Differentiation Of Neuroblastoma Cells Research Articles

CDK4/6 inhibitors display a class effect in inducing differentiation of neuroblastoma cells

Background Neuroblastoma is the most common extracranial solid tumour in infants and children, accounting for approximately 15% of paediatric cancer mortality. These tumours are unique in that a subset, namely stage MS, frequently undergo spontaneous regression or differentiation. Differentiation therapy, where cancer cells are re-routed back down their correct developmental pathway, is therefore a promising therapeutic avenue. We have previously shown that the CDK4/6 inhibitor palbociclib induces both decreased proliferation and enhanced neuronal differentiation of neuroblastoma cells in vitro. When combined with retinoic acid, already used clinically for maintenance therapy, this differentiation is enhanced. Methods Here, we investigate two additional CDK4/6 inhibitors, abemaciclib and ribociclib, to induce differentiation of the relapsed, high-risk MYCN-amplified neuroblastoma cell line SK-N-BE(2)C, with and without retinoic acid. We culture SK-N-BE(2)C cells in both adherent and three-dimensional culture and monitor proliferation and differentiation using readouts including live-imaging, immunocytochemistry, qRT-PCR and EdU incorporation. Results We find the CDK4/6 inhibitors palbociclib, abemaciclib and ribociclib all enhance retinoic acid-induced differentiation in both adherent SK-N-BE(2)C cells and 3D spheroids. Conclusions CDK4/6 inhibitors display a class effect in inducing neuronal differentiation together with retinoic acid, both in adherent neuroblastoma cell lines and three-dimensional tumour spheroids. This is an important consideration for potentially developing CDK inhibitor-induced differentiation as a therapy in the clinic.

Systematic Analysis of miR-506-3p Target Genes Identified Key Mediators of Its Differentiation-Inducing Function.

Background/Objectives: miR-506-3p has been demonstrated to be a strong inducer of neuroblastoma cell differentiation, highlighting the potential of applying miR-506-3p mimics to neuroblastoma differentiation therapy. However, the target genes of miR-506-3p that mediate its differentiation-inducing function have not been fully defined. This study aims to comprehensively investigate the targetome of miR-506-3p regarding its role in regulating neuroblastoma cell differentiation. Methods: We combined gene expression profiling and functional high-content screening (HCS) to identify miR-506-3p target genes that have differentiation-modulating functions. For evaluating the potential clinical relevance of the identified genes, we analyzed the correlations of gene expressions with neuroblastoma patient survival. Results: We identified a group of 19 target genes with their knockdown significantly inducing cell differentiation, suggesting that these genes play a key role in mediating the differentiation-inducing activity of miR-506-3p. We observed significant correlations of higher mRNA levels with lower patient survival with 13 of the 19 genes, suggesting that overexpression of these 13 genes plays important roles in promoting neuroblastoma development by disrupting the cell differentiation pathways. Conclusions: Through this study, we identified novel target genes of miR-506-3p that function as strong modulators of neuroblastoma cell differentiation. Our findings represent a significant advancement in understanding the mechanisms by which miR-506-3p induces neuroblastoma cell differentiation. Future investigations of the identified 13 genes are needed to fully define their functions and mechanisms in controlling neuroblastoma cell differentiation, the understanding of which may reveal additional targets for developing novel differentiation therapeutic agents.

A peptide against the N-terminus of myristoylated alanine-rich C kinase substrate promotes neuronal differentiation in SH-SY5Y human neuroblastoma cells.

Myristoylated alanine-rich protein kinase C substrate (MARCKS) plays crucial roles in neuronal functions and differentiation. However, specific effects of the myristoylated N-terminal sequence (MANS) peptide, a widely used MARCKS modulator comprising the initial 24 amino acids of MARCKS, on neuronal cells remain unclear. Therefore, in this study, we aimed to examine the effects and action mechanisms of the MANS peptide on SH-SY5Y human neuroblastoma cells, which served as the in vitro neuronal cell models. MANS treatment of SH-SY5Y cells resulted in significant neurite outgrowth within 24 hr, which was as prominent as that induced by seven days of treatment with all-trans retinoic acid, the most common agent used to induce SH-SY5Y cell differentiation. Levels of synaptophysin, a neuronal marker protein, were significantly increased in the MANS peptide-treated cells. Additionally, increased MARCKS levels and decreased MARCKS phosphorylation were observed in MANS peptide-treated cells. Notably, neurite outgrowth induced by the MANS peptide was significantly reduced in MARCKS-knocked-down cells. Overall, these results suggest the MANS peptide as a novel agent for SH-SY5Y cell differentiation, particularly for the analysis of MARCKS functions.

Alteration of mature neuronal marker of β-III tubulin expression in differentiated SH-SY5Y cells by refinement of foetal bovine serum concentration

BackgroundThe process of differentiating neuroblastoma cells (SH-SY5Y) is crucial for obtaining mature neuronal markers. However, there is variability in the concentrations of foetal bovine serum (FBS) used in the differentiation media, ranging from 1 to 10%. This inconsistency in FBS concentrations may contribute to the inconsistent differentiation of cells. To improve the utility of the SH-SY5Y cell line as a model for neuronal cell culture, we investigated the impact of FBS concentrations in the differentiation media using Dulbecco's modified eagle medium and 10 μM all-trans-retinoic acid (ATRA). The aim of this study was to optimise the concentrations of FBS in the differentiation media of SH-SY5Y cells. Our study focused on assessing the length of dendrites in neuronal cells and the expression of β-III tubulin, a marker indicative of mature neurons. SH-SY5Y cells were differentiated with 10 µM of ATRA for 7 days. Four treatment groups with different FBS concentrations (1%, 3%, 5%, and 10%) were examined to assess the alteration of cellular morphology and dendritic length. The expression of the mature neuron marker β-III tubulin was evaluated using immunocytochemistry technique.ResultsSH-SY5Y cells' dendrite length was significantly longer (p < 0.05) when there was a higher concentration of FBS in the differentiation medium. The result was confirmed with the significant increase of β-III tubulin expression (p < 0.001) of the differentiated SH-SY5Y cells that have been incubated with higher concentrations of FBS in the differentiation medium.ConclusionsWe concluded that optimised concentrations of FBS in the differentiation media display longer length of the dendrites and express higher production of β-III tubulin in the differentiated SH-SY5Y cells. The consistency of serum concentration used in the differentiation media is important to produce a sustainable in vitro neuronal model of SH-SY5Y cells for neurodegenerative studies.

Novel miRNA-inducing drugs enable differentiation of retinoic acid-resistant neuroblastoma cells.

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA-modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and BDNF-activating bufalin synergistically arrests proliferation, induces differentiation, and maintains the differentiated state of MES SK-N-AS cells for 8 weeks. RNA-seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained MES neuroblastoma GI-ME-N and SH-EP cell lines, as well as glioblastoma LN-229 and U-251 cell lines, for over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.

Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

KLF7 promotes neuroblastoma differentiation through the GTPase signaling pathway by upregulating neuroblast differentiation-associated protein AHNAKs and glycerophosphodiesterase GDPD5.

The arrest of neural crest-derived sympathoadrenal neuroblast differentiation contributes to neuroblastoma formation, and overriding this blocked differentiation is a clear strategy for treating high-risk neuroblastoma. A better understanding of neuroblast or neuroblastoma differentiation is essential for developing new therapeutic approaches. It has been proposed that Krueppel-like factor 7 (KLF7) is a neuroblastoma super-enhancer-associated transcription factor gene. Moreover, KLF7 was found to be intensely active in postmitotic neuroblasts of the developing nervous system during embryogenesis. However, the role of KLF7 in the differentiation of neuroblast or neuroblastoma is unknown. Here, we find a strong association between high KLF7 expression and favorable clinical outcomes in neuroblastoma. KLF7 induces differentiation of neuroblastoma cells independently of the retinoic acid (RA) pathway and acts cooperatively with RA to induce neuroblastoma differentiation. KLF7 alters the GTPase activity and multiple differentiation-related genes by binding directly to the promoters of neuroblast differentiation-associated protein (AHNAK and AHNAK2) and glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) and regulating their expression. Furthermore, we also observe that silencing KLF7 in neuroblastoma cells promotes the adrenergic-to-mesenchymal transition accompanied by changes in enhancer-mediated gene expression. Our results reveal that KLF7 is an inducer of neuroblast or neuroblastoma differentiation with prognostic significance and potential therapeutic value.

Regulatory roles of insulin growth factor binding protein family in neuroblastoma cell proliferation and differentiation: Potential prognostic biomarkers and therapeutic targets for neuroblastoma

AbstractNeuroblastoma (NB), as a representative of tumors of embryonic origin in children, has specific clinical features. On the one hand, a very small number of NBs may appear to regress on their own. On the other hand, highly malignant NBs can invade the surrounding blood vessels and organs and metastasize to distant bone, bone marrow, and lymph nodes in the early stages of the disease. Based on differential affinities to insulin growth factors (IGFs), insulin growth factor binding proteins (IGFBPs) are classified into two groups: IGF binding proteins (IGFBP1‐6) with high‐affinity and IGF low‐affinity binding proteins, such as IGFBP‐related proteins (IGFBP rP1‐10). IGFBP are crucial regulators of the bioavailability and function of IGF in metabolic signaling and as modulators of IGF signaling, and their role in NB is gaining increasing attention. In this study, we investigate the involvement of IGFBP family members in the growth and differentiation of NB cells, as well as the potential of IGFBPs as prognostic biomarkers and therapeutic targets for human NB.

ZMYND8 Is a Regulator of Sonic Hedgehog Signaling in ATRA-Mediated Differentiation of Neuroblastoma Cells.

Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, GLI1, and PTCH1 expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the GLI1 and PTCH1 gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.

Lipid levels correlate with neuronal and dopaminergic markers during the differentiation of SH-SY5Y cells

Parkinson's Disease (PD) is characterised by the loss of dopaminergic neurons and the deposition of protein inclusions called Lewy Bodies (LBs). LBs are heterogeneous structures composed of protein and lipid molecules and their main constituent is the presynaptic protein α-synuclein. SH-SY5Y cells are neuroblastoma cells commonly used to model PD because they express dopaminergic markers and α-synuclein and they can be differentiated into neuronal cells using established protocols. Despite increasing evidence pointing towards a role of lipids in PD, limited knowledge is available on the lipidome of undifferentiated and differentiated SH-SY5Y cells. Using a combination of lipidomics, proteomics, morphological and electrophysiological measurements, we identified specific lipids, including sphingolipids, whose levels are affected by the differentiation of SH-SY5Y neuroblastoma cells and found that the levels of these lipids correlate with those of neuronal and dopaminergic markers. These results provide a quantitative characterisation of the changes in lipidome associated with the differentiation of SH-SY5Y cells into more neuronal and dopaminergic-like phenotype and serve as a basis for further characterisation of lipid disruptions in association with PD and its risk factors in this dopaminergic-like neuronal cell model.

Effects of Anionic Liposome Delivery of All-Trans-Retinoic Acid on Neuroblastoma Cell Differentiation.

All-trans-retinoic acid (ATRA) has long been known to affect cell growth and differentiation. To improve ATRA's therapeutic efficacy and pharmacodynamics, several delivery systems have been used. In this study, free ATRA and anionic-liposome-encapsulated ATRA were compared for their effects on SK-N-SH human neuroblastoma cell growth and differentiation. Anionic liposomes made of L-α-phosphatidylcholine (PC) and L-α-phosphatidic acid (PA), empty (PC-PA) and loaded with ATRA (PC-PA-ATRA), were characterized by dynamic light scattering (DLS) and electrophoretic mobility measurements, and drug entrapment efficiency (EE%) was measured to evaluate the applicability of the new colloidal formulation. The results of brightfield microscopy and cell growth curves indicated that ATRA, whether free or encapsulated, reduced growth and induced differentiation, resulting in SK-N-SH cells changing from epithelioid to neuronal-like morphologies, and producing a significant increase in neurite growth. To further characterize the neuro-differentiation of SK-N-SH cells, the expression of βIII-Tubulin and synaptophysin and mitochondria localization were analyzed via immunofluorescence. Increased expression of neuronal markers and a peculiar localization of mitochondria in the neuritic extensions were apparent both in ATRA- and PC-PA-ATRA-differentiated cells. As a whole, our results strongly indicate that ATRA treatment, by any means, can induce the differentiation of parent SK-N-SH, and they highlight that its encapsulation in anionic liposomes increases its differentiation ability in terms of the percentage of neurite-bearing cells. Interestingly, our data also suggest an unexpected differentiation capability of anionic liposomes per se. This work highlights the importance of developing and carefully testing novel delivery nanocarriers, which are a necessary first "step" in the development of new therapeutic settings.

Abstract 2852: Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells

Abstract Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system derived from migratory neural crest cells that have committed to become sympathetic neuroblasts, but their definitive differentiation is blocked. Neuroblastoma cell identity depends on the adrenergic core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive cell proliferation and the oncogenic gene expression program. Retinoic acid is used as differentiation therapy for patients with high-risk neuroblastoma, with the goal to block proliferation and drive sympathetic neuronal differentiation. We recently showed that retinoic acid re-organizes the enhancer landscape of neuroblastoma and promotes a new retino-sympathetic cell state characterized by MYCN downregulation, proliferative arrest, and sympathetic neuronal differentiation. However, the effects of retinoic acid on epigenetic rewiring of the adrenergic CRC and suppression of MYCN expression are reversible in vitro and in vivo, leading to tumor re-growth when the retinoic acid is withdrawn. Therefore, we sought to determine if retinoid-induced differentiation can be forced to progress to irreversible neuronal maturation by the addition of inhibitors of epigenetic modifying enzymes. Here, we conducted a drug screen of epigenetic modifying drugs alone and in combination with retinoic acid to determine their effects on neuroblastoma cell growth and differentiation. In this screen, we found that an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, PF-9363, synergistically inhibits neuroblastoma cell growth in combination with retinoic acid. Moreover, retinoid-resistant neuroblastoma cell lines can be sensitized to retinoic acid when combined with a KAT6 inhibitor. Importantly, this combination treatment renders the differentiated cell state irreversible, such that it is maintained after retinoic acid is withdrawn, with continued suppression of the adrenergic CRC and MYCN expression. In conclusion, the retino-sympathetic differentiated cell state induced by retinoic acid in neuroblastoma becomes irreversible when the cells are also treated with an inhibitor of the KAT6A/B histone H3K23 acetyltransferases, implicating the essential role of these enzymes in restoring the proliferative immature progenitor phenotype in adrenergic neuroblastoma cells. Citation Format: Nina Weichert-Leahey, Alla Berezovskaya, Mark Zimmerman, Brian J. Abraham, Adam W. Durbin, A Thomas Look. Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2852.

A Potential Prognostic Gene Signature Associated with p53-Dependent NTRK1 Activation and Increased Survival of Neuroblastoma Patients.

Neuroblastoma is the most common extracranial solid tumour in children, comprising close to 10% of childhood cancer-related deaths. We have demonstrated that activation of NTRK1 by TP53 repression of PTPN6 expression is significantly associated with favourable survival in neuroblastoma. The molecular mechanisms by which this activation elicits cell molecular changes need to be determined. This is critical to identify dependable biomarkers for the early detection and prognosis of tumours, and for the development of personalised treatment. In this investigation we have identified and validated a gene signature for the prognosis of neuroblastoma using genes differentially expressed upon activation of the NTRK1-PTPN6-TP53 module. A random survival forest model was used to construct a gene signature, which was then assessed across validation datasets using Kaplan-Meier analysis and ROC curves. The analysis demonstrated that high BASP1, CD9, DLG2, FNBP1, FRMD3, IL11RA, ISGF10, IQCE, KCNQ3, and TOX2, and low BSG/CD147, CCDC125, GABRB3, GNB2L1/RACK1 HAPLN4, HEBP2, and HSD17B12 expression was significantly associated with favourable patient event-free survival (EFS). The gene signature was associated with favourable tumour histology and NTRK1-PTPN6-TP53 module activation. Importantly, all genes were significantly associated with favourable EFS in an independent manner. Six of the signature genes, BSG/CD147, GNB2L1/RACK1, TXNDC5, FNPB1, B3GAT1, and IGSF10, play a role in cell differentiation. Our findings strongly suggest that the identified gene signature is a potential prognostic biomarker and therapeutic target for neuroblastoma patients and that it is associated with neuroblastoma cell differentiation through the activation of the NTRK1-PTPN6-TP53 module.

Aagab is required for zebrafish larval development by regulating neural activity

Clathrin-mediated endocytosis has been implicated in various physiological processes, including nutrient uptake, signal transduction, synaptic vesicle recycling, maintenance of cell polarity, and antigen presentation. Despite prior knowledge of its importance as a key regulator in promoting clathrin-mediated endocytosis, the physiological function of α- and γ-adaptin binding protein (aagab) remains elusive. In this study, we investigate the biological function of aagab during zebrafish development. We establish a loss-of-function mutant of aagab in zebrafish, revealing impaired swimming and early larval mortality. Given the high expression level of aagab in the brain, we probe into its physiological role in the nervous system. aagab mutants display subdued calcium responses and local field potential in the optic tectal neurons, aligning with reduced neurotransmitter release (e.g., norepinephrine) in the tectal neuropil of aagab mutants. Overexpressing aagab mRNA or nervous stimulant treatment in mutants restores neurotransmitter release, calcium responses, swimming ability, and survival. Furthermore, our observations show delayed release of FM 1–43 in AAGAB knockdown differentiated neuroblastoma cells, pointing towards a probable link to defective clathrin-mediated synaptic vesicle recycling. In conclusion, our study underscores the significance of Aagab in neurobiology and suggests its potential impacts on neurological disorders.

Development of a molecular glue-based Lin28 degrader to regulate cellular proliferation and stemness.

Let-7 microRNAs (miRNAs) regulate cellular processes including stemness and proliferation. Lin28, an RNA-binding protein, controls let-7 miRNA biogenesis and is a key factor in maintaining stem cell properties. We developed SB1349, a novel molecular glue-based degrader targeting Lin28. SB1349 induces Lin28 degradation through a proteasome-dependent pathway, enhances let-7 miRNA levels, and downregulates oncogenes c-Myc and IMP1. SB1349 also promotes the differentiation in neuroblastoma cells, highlighting its potential as a therapeutic agent for various diseases.

Identification of CDKN3 as a Key Gene that Regulates Neuroblastoma Cell Differentiation.

We conducted a high-content screening (HCS) in neuroblastoma BE(2)-C cells to identify cell cycle regulators that control cell differentiation using a library of siRNAs against cell cycle-regulatory genes. We discovered that knocking down expression of cyclin dependent kinase inhibitor 3 (CDKN3) showed the most potent effect in inducing neurite outgrowth, the morphological cell differentiation marker of neuroblastoma cells. We then demonstrated that CDKN3 knockdown increased expression of neuroblastoma molecular differentiation markers, neuron specific enolase (NSE), βIII-tubulin and growth associated protein 43 (GAP43). We further showed that CDKN3 knockdown reduced expression of cell proliferation markers Ki67 and proliferating cell nuclear antigen (PCNA), and reduced colony formation of neuroblastoma cells. More importantly, we observed a correlation of high tumor CDKN3 mRNA levels with poor patient survival in the investigation of public neuroblastoma patient datasets. In exploring the mechanisms that regulate CDKN3 expression, we found that multiple strong differentiation-inducing molecules, including miR-506-3p and retinoic acid, down-regulated CDKN3 expression. In addition, we found that N-Myc promoted CDKN3 expression at the transcriptional level by directly binding to the CDKN3 promoter. Furthermore, we found that CDKN3 and two additional differentiation-regulating cell cycle proteins identified in our HCS, CDC6 and CDK4, form an interactive network to promote expression of each other. In summary, we for the first time discovered the function of CDKN3 in regulating neuroblastoma cell differentiation and characterized the transcriptional regulation of CDKN3 expression by N-Myc in neuroblastoma cells. Our findings support that CDKN3 plays a role in modulating neuroblastoma cell differentiation and that overexpression of CDKN3 may contribute to neuroblastoma progression.

Nordihydroguaiaretic Acid Affects Undifferentiated and Differentiated Neuroblastoma Cells Differently through Mechanisms that Impact on Cell Viability.

We aimed to investigate the mechanisms involved in the neurotoxic effects of NDGA on differentiated and undifferentiated human neuroblastoma cells (MSN), assessing cell viability, changes in the actin cytoskeleton, cell migration and the expression of the 5-LOX enzyme and the inhibitor of cell cycle progression p21WAF1/CIP1. High expression and activity of the lipoxygenase enzyme (LOX) have been detected in several tumors, including neuroblastoma samples, suggesting the use of LOX inhibitors as potential therapy molecules. Among these, the natural compound nordihydroguaiaretic acid (NDGA) has been extensively tested as an antiproliferative drug against diverse types of cancer cells. In this study, we analyzed the toxic effect of NDGA on neuroblastoma cells at a dose that did not affect cell survival when they differentiated to a neuron-like phenotype and the potential mechanisms involved in the anticancer properties. We exposed human neuroblastoma cells (MSN) to different concentrations of NDGA before and after a differentiation protocol with retinoic acid and nerve growth factor and analyzed cell viability, cell migration, actin cytoskeleton morphology and the levels of the cell cycle inhibitor p21WAF1/CIP1 and 5-LOX. We found that differentiated human neuroblastoma cells are more resistant to NDGA than undifferentiated cells. The toxic effects of NDGA were accompanied by reduced cell migration, changes in actin cytoskeleton morphology, induction of p21WAF1/CIP1 and decreased levels of the 5-LOX enzyme. This study provides new evidence regarding the potential use of NDGA to induce cell death in human neuroblastoma.

Dyngo-4a Induces Neuroblastoma Cell Differentiation Through The AKT and ERK1/2 Pathway.

The aim of the study is to check whether dyngo-4a can inhibit neuroblastoma (NB) proliferation and induce NB cell differentiation Background: Dynamin plays a role in regulating neurotransmission, signaling pathways, nutrient uptake, and pathogen infection, enhancing cell proliferation, tumor invasion, and metastasis. Studies have reported that dyngo-4a, a dynamin inhibitor, can be used to identify potential biomarkers and promising novel therapeutic targets for cancer treatment. To our knowledge, no published reports are showing that dynamin inhibitors can reduce NB cell proliferation and induce differentiation. In this study, we report that dyngo-4a can inhibit NB proliferation and induce NB cell differentiation. In this study, mouse neuroblastoma (Neuro-2a) cells were cultured in the presence or absence of dyngo-4a or retinoic acid (RA), or in the presence of both dyngo-4a and RA, or in the presence of sequential administration of dyngo-4a and RA to compare the effects on the inhibition of cell proliferation and effects on neuroblastoma cell differentiation induction. The neural cell markers, Nestin and Tuj 1 (Neuron-specific class III beta-tubulin), were used to demonstrate that the differentiated cells have neuronal cell features. The phosphorylation of Protein Kinase B (AKT), extracellular signalregulated kinases1/2 (ERK1/2), and epidermal growth factor receptor (EGFR) were determined to examine the potential mechanisms of induced differentiation. Dyngo-4a or RA or dyngo-4a with subsequent RA administration induced Neuro-2a cell differentiation. However, RA with subsequent dyngo-4a administration results in almost total death of the Neuro-2a cells. The differentiation rate induced by dyngo-4a was significantly higher than the rate by RA treatment (72.5 ± 1.4% vs. 52.9 ± 3.1% with neuron features, P<0.05; 39.0 ± 0.8% vs. 29.9 ± 1.8% for axons under light microscopy, p<0.05). The differentiation rate of cells treated with dyngo-4a first, followed by RA, was greater than when they were added together (74.8 ± 3.8% vs. 10.6 ± 3.6%; 45.5 ± 1.6% vs. 12.4 ± 0.6%, p<0.01). Co-administration of dyngo-4a and RA at the same time diminished differentiation efficacy significantly. Dyngo-4a induced Neuro-2a cell differentiation and increased Tuj-1 positive staining by the 6th day post- treatment. Dyngo-4a also inhibited Neuro-2a cell proliferation in a dose-dependent manner. Regarding the mechanism, dyngo-4a treatment showed a significant increase in p-AKT and p-ERK1/2 but not in p-EGFR. At a level comparable to RA, dynamin inhibition with dyngo-4a lowers proliferation and causes differentiation of Neuro-2a mouse NB cells in vitro. The AKT pathway is activated by dynago- 4a, which results in differentiation. The combination of RA with dynago-4a reduces the efficiency of differentiation. The application of dynago-4a followed by RA, on the other hand, enhances the differentiating effect, implying alternative mechanistic roles in the process.

Differentiated neuroblastoma cells remain epigenetically poised for de-differentiation to an immature state.

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for a significant share of childhood cancer deaths. Prior studies utilizing RNA sequencing of bulk tumor populations showed two predominant cell states characterized by high and low expression of neuronal genes. Although cells respond to treatment by altering their gene expression, it is unclear whether this reflects shifting balances of distinct subpopulations or plasticity of individual cells. Using mouse and human neuroblastoma cell lines lacking MYCN amplification, we show that the antigen CD49b (also known as ITGA2) distinguishes these subpopulations. CD49b expression marked proliferative cells with an immature gene expression program, whereas CD49b-negative cells expressed differentiated neuronal marker genes and were non-cycling. Sorted populations spontaneously switched between CD49b expression states in culture, and CD49b-negative cells could generate rapidly growing, CD49b-positive tumors in mice. Although treatment with the chemotherapy drug doxorubicin selectively killed CD49b-positive cells in culture, the CD49b-positive population recovered when treatment was withdrawn. We profiled histone 3 (H3) lysine 27 acetylation (H3K27ac) to identify enhancers and super enhancers that were specifically active in each population and found that CD49b-negative cells maintained the priming H3 lysine 4 methylation (H3K4me1) mark at elements that were active in cells with high expression of CD49b. Improper maintenance of primed enhancer elements might thus underlie cellular plasticity in neuroblastoma, representing potential therapeutic targets for this lethal tumor.

Optimization of 3D Synthetic Scaffolds for Neuronal Tissue Engineering Applications.

The increasing prevalence of neurodegenerative diseases has spurred researchers to develop advanced 3D models that accurately mimic neural tissues. Hydrogels stand out as ideal candidates as their properties closely resemble those of the extracellular matrix. A critical challenge in this regard is to comprehend the influence of the scaffold's mechanical properties on cell growth and differentiation, thus enabling targeted modifications. In light of this, a synthesis and comprehensive analysis of acrylamide-based hydrogels incorporating a peptide has been conducted. Adequate cell adhesion and development is achieved due to their bioactive nature and specific interactions with cellular receptors. The integration of a precisely controlled physicochemical hydrogel matrix and inclusion of the arginine-glycine-aspartic acid peptide sequence has endowed this system with an optimal structure, thus providing a unique ability to interact effectively with biomolecules. The analysis fully examined essential properties governing cell behavior, including pore size, mechanical characteristics, and swelling ability. Cell-viability experiments were performed to assess the hydrogel's biocompatibility, while the incorporation of grow factors aimed to promote the differentiation of neuroblastoma cells. The results underscore the hydrogel's ability to stimulate cell viability and differentiation in the presence of the peptide within the matrix.