy controls, and characterized EV phenotypes by electron microscopy, nanoparticle tracking analysis, protein concentration, and proteomics. We evaluated GBM EV functions by determining their cytotoxicity in primary neurons and the neuroblastoma cell line SH-SY5Y. In addition, we determined levels of IgG antibodies in the plasma in GBM (n = 82), MMA (n = 83), and controls (non-tumor CNS disorders and healthy donors, n = 50) with capture ELISA. We discovered that GBM plasma EVs are smaller in size and had no relationship between size and concentration. Importantly, GBM EVs purified from both plasma and tumor cell lines produced IgG-mediated, complement-dependent apoptosis and necrosis in primary human neurons, mouse brain slices, and neuroblastoma cells. The unique phenotype of GBM EVs may contribute to its neuronal cytotoxicity, providing insight into its role in tumor pathogenesis.

Alzheimer's Disease Prevention through Natural Compounds: Cell-Free, In Vitro, and In Vivo Dissection of Hop (Humulus lupulus L.) Multitarget Activity.

The relevant social and economic costs associated with aging and neurodegenerative diseases, particularly Alzheimer's disease (AD), entail considerable efforts to develop effective preventive and therapeutic strategies. The search for natural compounds, whose intake through diet can help prevent the main biochemical mechanisms responsible for AD onset, led us to screen hops, one of the main ingredients of beer. To explore the chemical variability of hops, we characterized four hop varieties, i.e., Cascade, Saaz, Tettnang, and Summit. We investigated the potential multitarget hop activity, in particular its ability to hinder Aβ1-42 peptide aggregation and cytotoxicity, its antioxidant properties, and its ability to enhance autophagy, promoting the clearance of misfolded and aggregated proteins in a human neuroblastoma SH-SY5Y cell line. Moreover, we provided evidence of in vivo hop efficacy using the transgenic CL2006Caenorhabditis elegans strain expressing the Aβ3-42 peptide. By combining cell-free and in vitro assays with nuclear magnetic resonance (NMR) and MS-based metabolomics, NMR molecular recognition studies, and atomic force microscopy, we identified feruloyl and p-coumaroylquinic acids flavan-3-ol glycosides and procyanidins as the main anti-Aβ components of hop.

A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

The human SH-SY5Y neuroblastoma cell line is widely used in neuroscience research as a neuronal cell model. Following differentiation to a neuron-like state, SH-SY5Y cells become more morphologically similar to neurons and form functional synapses. Previous studies have managed to differentiate SH-SY5Y cells towards cholinergic, dopaminergic and adrenergic fates. However, their application in disease modeling remains limited as other neuronal subtypes (e.g., glutamatergic, GABAergic) are also implicated in neurological disorders, and no current protocols exist to generate these subtypes of differentiated SH-SY5Y cells. Our study aimed to evaluate the use of a xeno-free version of B-27, a supplement commonly used in neuronal culture, for SH-SY5Y maintenance and differentiation. To evaluate the proliferative capacity of SH-SY5Y cells cultured in B-27, we performed growth curve analyses, immunocytochemical staining for Ki-67 and qRT-PCR to track changes in cell cycle progression. SH-SY5Y cells cultured in FBS or under serum-starved conditions were used as controls. We observed that SH-SY5Y cells show reduced growth and proliferation rates accompanied by decreased CDK6 and CDK1 expression following 4-day exposure to B-27, suggesting B-27 induces a quiescent state in SH-SY5Y cells. Importantly, this reduced growth rate was not due to increased apoptosis. As cell cycle exit is associated with differentiation, we next sought to determine the fate of SH-SY5Y cells cultured in B-27. B-27-cultured SH-SY5Y cells show changes in cell morphology, adopting pyramidal shapes and extending neurites, and upregulation of neuronal differentiation markers (GAP43, TUBB3, and SYP). B-27-cultured SH-SY5Y cells also show increased expression of glutamatergic markers (GLUL and GLS). These findings suggest that B-27 may be a non-toxic inducer of glutamatergic SH-SY5Y differentiation. Our study demonstrates a novel way of using B-27 to obtain populations of glutamatergic SH-SY5Y cells. As dysregulated glutamatergic signaling is associated with a variety of neuropsychiatric and neurodegenerative disorders, the capability to generate glutamatergic neuron-like SH-SY5Y cells creates endless disease modeling opportunities. The ease of SH-SY5Y culture allows researchers to generate large-scale cultures for high-throughput pharmacological or toxicity studies. Also compatible with the growing popularity of animal-component-free studies, this xeno-free B-27/SH-SY5Y culture system will be a valuable tool to boost the translational potential of preliminary studies requiring glutamatergic neuronal cells of human origin.

Anticancer activity, hTERT expression and telomere length analysis in SH-SY5Y neuroblastoma cell lines applied to docetaxel

Neuroblastoma is the most common extracranial solid tumor of infancy in a broad range of clinical courses, ranging from spontaneous regression to fatal progression. Telomere maintenance plays an important role in genome stability and cell proliferation. Telomerase reverse transcriptase (hTERT in humans) is a catalytic subunit of the enzyme telomerase. In this study, it was aimed to determine the anticancer activity of the docetaxel chemotherapeutic agent in neuroblastoma cell line (SH-SY5Y) and to investigate its effect on hTERT gene expression level and telomere length. Molecular docking studies were performed on docetaxel with the crystal structure of telomerase. The electronic properties of docetaxel were calculated using the density functional theory (DFT) method. SH-SY5Y cells were treated for 24, 48 and 72 h with specific concentrations of docetaxel drug ranging from 1 to 100 µg/ml. IC50 doses of docetaxel were determined and administered to SH-SY5Y cells, followed by RNA isolation. hTERT and MYC gene expression levels and telomere length were measured in the docetaxel-treated sample using the RT-PCR method. In addition, theoretical analyzes were made. The IC50 values of docetaxel after 24, 48 and 72 h were 8.32±1.45 μg/ml, 7.67±2.56 μg/ml and 5.51±1.24 μg/ml, respectively. According to the results obtained, docetaxel was found to have the highest activity in 72 h of incubation. It was determined that the docetaxel drug decreased the expression level of the hTERT gene in SH-SY5Y cells. Telomere lengths were significantly reduced in the docetaxel treated SH-SY5Y cell line compared to the control group (p < 0.05). Molecular docking analysis results were in agreement with the experiments. Analysis results indicated a good interaction between docetaxel and the active site of telomerase. The results of this study, reinforced by molecular docking analyzes, might be proved valuable for the development of potent telomerase inhibitors.

A Multifunctional Conjugated Polymer Developed as an Efficient System for Differentiation of SH-SY5Y Tumour Cells.

Polymer-based systems have been demonstrated in novel therapeutic and diagnostic (theranostic) treatments for cancer and other diseases. Polymers provide a useful scaffold to develop multifunctional nanosystems that combine various beneficial properties such as drug delivery, bioavailability, and photosensitivity. For example, to provide passive tumour targeting of small drug molecules, polymers have been used to modify and functionalise the surface of water-insoluble drugs. This approach also allows the reduction of adverse side effects, such as retinoids. However, multifunctional polymer conjugates containing several moieties with distinct features have not been investigated in depth. This report describes the development of a one-pot approach to produce a novel multifunctional polymer conjugate. As a proof of concept, we synthesised polyvinyl alcohol (PVA) covalently conjugated with rhodamine B (a tracking agent), folic acid (a targeting agent), and all-trans retinoic acid (ATRA, a drug). The obtained polymer (PVA@RhodFR) was characterised by MALDI-TOF mass spectrometry, gel permeation chromatography, thermal analysis, dynamic light-scattering, NMR, UV-Vis, and fluorescence spectroscopy. Finally, to evaluate the efficiency of the multifunctional polymer conjugate, cellular differentiation treatments were performed on the neuroblastoma SH-SY5Y cell line. In comparison with standard ATRA-based conditions used to promote cell differentiation, the results revealed the high capability of the new PVA@RhodFR to induce neuroblastoma cells differentiation, even with a short incubation time and low ATRA concentration.

High-resolution atomic force microscopy as a tool for topographical mapping of surface budding.

Extracellular vesicles (EVs) are membranous nanoparticles secreted by almost all cell types. Reflecting the physiopathological state of the parental cell, EVs circulate in all body fluids, reaching distant cell targets and delivering different bioactive cargoes. As biological carriers, EVs influence their microenvironment altering cellular responses, being considered promising biomarkers for both physiological and pathological conditions. EVs are heterogeneous in terms of size and composition, depending on cell type and exposure to stimuli, and different methods have been developed to characterize their morphological, biophysical, and biochemical features. Among them, electron microscopy (EM) is the main technique used, however, the lack of standardized protocols makes it difficult to characterize EVs with a good reproducibility, thus using multiple approaches may represent a way to obtain more precise information. Furthermore, the relationship between architecture and function, not only in a molecular, but also in a cellular level, is gaining growing emphasis, characterizing morphometric parameters may represent a distinct, but effective approach to study the physiopathological state of the cell. Atomic force microscopy (AFM), may represent a promising method to study in detail EVs dynamics throughout the cell surface and its variations related to the physiological state, overcoming the limits of EM, and providing more reliable information. In this study, human neuroblastoma SH-SY5Y cell line, a cellular model to investigate neurodegeneration and oxidative stress, has been used to perform a comparative morphological and quantitative analysis of membrane budding and isolated large vesicles-enriched (microvesicles-like vesicles; MVs) fraction from control or oxidative stressed cells. Our main goal was to build up a methodology to characterize EVs morphology and spatial distribution over the cell surface in different physiological conditions, and to evaluate the efficacy of AFM against conventional EM. Interestingly, both microscopy techniques were effective for this analysis, but AFM allowed to reveal a differential profiling of plasma membrane budding between the physiological and the stress condition, indicating a potential relationship between mechanical characteristics and functional role. The results obtained may provide interesting perspectives for the use of AFM to study EVs, validating a morphometric approach to understand the pathophysiological state of the cell related to EVs trafficking.

In vitro cytotoxic effects, in silico studies, some metabolic enzymes inhibition, and vibrational spectral analysis of novel β-amino alcohol compounds

In this study, an efficient single-step method for the preparation of β-amino alcohols (1–3) in aqueous media was applied. The aim was to investigate the cytotoxic activity of Compounds 1, 2 and 3 in neuroblastoma SH-SY5Y cell line and mouse fibroblast l-929 cell lines. Cytotoxic activities of compounds 1, 2 and 3 in this cell lines were also determined by MTT method. Cells were incubated with different concentrations of Compound 3 showed the highest cytotoxic activity in SHY5Y cells at an IC50 dose of 13.01±0.87 µM at 72 h compared to other compounds. Compound 3 was determined to have lower cytotoxic activity in l-929 cells. The chemical activities of the molecules against the B3LYP, HF, M062X level 3–21 g, 6–31 g, and SDD basis set with the Gaussian package program and biologically against the adenosine A(2A) receptor (PDB ID: 3PWH and 5NM4) proteins for neuroblastoma tumors cell with the Maestro Molecular modeling platform by Schrödinger were compared. Both experimental and theoretical NMR, UV–vis, and IR spectra of the studied molecules were compared. ADME/T analysis was performed to examine the drug properties of the molecules. Finally, these assayed for their activities against metabolic enzymes acetylcholinesterase and α-glucosidase. The most potent compounds against AChE were order compounds 3, 2 and 1 with Ki values of 35.88±6.61, 43.75±8.28, and 45.34±3.50 µM against AChE, respectively. The results indicated that all the synthesized compounds exhibited excellent inhibitory activities against mentioned enzymes as compared with standard inhibitors. These inhibitors may be candidates for drug design.

Spermidine activates authophagy but does not rescue human neuroblastoma SH-SY5Y cells from effects of arginine starvation

Background. Neuroblastoma is a malignant tumor of the sympathetic nervous system common in early childhood. Autophagy is manifested in neuroblastoma cells at basal levels, but is often upregulated in cells of the aggressive neuroblastoma forms. The aim of the study was to evaluate effects of polyamine spermidine and deficiency of arginine on cell viability and autophagy regulation in cells of human neuroblastoma. Materials and Methods. The human neuroblastoma SH-SY5Y cell line was an experimental model for the MTT assay of metabolic activity and cell viability upon exposure to different concentrations of spermidine in complete and arginine-free media. Assessing autophagy induction under arginine deficiency and spermidine treatment was carried out using fluorescence microscopy of neuroblastoma cells labeled with autophagic lysosomes-staining dye monodancylcadaverine. Results and Discussion. It was found that arginine withdrawal abrogates proliferation of SH-SY5Y cells. In the presence of arginine precursor, citrulline, in arginine-free medium, SH-SY5Y cells could not proliferate and, therefore, are auxotrophic for arginine. SH-SY5Y cells were more sensitive to arginine starvation than to starvation for indispensible amino acids lysine or leucine. It was also revealed that spermidine at low concentrations of 5-10 μM did not affect SH-SY5Y cells viability irrespective of arginine availability. However, at 50 and higher concentrations this polyamine was highly cytotoxic in arginine-sufficient or deficient media. Analysis of autophagy induction by spermidine and under arginine starvation revealed an increase in the number of autophagic lysosomes in SH-SY5Y cells and additive effect of the two stimuli. Conclusion. The described experiments revealed that arginine deprivation abrogated proliferation, led to a decrease in cell viability and induced autophagy in human neuroblastoma SH-SY5Y cells. Spermidine at the concentrations of 5-10 μM, while upinducing autophagy, did not improve survival of SH-SY5Y cells under arginine deprivation, whereas at a concentration above 50 μM spermidine had a strong cytotoxic effect. The main observation of this study is that autophagy can be readily manipulated in neuroblastoma cells by simultaneous deprivation for arginine and spermidine treatment.

Anti-Proliferative Effect of Potential LSD1/CoREST Inhibitors Based on Molecular Dynamics Model for Treatment of SH-SY5Y Neuroblastoma Cancer Cell Line.

Lysine-specific demethylase is a demethylase enzyme that can remove methyl groups from histones H3K4me1/2 and H3K9me1/2. It is expressed in many cancers, where it impedes differentiation and contributes to cancer cell proliferation, cell metastasis and invasiveness, and is associated with inferior prognosis. LSD1 is associated with its corepressor protein CoREST, and utilizes tetrahydrofolate as a cofactor to accept CH2 from the demethylation process. The fact that the cofactor is best bound to the active site inspired us to explore its interactions to LSD1/CoREST enzyme complex utilizing molecular dynamics simulation, which aids designing novel and potent inhibitors. In this study we minted to identify a new potential LSD1/CoREST inhibitors and test the potency and the safety of such inhibitors against human neuroblastoma and fibroblast cells lines. We have implemented a previously derived model from the molecular dynamics simulation study and the key contacts to the active site in a subsequent structure based drug design and in-silico screening, which revealed a number of potential inhibitors toward LSD1/CoREST complex. The anti-proliferative activities of the identified compounds will be tested against neuroblastoma SH-SY5Y cancer cell line which known to highly express LSD1/CoREST complex. In-silico mining on National Cancer Institute (NCI) database identified 55 promising and structurally diverse inhibitors. Applying the abovementioned molecular modeling procedure yielded four compounds of LSD1/CoREST inhibiters with IC50 < 2µM. The four lead compounds were tested against SH-SY5Y neuroblastoma cell line that known to express high level of LSD1 and illustrated a potent activity with an IC50 ranging from 0.195 to 1.52µM. To estimate the toxicity of the selective leads, they were tested against normal fibroblast cells and scored a relatively high IC50 ranging from 0.303 to ≥ 100µM. Our model revealed promising inhibitors that can be used in treating cancers that overexpress the LSD1 enzyme such as the SH-SY5Y neuroblastoma.

Bub1 as a potential oncogene and a prognostic biomarker for neuroblastoma

BackgroundNeuroblastoma is the most common malignant extracranial tumor for children. Molecular mechanisms underpinning the pathogenesis of this disease are yet to be fully clarified. This study aimed to identify a novel oncogene that could be used as a biomarker informing the prognosis of neuroblastoma, and to predict its biological functions, using bioinformatics and molecular biology tools.MethodsThree data sets from the TARGET, GSE62564, and GSE85047 databases were used for analysis. Survivals of patients with high or low expression of bub1 were compared, using the Kaplan-Meier curve and log-rank test. Immune infiltration was evaluated using ESTIMATE and MCP-counter algorithms. Synthetic small interfering RNAs (siRNAs) were employed to silence bub1 expression in neuroblastoma cell lines SH-SY5Y and SK-N-SH, in order to characterize its biological functions. Gene enrichment analyses of bub1 were carried out, using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.ResultsExpression of bub1 was found to significantly affect overall survival and event-free survival of patients with neuroblastoma, positively correlate with the expressions of tpx2 and the ASPM gene, and negatively correlate with host immune infiltration. Expression of bub1 was elevated in patients with neuroblastoma. Silencing bub1 expression using siRNAs in SH-SY5Y and SK-N-SH resulted in decreased cell growth (p < 0.05), reduced migration (p < 0.05), and increased apoptosis (p < 0.05). Function analysis of bub1 revealed cancer-promoting effects, probably via regulating several important downstream molecules, including that related to the apoptosis process and epithelial-mesenchymal transition.ConclusionWe identified a potential tumor-promoting gene bub1 for neuroblastoma that could also serve as a prognostic biomarker.

The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines.

Sequence variants or mutations in the GBA gene are numerically the most important risk factor for Parkinson disease (PD). The GBA gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GBA mutations often reduce GCase activity and lead to the impairment of the autophagy-lysosomal pathway, which is important in the turnover of alpha-synuclein, accumulation of which is a key pathological hallmark of PD. Although the E326K variant is one of the most common GBA variants associated with PD, there is limited understanding of its biochemical effects. We have characterized homozygous and heterozygous E326K variants in human fibroblasts. We found that E326K variants did not cause a significant loss of GCase protein or activity, endoplasmic reticulum (ER) retention or ER stress, in contrast to the L444P GBA mutation. This was confirmed in human dopaminergic SH-SY5Y neuroblastoma cell lines overexpressing GCase with either E326K or L444P protein. Despite no loss of the GCase activity, a significant increase in insoluble alpha-synuclein aggregates in E326K and L444P mutants was observed. Notably, SH-SY5Y overexpressing E326K demonstrated a significant increase in the lipid droplet number under basal conditions, which was exacerbated following treatment with the fatty acid oleic acid. Similarly, a significant increase in lipid droplet formation following lipid loading was observed in heterozygous and homozygous E326K fibroblasts. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to the common loss of function GBA mutations; however, lipid dyshomeostasis and alpha-synuclein pathology are still evident.

Neuroblastoma SH-SY5Y Cell Differentiation to Mature Neuron by AM580 Treatment

Neuroblastoma is a type of developmental childhood cancer that arises from the neural crest. It is the most common pediatric solid tumor in the world. AM580 is a powerful cyto-differentiating molecule on acute promyelocytic leukemia cells and induced pluripotent stem cells, but its effect on neuroblastoma is still unknown. In this study, the neuronal differentiation impact of AM580 was investigated using the human neuroblastoma cell line SH-SY5Y as a model. AM580 successfully stimulated the SH-SY5Y cells to develop into neuron-like cells. Functional enrichment analysis of RNAseq data revealed that differentially expressed genes (DEGs) were substantially enriched for GO keywords and KEGG pathways linked to neuron development. Some potassium ion channel genes associated with neuronal excitation, such as KCNT1, were shown to be upregulated. Through the MEA tests, we found the AM580-induced neurons possessed electrical spikes as mature neurons. AM580 also induced the neuronal marker β-tubulin III and mature neurons marker Neurofilament H. Our study proved that AM580 can promote the differentiation of neurons and has the potential to treat neuroblastoma, neurodevelopmental and neurodegenerative diseases.

P16-09 Synthetic opioids induce oxidative stress and DNA damage in human neuroblastoma SH-SY5Y cell line

P16-09 Synthetic opioids induce oxidative stress and DNA damage in human neuroblastoma SH-SY5Y cell line