Human Neuroblastoma Cell Line Research Articles

A multi-functional platform for stimulating and recording electrical responses of SH-SY5Y cells.

In recent years, multifunctional cell regulation on a single chip has become an imperative need for cell research. In this study, a novel multi-functional micro-platform integrating wireless electrical stimulation, mechanical stimulation and electrical response recording of cells was proposed. Controlling cell fate by photoexcited radio stimulation of cells on photosensitive films can precisely orchestrate biological activities. This is the first report of combining hydrogenated amorphous silicon (a-Si:H) photosensitive films and a 532 nm green laser for cell stimulation and electrical response recording. Remote wireless electrical stimulation of nerve cells through photoelectric effects based on photosensitive films evoked a change in membrane potential and promoted the neurite growth and neuronal differentiation. These effects were confirmed in a cell model of the human neuroblastoma cell line. The electrical response of cells demonstrated that the photocurrent generated by laser irradiation of the photosensitive film induced a redistribution of ions inside and outside the cell. Furthermore, a mechanical stimulus was applied to cells using a probe placed above them. The chip was used as a signal output to simultaneously obtain the electrical response of cells. The ability of photosensitive films to precisely excite cellular activity offers a novel prospect for wireless electrical stimulation. This work provides a promising strategy for the design of multi-functional biological devices based on a single chip.

Norlignans and Phenolics from Curculigo capitulata and Their Neuroprotection Against Glutamate-Induced Oxidative Injury in SH-SY5Y Cells

The herb Curculigo capitulata (Lour.) Ktze is widely distributed in southern and southwestern China. The Curculigo genus and its primary chemical constituents exhibit remarkable antidepressant activities. To investigate the chemical constituents and potential health benefits of C. capitulata, a phytochemical study was conducted. In this study, seven new compounds (capitugenin A–G), including three new norlignans (1–3), a new chalcone dimer (4), a new hemiacetal (5), two novel pyrrolidine-based compounds (6 and 7), including one identified as a natural product (7), and nineteen known compounds (8–26), were isolated from C. capitulata. The chemical structures and absolute configurations of Compounds 1–7 were elucidated via comprehensive spectroscopic data analyses. The neuroprotective effects of Compounds 1–26 against glutamate-induced cell death were tested in the human neuroblastoma cell line SH-SY5Y. Compounds 1, 3, 6, 8, 11, and 17 showed significant neuroprotective effects, with protection rates ranging from 29.4 to 52.8% at concentrations ranging from 5 to 40 μM. Western blot analysis indicated that Compound 3 exerted a protective effect by regulating the expression of Nrf2/HO-1.

Validation of the biological function and prognostic significance of AURKA in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children, and the AURKA gene encodes a protein kinase involved in cell cycle regulation that plays an oncogenic role in a variety of human cancers. The aim of this study was to validate the biological function and prognostic significance of AURKA in NB using basic experiments and bioinformatics. Data obtained from Target and GEO databases were analyzed using various bioinformatic techniques. The expression of AURKA in 77 NB samples was detected by immunohistochemistry (IHC) method. The lentiviral RNA interference technique was employed to downregulate AURKA gene expression in NB cell lines. Additionally, cell counting kit-8 and flow cytometry analysis were conducted to investigate the impact of AURKA expression on cell proliferation, cell cycle progression, and apoptosis. A bioinformatic analysis showed that patients with NB in the AURKA-high-expression group had shorter OS (Overall Survival). Immune cell infiltration analysis showed that only activated CD4 T cell and type 2 T helper cell infiltration levels were higher in the AURKA-high-expression group than in the AURKA-low-expression group, with the infiltration levels of most other immune cells and cytokines lower in the high-expression group. Furthermore, the enhanced infiltration of activated CD4 T cells was associated with worse OS in patients with NB. IHC results showed that the AURKA expression was correlated with MYCN status and INSS stage. Log-rank test showed that pathological type, MYCN status, INSS stage, COG risk group, and AURKA expression was related to PFS (Progression-free survival) of NB patients, but COX regression analysis showed that none of the above factors were independently prognostic for PFS. In vitro, shRNA delivered via an AURKA-specific lentivirus significantly and consistently silenced endogenous AURKA expression in the human NB cell line SK-N-AS. This inhibited tumor cell proliferation, induced apoptosis, and caused G2/M-phase cell cycle arrest. Moreover, western blot assay showed significant reductions in the levels of mTOR, p70S6K, and 4E-BP1 phosphorylation in the AURKA-knockdown group. I found in subsequent experiments that NFYB can bind to the AURKA promoter and thus promote AURKA expression. High-level AURKA expression in NB is associated with poor patient prognosis. Silencing AURKA inhibited tumor cell proliferation, induced tumor cell apoptosis, and led to cell cycle arrest in the G2/M phase. Mechanistically, AURKA knockdown inhibited the phosphorylation and the activation of the mTOR1/p70S6K/4E-BP1 signaling pathway. In addition, AURKA was observed to regulate the infiltration levels of various immune cells in the NB tumor microenvironment, resulting in remodeling of the immunosuppressive tumor microenvironment.

The neuroprotective effect of 1,25-dyhydroxyvitamin D3 (calcitriol) and probiotics on the rotenone-induced neurotoxicity model in SH-SY5Y cells

This study aimed to investigate the neuroprotective role of probiotics and 1,25-dyhydroxyvitamin D3 (calcitriol) against neurotoxicity on rotenone-induced human neuroblastoma cell line SH-SY5Y. Rotenone was administered to induce neurotoxic effects in SH-SY5Y cells. Calcitriol and probiotics were administered at different concentrations as pre- and post-treatment. The thiazolyl blue tetrazolium bromide (MTT) assay was performed to measure cell viability. Intracellular protein levels of antioxidant enzymes (protein tyrosine kinase (PTK), superoxide dismutase (SOD), glutathione peroxidase (GSH), glutathione reductase (GSR), and catalase (CAT)) were determined by the enzyme-linked immunosorbent assay (ELISA). Rotenone (150 nM) reduced (p < 0.001) cell viability compared to control cells. Single and combined pretreatments with probiotics (0.01 mg/ml, 0.05 mg/ml, and 0.1 mg/ml) and calcitriol (1.25 µM, 2.5 µM, and 5 µM) increased (p < 0.05) cell viability compared to rotenone group. In the pre- and post-treatment design, all treatment groups increased the SOD and GSH levels and decreased the GSR levels compared to rotenone. None of the pretreatments reversed the PTK levels (except probiotics: 0.01 mg/ml). Calcitriol (2.5 µM) increased the CAT levels in pretreatment design, and probiotics (0.05 mg/ml and 0.1 mg/ml) increased CAT levels in post-treatment design compared to rotenone group. Calcitriol and probiotics protect against rotenone-induced neurotoxicity in SH-SY5Y cells by decreasing reactive oxygen species (ROS) and increasing antioxidant enzyme parameters. These neuroprotective effects of calcitriol and probiotics against rotenone-induced dopaminergic neurotoxicity provide an experimental basis for their potential clinical use in the treatment of Parkinson’s disease (PD).

The biotoxin BMAA promotes mesenchymal transition in neuroblastoma cells.

Mesenchymal-like cancer cells are an indicator of malignant tumors as they exhibit tumorigenic properties including downregulation of differentiation markers, and increased colony-forming potential, motility, and chemoresistance. We have previously demonstrated that the cyanobacterial biotoxin beta-methylamino-L-alanine (BMAA) is capable of influencing neural cell differentiation state through mechanisms involving the Wnt signaling pathway, suggesting the possibility that BMAA may play a role in influencing other Wnt related differentiation processes including mesenchymal transition. In this study we present evidence characterizing the effects of BMAA on mesenchymal transition in a human neuroblastoma cell line and provide support for the hypothesis that the biotoxin can promote this process in these cells by altering differentiation state, inducing changes in gene expression, and changing cellular function in manners consistent with cellular mesenchymal transition. Results of this study indicate that BMAA exposure may promote carcinogenesis through its effects on cell differentiation state in certain contexts. These results suggest that exposure to the biotoxin BMAA may be an influencing factor in chemotherapy resistance and cancer relapse in neuroblastoma.

New insight into the molecular etiopathogenesis of konzo: Cyanate could be a plausible neurotoxin contributing to konzo, contrary to thiocyanate

IntroductionChronic cassava-derived cyanide poisoning is associated with the appearance of konzo, a tropical spastic paraparesis due to selective upper motor neuron damage. Whether the disease is caused by a direct action of cyanide or its metabolites is still an open question. This preliminary study assessed the neurotoxic effects of thiocyanate (SCN) and cyanate (OCN), two cyanide metabolites hypothesized to be plausible toxic agents in konzo. MethodsCultured mouse neuroblastoma (Neuro-2A) and human neuroblastoma (SH-SY5Y) cell lines were incubated (24, 48, and 72hours) in sodium OCN or sodium SCN in a disease-relevant concentration range. Cell viability, caspase (3, 8, and 9) activities, and reactive oxygen species (ROS) generation were evaluated using appropriate assay kits. Additionally, electrophysiological responses induced by OCN and SCN in primary spinal cord neurons (from Sprague Dawley rats) were assessed by whole-cell patch-clamp techniques. ResultsBoth OCN and SCN were toxic in a dose-dependent way, even if SCN toxicity appeared at very high concentrations (30mM, corresponding to more than 100-fold higher than normal plasmatic levels), contrary to OCN (0.3-3mM). OCN was markedly more toxic in a poor culture medium (MEM; IC50 = 3.2mM) compared to a glucose- and amino acid-rich medium (DMEM; IC50=7.6mM). OCN treatment increased the ROS generation by 8.9 folds, as well as the Caspase-3, Caspase-8, and Caspase-9 activities by 3.2, 2.5, and 2.6 folds, respectively. Finally, OCN (and SCN to a lesser extent) induced depolarizing currents in primary spinal cord neurons, through an activation of ionotropic glutamate receptors. ConclusionOur results suggest OCN as the most plausible neurotoxic agent involved in konzo, while SCN toxicity could be questioned at such high concentrations. Also, they support apoptosis, oxidative stress, and excitotoxicity as probable mechanisms of OCN neurotoxicity.

Evaluation of Selected Plant Phenolics via Beta-Secretase-1 Inhibition, Molecular Docking, and Gene Expression Related to Alzheimer's Disease.

Background: The goal of the current study was to investigate the inhibitory activity of six phenolic compounds, i.e., rosmarinic acid, gallic acid, oleuropein, epigallocatechin gallate (EGCG), 3-hydroxytyrosol, and quercetin, against β-site amyloid precursor protein cleaving enzyme-1 (BACE1), also known as β-secretase or memapsin 2, which is implicated in the pathogenesis of Alzheimer's disease (AD). Methods and Results: The inhibitory potential against BACE1, molecular docking simulations, as well as neurotoxicity and the effect on the AD-related gene expression of the selected phenolics were tested. BACE1 inhibitory activity was carried out using the ELISA microplate assay via fluorescence resonance energy transfer (FRET) technology. Molecular docking experiments were performed in the human BACE1 active site (PDB code: 2WJO). Neurotoxicity of the compounds was carried out in SH-SY5Y, a human neuroblastoma cell line, by the Alamar Blue method. A gene expression analysis of the compounds on fourteen genes linked to AD was conducted using the real-time polymerase chain reaction (RT-PCR) method. Rosmarinic acid, EGCG, oleuropein, and quercetin (also used as the reference) were able to inhibit BACE1 with their respective IC50 values 4.06 ± 0.68, 1.62 ± 0.12, 9.87 ± 1.01, and 3.16 ± 0.30 mM. The inhibitory compounds were observed to occupy the non-catalytic site of the BACE1. However, hydrogen bonds were found to be present between rosmarinic acid and EGCG and aspartic amino acid D228 in the catalytic site. Oleuropein and quercetin effectively suppressed the expression of PSEN, APOE, and CLU, which are recognized to be linked to the pathogenesis of AD. Conclusions: The outcomes of the work bring quercetin, EGCG, and rosmarinic acid to the forefront as promising BACE1 inhibitors.

An integrated proteomics and metabolomics analysis of methylglyoxal-induced neurotoxicity in a human neuroblastoma cell line

This study aimed to highlight the molecular and biochemical changes induced by methylglyoxal (MGO) exposure in SH-SY5Y human neuroblastoma cells, and to explore how these changes contribute to its neurotoxicity, utilizing an integrated proteomics and metabolomics approach. Using label-free quantitative nanoLC-MS/MS proteomics and targeted LC-TQ-MS/MS-based metabolomics, the results revealed that MGO exposure, particularly at cytotoxic levels, significantly altered the proteome and metabolome of SH-SY5Y cells. Analysis of proteomics data showed significant alterations in cellular functions including protein synthesis, cellular structural integrity, mitochondrial function, and oxidative stress responses. Analysis of metabolomics and integration of metabolomics and proteomics data highlighted significant changes in key metabolic pathways including arginine biosynthesis, glutathione metabolism, cysteine and methionine metabolism, and the tricarboxylic acid cycle. These results suggest that MGO exposure induced both toxic effects and adaptive responses in cells. MGO exposure led to increased endoplasmic reticulum stress, disruptions in cellular adhesion and extracellular matrix integrity, mitochondrial dysfunction, and amino acid metabolism disruption, contributing to cellular toxicity. Conversely, cells exhibited adaptive responses by upregulating protein synthesis, activating the Nrf2 pathway, and reprogramming metabolism to counteract dicarbonyl stress and maintain energy levels. Furthermore, a set of key proteins and metabolites associated with these changes were shown to exhibit a significant concentration-dependent decrease or increase in their expression levels with increasing MGO concentrations, suggesting their potential as biomarkers for MGO exposure. Taken together, these findings provide insight into the molecular mechanisms underlying MGO-induced neurotoxicity and potential targets for therapeutic intervention.

Exploring Nanocarriers for Boosting Entacapone Bioavailability: A Journey through System Characterization and Assessment of Toxicity and Pharmacological and 2D Permeability Paybacks.

Catechol-O-methyltransferase inhibitors (iCOMT), such as entacapone, have been successfully employed to treat tremor-related symptoms of Parkinson's disease. However, iCOMT has been associated with a short half-life and poor oral bioavailability. Nanobased drug delivery systems have often been used to overcome this type of setbacks. Therefore, entacapone was encapsulated in PEGylated poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) via a nanoprecipitation process, as well as in PEGylated nanostructured lipid carriers (NLCs) using a solvent emulsification/evaporation method. Both nanoformulations presented sub-200 nm populations, with zeta-potential (ZP) values close to -30 mV, and showed stability at different pHs, while maintaining their physicochemical properties mostly intact, presenting only a change in their superficial charge (ZP values), indicating their interaction. Both nanoformulations presented interaction with mucins, which anticipates good permeation and bioavailability for oral and topical administration. No cytotoxic effects were observed for lyophilized PLGA NPs encapsulating entacapone, in which 2-hydroxypropyl-ß-cyclodextrin (HPβCD) was used as a cryoprotectant at 3% concentration (HP-PLGA@Ent), in human hepatocellular carcinoma (HepG2), human neuroblastoma (SH-SY5Y), or human epithelial colorectal adenocarcinoma (Caco-2) cell lines. Conversely, NLCs encapsulating entacapone (W-NLCs@Ent) presented cytotoxic effects on the HepG2 cell line, likely due to intracellular lipid accumulation or storage. Both nanoformulations maintained a COMT inhibition effect in HepG2 cells, using 3-BTD as the COMT probe. An increase of entacapone permeability in both monolayer and coculture models (Caco-2 and Caco-2/HT29-MTX, respectively) was observed for the developed nanoformulations. Overall, this work shows that encapsulated entacapone in different nanocarriers could be a stimulating alternative to solve entacapone setbacks, since they improve its physicochemical properties and permeability while still maintaining the COMT inhibitory activity.

New Perspectives on the Old Uses of Traditional Medicinal and Edible Herbs: Extract and Spent Material of Persicaria hydropiper (L.) Delarbre

Background/Objectives:Persicaria hydropiper (L.) Delarbre, commonly known as water pepper, possesses multifunctional potential. Our research focuses on its complex phenolic composition, bioactivity, safety evaluation and utilization in a sustainable manner. Moreover, a survey was conducted among the Serbian population to gain insight into the attitude towards traditional wild-growing herbs (i.e., P. hydropiper), the level of familiarity with their zero-waste culture, and to assess eating behaviors. Methods: A survey was conducted with 168 participants to assess attitudes towards traditional herbs, zero-waste culture, and eating behaviors, while cytotoxicity, in vivo toxicity, chemical analysis of secondary metabolites, and probiotic viability assays were performed to evaluate the effects of the PH extract. Results: Notably, P. hydropiper extract (PH) exhibits a diverse phenolic profile, including quinic acid (3.68 ± 0.37 mg/g DW), gallic acid (1.16 ± 0.10 mg/g DW), quercetin (2.34 ± 0.70 mg/g DW) and kaempferol-3-O-glucoside (4.18 ± 0.17 mg/g DW). These bioactive compounds have been linked to anticancer effects. The tested extract demonstrated a cytotoxic effect on the human neuroblastoma cell line, opening questions for the further exploration of its mechanisms for potential therapeutic applications. Based on the toxicity assessment in the Artemia salina model, the PH could be characterized with good safety, especially for the lower concentrations (LC50 = 0.83 mg/mL, 24 h). The utilization of the spent PH material supports the viability of psychobiotic strains (up to 9.26 ± 0.54 log CFU/mL). Based on the conducted survey, 63.7% (n = 107) of respondents mainly prefer traditional instead of imported herbs. The respondents were skeptical about zero-waste edibles; 51.2% (n = 86) would not try them, and a bit more than half were not familiar with zero-waste culture (57.7%; n = 97). Only 8.3% (n = 14) followed a flexitarian diet as a dietary pattern. Conclusions: The use of underutilized traditional plants and their spent material could potentially contribute to the acceptance of a zero-waste culture in Serbia. Reinventing the use of neglected traditional plants and addressing ways for spent material valorization could contribute to the acceptance of a zero-waste strategy and encourage healthier eating behavior.

Synthesis of new phenothiazine derivatives: Molecular docking, assessment of cytotoxic activity and oxidant-antioxidant properties on PCS-201-012, HT-29, and SH-SY5Y cell lines.

Phenothiazine (PTZ) derivatives have been acknowledged as versatile compounds with significant implications across various areas of medicine, particularly, in cancer research. The cytotoxic effects of synthesized compounds on both normal and cancerous cells, along with their oxidant-antioxidant properties, are pivotal factors in cancer treatment strategies. In the current study, eight new PTZ derivatives were synthesized and the compounds' cytotoxic activities were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay while the oxidant-antioxidant properties were evaluated by oxidative stress index (OSI) calculation in SH-SY5Y (a human neuroblastoma cell line), HT-29 (a human colorectal adenocarcinoma cell line), and PCS-201-012 (a human primary dermal fibroblast cell line) cells. Consequently, the half-maximal inhibitory concentration (IC50) values of compound 3a were determined to be 218.72, 202.85, and 227.86 μM while the IC50 values of compound 3b were defined to be 227.42, 199.27, and 250.11 μM in PCS-201-012, HT-29, and SH-SY5Y cells, respectively. Additionally, it was determined that the synthesized compounds demonstrated the lowest OSI in PCS-201-012 cells as compared to the other cell lines.

Electrospun nanofibers for localized drug release of a neuroprotective natural extract of Usnea ghattensis

This research is based on the incorporation of the methanolic extract of the Usnea ghattensis into poly (caprolactone) (PCL) nanofibers (NFs) to investigate the capacity in reducing reactive oxygen species (ROS). PCL-NFs were fabricated by the electrospinning technique and are investigated as potential dressing material focused on the release of usnic acid (PCL-USNIC NFs), and its encapsulation efficiency and kinetic release were analyzed by high performance liquid chromatography (HPLC). This investigation was performed by analyzing the usnic acid concentration as a function of the distance from the mat center point. The kinetic release analysis is also developed with the usnea ghattensis extract (PCL-USNEA NFs), performing a metabolomic analysis of the released molecules as a function of time by nuclear magnetic resonance (NMR). Usnic acid was revealed as the most relevant compound together with other molecules, such as sucrose, mannitol, arabitol or glycerol that generate a positive matrix effect on the release of usnic acid. Finally, we analize the cytotoxicity and the neuroprotective effect of PCL-USNEA and PCL-USNIC NFs using a human neuroblastoma cell line model. Negligible toxicity was appreciated for both polymeric systems, showing high protective effects in presence of highly oxidative environment (e.g. in presence of H2O2).

Nilotinib as a Prospective Treatment for Alzheimer's Disease: Effect on Proteins Involved in Neurodegeneration and Neuronal Homeostasis.

Nilotinib, a tyrosine kinase inhibitor that targets the Abelson tyrosine kinase (c-Abl) signaling pathway, is FDA-approved to treat chronic myeloid leukemia. Nilotinib has properties indicative of a possible utility in neuroprotection that have prompted exploration of repurposing the drug for the treatment of Alzheimer's disease (AD) and Parkinson's disease (PD). AD is a progressive age-related neurodegenerative disorder characterized by the deposition of extracellular amyloid-β plaques and intracellular neurofibrillary tangles. It is incurable and affects approximately 50 million patients worldwide. Nilotinib reduces c-Abl phosphorylation, amyloid-β levels, and dopaminergic neuron degeneration in preclinical AD models. This study explores the effects of nilotinib on amyloid processing and mitochondrial functioning in the SH-SY5Y human neuroblastoma cell line. SH-SY5Y cells were exposed to nilotinib (1, 5, and 10 µM). Real-time PCR and immunoblot analysis were performed to quantify the expression of genes pertaining to amyloid-β processing and neuronal health. Nilotinib did not significantly change APP, BACE1, or ADAM10 mRNA levels. However, BACE1 protein was significantly increased at 1 µM, and ADAM10 was increased at 10 µM nilotinib without affecting APP protein expression. Further, nilotinib treatment did not affect the expression of genes associated with neuronal health and mitochondrial functioning. Taken together, our findings do not support the efficacy of nilotinib treatment for neuroprotection.

Ethosuximide-loaded bismuth ferrite nanoparticles as a potential drug delivery system for the treatment of epilepsy disease.

Encapsulating antiepileptic drugs (AEDs), including ethosuximide (Etho), into nanoparticles shows promise in treating epilepsy. Nanomedicine may be the most significant contributor to addressing this issue. It presents several advantages compared to traditional drug delivery methods and is currently a prominent area of focus in cancer research. Incorporating Etho into bismuth ferrite (BFO) nanoparticles within diverse controlled drug delivery systems is explored to enhance drug efficacy. This approach is primarily desired to aid in targeted drug delivery to the brain's deepest regions while limiting transplacental permeability, reducing fetal exposure, and mitigating associated adverse effects. In this investigation, we explored Etho, an antiepileptic drug commonly employed for treating absence seizures, as the active ingredient in BFO nanoparticles at varying concentrations (10 and 15 mg). Characterization of the drug-containing BFO nanoparticles involved scanning electron microscopy (SEM) and elemental analysis. The thermal properties of the drug-containing BFO nanoparticles were evaluated via differential scanning calorimetry (DSC) analysis. Cytotoxicity evaluations using the MTT assay were conducted on all nanoparticles, and human neuroblastoma cell line cultures (SH-SY5Y) were treated with each particle over multiple time intervals. Cell viability remained at 135% after 7 days when exposed to 15 mg of Etho in BFO nanoparticles. Additionally, in vitro drug release kinetics for Etho revealed sustained release lasting up to 5 hours with a drug concentration of 15 mg.

LncRNA BASP1-AS1 is a positive regulator of stemness and pluripotency in human SH-SY5Y neuroblastoma cells

Neuroblastoma is the most common extra-cranial solid tumor diagnosed mostly in children below the age of five years and comprises of about 15 % of all paediatric cancer deaths. Tumor initiating cancer stem cells (CSCs) can be targeted for better treatment approaches. BASP1-AS1 is a long non coding (Lnc) RNA that is a divergent LncRNA for its coding gene brain abundant membrane attached signal protein 1 (BASP1). We had earlier demonstrated it to be expressed in foetus derived human neural progenitor cells (hNPCs), where it was a positive regulator of BASP1 and was critical for neural differentiation. In this study, we have investigated the role of BASP1-AS1 in CSCs derived from the human neuroblastoma cell line SH-SY5Y. We cultured SH-SY5Y cells on Poly-d-Lysine coated flasks in serum free media supplemented with growth factors, which led to the enrichment of CSCs as determined by marker expression. When grown on ultra-low attachment flasks, these cells formed CSCs enriched neurospheres. We examined the effects of BASP1-AS1 siRNA mediated knockdown on CSCs enriched SH-SY5Y cells and SH-SY5Y derived neurospheres. BASP1-AS1 knockdown decreased the levels of the corresponding gene BASP1 and the rate of cell proliferation of CSCs enriched cells along with low expression of Ki67. It also reduced the mRNA levels of stem cell and pluripotency gene markers (CD133, CD44, c-KIT, SOX2, OCT4 and NANOG), as also Wnt 2 and the Wnt pathway effector β catenin. It also abrogated the formation of neurospheres in ultra-low attachment flasks. A similar effect on proliferation and stemness related properties was seen on BASP1 knockdown. BASP1-AS1 and its related pathways may provide a point of intervention for the CSCs population in neuroblastoma.

Soft Corals-derived Dihydrosinularin Attenuates Neuronal Apoptosis in 6-hydroxydopamine-induced Cell Model of Parkinson's Disease by Regulating the PI3K Pathway.

Parkinson's disease (PD) is an irreversible, progressive disorder that profoundly impacts both motor and non-motor functions, thereby significantly diminishing the individual's quality of life. Dihydrosinularin (DHS), a natural bioactive molecule derived from soft corals, exhibits low cytotoxicity and anti-inflammatory properties. However, the therapeutic effects of DHS on neurotoxins and PD are currently unknown. This study investigated whether DHS could mitigate 6-hydroxydopamine (6- OHDA)-induced neurotoxicity and explored the role of neuroprotective PI3K downstream signaling pathways, including that of AKT, ERK, JNK, BCL2, and NFκB, in DHS- mediated neuroprotection. We treated the human neuroblastoma cell line, SH-SY5Y, with the neurotoxin 6-OHDA to establish a cellular model of PD. Meanwhile, we assessed the anti-apoptotic and neuroprotective properties of DHS through cell viability, apoptosis, and immunostaining assays. Furthermore, we utilized the PI3K inhibitor LY294002 to validate the therapeutic target of DHS. Based on the physicochemical properties of DHS, it can be inferred that it has promising oral bioavailability and permeability across the blood-brain barrier (BBB). It was demonstrated that DHS upregulates phosphorylated AKT and ERK while downregulating phosphorylated JNK. Consequently, this enhances the expression of BCL2, which exerts a protective effect on neuronal cells by inhibiting caspase activity and preventing cell apoptosis. The inhibition of PI3K significantly reduced the relative protective activity of DHS in 6-OHDA-induced neurotoxicity, suggesting that the neuroprotective effects of DHS are mediated through the activation of PI3K signaling. By investigating the mechanisms involved in 6-OHDA-induced neurotoxicity, we provided evidence concerning the therapeutic potential of DHS in neuroprotection. Further research into DHS and its mechanisms of action holds promise for developing novel therapeutic strategies for PD.

Differentiation of a Human Neuroblastoma Cell Line into Mature Neurons

Differentiation of a Human Neuroblastoma Cell Line into Mature Neurons

Differentiation of a Human Neuroblastoma Cell Line into Mature Neurons

Differentiation of a Human Neuroblastoma Cell Line into Mature Neurons

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake

ObjectiveGlucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain. MethodsThe effect of PGM2L1 on neuronal post-ischemic viability was assessed by siRNA-mediated knockdown of PGM2L1 in primary mouse neurons. Acute mouse brain slices were used to correlate the reduction in G-1,6-BP upon ischemia to changes in carbon metabolism by 13C6-glucose tracing. A drug affinity responsive target stability assay was used to test if G-1,6-BP interacts with the mitochondrial pyruvate carrier (MPC) subunits in mouse brain protein extracts. Human embryonic kidney cells expressing a MPC bioluminescence resonance energy transfer sensor were used to analyze how PGM2L1 overexpression affects MPC activity. The effect of G-1,6-BP on mitochondrial pyruvate uptake and oxygen consumption rates was analyzed in isolated mouse brain mitochondria. PGM2L1 and a predicted upstream kinase were overexpressed in a human neuroblastoma cell line and G-1,6-BP levels were measured. ResultsWe found that G-1,6-BP in mouse brain slices was quickly degraded upon ischemia and reperfusion. Knockdown of PGM2L1 in mouse neurons reduced post-ischemic viability, indicating that PGM2L1 plays a neuroprotective role. The reduction in G-1,6-BP upon ischemia was not accompanied by alterations in glycolytic rates but we did see a reduced 13C6-glucose incorporation into citrate, suggesting a potential role in mitochondrial pyruvate uptake or metabolism. Indeed, G-1,6-BP interacted with both MPC subunits and overexpression of PGM2L1 increased MPC activity. G-1,6-BP, at concentrations found in the brain, enhanced mitochondrial pyruvate uptake and pyruvate-induced oxygen consumption rates. Overexpression of a predicted upstream kinase inhibited PGM2L1 activity, showing that besides metabolism, also signaling pathways can regulate G-1,6-BP levels. ConclusionsWe provide evidence that G-1,6-BP positively regulates mitochondrial pyruvate uptake and post-ischemic neuronal viability. These compelling data reveal a novel mechanism by which neurons can couple glycolysis-derived pyruvate to the tricarboxylic acid cycle. This process is sensitive to the glycolytic flux, the cell's energetic state, and upstream signaling cascades, offering many regulatory means to fine-tune this critical metabolic step.

MicroRNA-32-5p inhibits metastasis by directly targeting VPS4B and increases sensitivity to dihydroartemisinin in neuroblastoma

Abstract Background Neuroblastoma (NB) is a malignant pediatric tumor requiring new therapies. Accumulating evidence has confirmed that microRNAs play critical roles in NB metastasis. Dihydroartemisinin (DHA) is capable of inhibiting the growth of NB cells. The primary objective of the current investigation was to characterize a newly discovered microRNA, miR-32-5p, in terms of the functional role, underlying mechanism of action, and potential synergistic therapeutic impact in the context of NB metastasis. Materials and methods Real-time quantitative polymerase chain reaction and Western blotting were employed to assess the expression levels of miR-32-5p and its target, vacuolar protein sorting 4B (VPS4B). Furthermore, Transwell assay was utilized to evaluate in vitro cell migration and invasion, whereas a metastasis xenograft model was established in nude mice via caudal vein injections. Results Gene Expression Omnibus database and real-time quantitative polymerase chain reaction analysis showed that miR-32-5p was downregulated in human NB samples and NB cell lines, in comparison with the normal tissue and cell lines. Inhibiting miR-32-5p induced the migration and invasion of NB cells, whereas overexpression of miR-32-5p prevented the migration and invasion in NB cell lines. Furthermore, VPS4B was identified as the direct target of miR-32-5p and the miR-32-5p reduction associated with NB metastasis upregulated the expression of VPS4B. Conversely, overexpression of VPS4B reversed the suppressive effects of miR-32-5p on NB cells. Moreover, miR-32-5p increased the sensitivity to DHA both in NB cells and in the metastasis xenograft model of nude mice. Conclusions The downregulation of miR-32-5p in NB regulates NB metastasis by targeting VPS4B. Moreover, miR-32-5b can improve the sensitivity of DHA in the xenograft mouse model. Our findings have important implications for the combined application of miR-32-5p and DHA in the treatment of NB.