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Purpose: Long non-coding RNAs (lncRNAs) play important roles in progression of neuroblastoma (NB). LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been shown to affect the development of multiple tumors. However, the effect of NEAT1 on NB remain unclear. In this study, the new mechanisms whereby how NEAT1 influences tumor progression in NB was investigated., Methods: RT-qPCR, western blot, bioinformatics, cell growth, Transwell, and flow cytometric analyses were performed to determine how NEAT1 synchronously regulates the miR-873-5p/MYCN proto-oncogene(MYCN) and miR-873-5p/polypeptide N-acetylgalactosaminyltransferase 1(GalNAcT-I) axes to accelerate the progression of NB. NB-bearing animal models were established to evaluate the function of NEAT1 in NB. The relationships between transcription factor coactivatorassociated arginine methyltransferase 1 (CARM1) and NEAT1, NEAT1 and miR-873-5p, miR-873-5p and GalNacT-I or MYCN, were verified using luciferase reporter gene assay, respectively., Results: Our study revealed elevated levels of NEAT1 expression in NB cells and tissues which was associated with an advanced pathological stage and poor prognostic outcomes. According to in vitro gain- and loss- of function experiments, NEAT1 enhances progression of NB. NEAT1 silencing was found to inhibit NB proliferation in vivo. Mechanistically, to achieve upstream regulation, epigenetic downregulation of NEAT1 was achieved via the inhibition of CARM1. NEAT1 was found to positively regulate MYCN and GalNAcT-I levels as a competitive sponge of miR-873-5p., Conclusion: Activity of the lncRNA NEAT1 can be triggered via CARM1, which synchronously promotes NB development via the miR-873-5p/MYCN and miR-873-5p/GalNAcT-I axes. These findings shed light on the novel molecular mechanisms underlying NB progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Dysregulated deubiquitinating enzymes (DUBs) execute as intrinsic oncogenes or tumor suppressors and are involved in chemoresistance in cancers. However, the functions and exact molecular mechanisms remain largely unclear in neuroblastoma., Methods: Here, an R2 screening strategy based on the standard deviation values was used to identify the most important DUB, USP44, in neuroblastoma with stage 4. We validated the role of USP44 regulation upon cisplatin treatment in vitro and in vivo experiments, revealing the molecular mechanisms associated with USP44 regulation and cisplatin sensitivity in neuroblastoma., Results: We found that low USP44 expression was associated with an inferior prognosis in neuroblastoma patients. Overexpression of USP44 enhanced neuroblastoma cell sensitivity to cisplatin in vitro and in vivo. Mechanistically, USP44 recruited and stabilized the E3 ubiquitin ligase STUB1 by removing its K48-linked polyubiquitin chains at Lys30, and STUB1 further reinforced the K48-linked polyubiquitination of LRPPRC at Lys453 and promoted its protein degradation, thus enhancing the accumulation of mitochondrial reactive oxygen species (mROS), in turn facilitating neuroblastoma cell apoptosis and cisplatin sensitivity. Additionally, overexpression of LRPPRC reversed the promoting effect of USP44 on cell apoptosis in cisplatin-treated neuroblastoma cells., Conclusions: Our findings demonstrate that the USP44-STUB1-LRPPRC axis plays a pivotal role in neuroblastoma chemoresistance and provides potential targets for neuroblastoma therapy and prognostication., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Neuroblastoma (NB) is a prevalent extracranial malignant neuroendocrine tumor in children, originating from the sympathetic nervous system. This study aims to investigate new therapeutic targets for NB. The differentially expressed genes were screened by analyzing the GSE35133 and GSE90689 datasets. Hub genes were identified by constructing a protein-protein interaction network. The diagnostic value of the hub genes was assessed through the analysis of receiver operating characteristic (ROC) curves and the expression, prognosis, and immune infiltration of IFI27 in pan-cancer were analyzed on the online website Sangerbox. The hub gene expression levels were validated by performing real-time reverse transcriptase-polymerase chain reaction. The functions of IFI27 in NB were investigated by Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, wound healing, and Transwell assays. Six candidate genes (IFI27, TNFSF10, IFI44, DDX58, HIST1H1C, and HIST1H1E) were identified as potential diagnostic biomarkers for NB. The expression levels of IFI27, TNFSF10, IFI44, and DDX58 were significantly decreased, while HIST1H1C and HIST1H1E were elevated. Notably, IFI27 displayed correlations with prognosis and immune infiltration in multiple tumors. In vitro, functional assays demonstrated that the knockdown of IFI27 promoted the proliferation, migration, and invasion of U251 cells. Conversely, in SK-N-AS cells, IFI27 overexpression inhibited cell proliferation, migration, and invasion. IFI27 was lowly expressed in NB and participated in the progression of NB, which provides a new insight into the pathogenic mechanism and novel therapeutic strategy for NB., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests. Ethical approval: Not applicable. Consent for publication: Not applicable. Consent to participate: Not applicable., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Heterogeneous clinical features and prognosis in neuroblastoma (NB) children are frequently dominated by immune elements. Dysfunction and apoptosis in immune cells result from the exposure to continuous tumor-related antigen stimulation and coinhibitory signals. To date, key factors pointing to the restriction of NB-specific CD8 + T cells remain elusive., Methods: We performed bulk-RNA sequencing and lipidomic analyses of children with mediastinal NB. Bioinformatics analysis and biological validation were applied to uncover the underlying mechanism., Results: Three subtypes were identified using nonnegative matrix factorization (NMF), among which we highlighted an apoptotic status of infiltrated CD8 + T cells, along with the highest CD52 and EDF1 expression in Cluster3 (C3) subtypes. It was verified that high EDF1 expression in NB cells led to Lactosylceramide (LacCer) accumulation, as well as downstream ganglioside-GD3, which subsequently increased the expression of CD52 and immune checkpoint genes, chemotaxis, and apoptosis-related events in activated CD8 + T cells. Mechanistically, EDF1 was recruited as a coactivator to form the NF-κB/RelA/EDF1 complex, which further prevented the promoter region methylation of ST8SIA1, to elevate its transcription., Conclusion: These findings characterize abundant GD3 in NB cells, which regulated by the EDF1/RelA/ST8SIA1 axis, is responsible for CD8 + T cell dysfunction. Inhibition of EDF1 may reduce suppressive factors and prevent immune escape of NB cells. Modulating NB-associated GD3 levels through metabolic intervention is beneficial for tuning the depth and duration of responses to current NB therapies. The integration of transcriptomic and lipidomic data offers a more comprehensive understanding of the interaction between LacCer metabolites and the immune status in NB., Competing Interests: Declarations. Ethics approval and consent to participate: The research was approved by the Ethics Committee of Guangzhou Women and Children’s Medical Center(2023-120A01). Consent for publication: All subjects have written informed consent. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

c-MYC is an important driver of high-risk neuroblastoma. A lack of c-MYC-driven genetically engineered mouse models (GEMM) has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and to develop effective therapies. In this study, we showed that conditional c-MYC induction via Cre recombinase driven by a tyrosine hydroxylase promoter led to a preponderance of PDX1+ somatostatinoma, a type of pancreatic neuroendocrine tumor. However, c-MYC activation via an improved Cre recombinase driven by a dopamine β-hydroxylase promoter resulted in neuroblastoma development. The c-MYC murine neuroblastoma tumors recapitulated the pathologic and genetic features of human neuroblastoma and responded to anti-GD2 immunotherapy and difluoromethylornithine, an FDA-approved inhibitor targeting the MYC transcriptional target ODC1. Thus, c-MYC overexpression results in different but related tumor types depending on the targeted cell. The GEMMs represent valuable tools for testing immunotherapies and targeted therapies for these diseases. Significance: The development of c-MYC-driven genetically engineered neuroblastoma and somatostatinoma mouse models provides useful tools for understanding the tumor cell origin and investigating treatment strategies., (©2024 The Authors; Published by the American Association for Cancer Research.)

Previously considered junk or non-functional, long non-coding RNAs (lncRNAs) have emerged over the past few decades as pivotal components in both physiological and pathological processes, including cancer. Neuroblastoma-associated transcript-1 (NBAT-1) was initially discovered a decade ago as a risk-associated tumor suppressor lncRNA in neuroblastoma (NB). Subsequent studies have consistently demonstrated that NBAT-1 serves as a dedicated tumor suppressor in many cancers. NBAT-1 is significantly downregulated in cancer, which is closely linked to higher histological grades, increased metastasis, and poor survival in cancer patients suggesting NBAT-1's potential as a prognostic biomarker. In this review, we delve into the current body of literature, elucidating the tumor-suppressive roles of NBAT-1 and the underlying regulatory mechanisms in the context of human malignancies. Additionally, we shed light on the mechanisms contributing to the diminished expression of NBAT-1 and its potential as both a prognostic biomarker and a promising therapeutic target in cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Current treatment protocols have limited success against MYCN-amplified neuroblastoma. Adoptive T cell therapy presents an innovative strategy to improve cure rates. However, L1CAM-targeting CAR T cells achieved only limited response against refractory/relapsed neuroblastoma so far. We investigated how oncogenic MYCN levels influence tumor cell response to CAR T cells, as one possible factor limiting clinical success. A MYCN-inducible neuroblastoma cell model was created. L1CAM-CAR T cell effector function was assessed (activation markers, cytokine release, tumor cytotoxicity) after coculture with the model or MYCN-amplified neuroblastoma cell lines. RNA sequencing datasets characterizing the model were compared to publicly available RNA/proteomic datasets. MYCN-directed L1CAM regulation was explored using public ChIP-sequencing datasets. Synergism between CAR T cells and the indirect MYCN inhibitor, MLN8237, was assessed in vitro using the Bliss model and in vivo in an immunocompromised mouse model. Inducing high MYCN levels in the neuroblastoma cell model reduced L1CAM expression and, consequently, L1CAM-CAR T cell effector function in vitro. Primary neuroblastomas possessing high MYCN levels expressed lower levels of both the L1CAM transcript and L1CAM tumor antigen. MLN8237 treatment restored L1CAM tumor expression and L1CAM-CAR T cell effector function. Combining MLN8237 and L1CAM-CAR T cell treatment synergistically enhanced MYCN-overexpressing tumor cytotoxicity in vitro and in vivo concomitant with severe in vivo toxicity. We identify target antigen downregulation as source of resistance against L1CAM-CAR T cells in MYCN-driven neuroblastoma cells. These data suggest that L1CAM-CAR T cell therapy combined with pharmacological MYCN inhibition may benefit patients with MYCN-amplified neuroblastoma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lead is a ubiquitous environmental chemical with various toxic damage to human body. This investigation aimed to explore the intervention effect of human umbilical cord mesenchymal stem cells derived exosomes (HUC-MSC-exo) on the neurotoxicity of lead and the relevant mechanism. Differential gradient ultracentrifugation was adopted to isolate HUC-MSC-exo. Nanoparticle tracking assay (NTA), Transmission electron microscope (TEM) technology and exosomal specific biomarkers CD9, CD63 and CD81 were adopted for exosomal characterization. Human neuroblastoma cell (SH-SY5Y) was used as the recipient cell. Confocal laser scanning microscope analysis was conducted to confirm the intake of HUC-MSC-exo by SH-SY5Y cells. Cell migration ability, apoptosis, IL-6, IL-1β and TNF-α were analyzed. The role of miR-26a-5p/PTEN axis was assessed. The result showed that the exposure of SH-SY5Y cells to lead activated the miR-26a-5p/PTEN pathway by down-regulating miR-26a-5p and up-regulating PTEN expression, which was related to the significantly decreased cell migration and increased apoptosis, as well as significantly enhanced levels of inflammatory cytokine as compared with the control. While HUC-MSC-exo could significantly alleviate the cytotoxicity, apoptosis and inflammatory effects induced by lead on SH-SY5Y cells via partially restoring miR-26a-5p/PTEN pathway. Herein, we conclude that HUC-MSC-exo can alleviate lead-induced toxic effects on SH-SY5Y cells partially through miR-26a-5p/PTEN pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Neuroblastoma (NB) is a predominantly pediatric cancer with greater than 90% of cases arising in children under the age of five. More than half of patients have metastases detected at diagnosis, and high-risk disease is associated with five-year survival rates of only 50-60 %. Standard therapy involves highly toxic chemotherapy, surgery, radiation, and immunotherapy, and less toxic, more specific targeted therapies are urgently needed. Genomic studies have identified common driver aberrations in high-risk NB, such as MYCN amplification. In addition, a proportion of high-risk patients harbor amplification or activating mutations in anaplastic lymphoma kinase (ALK), and co-occurrence of ALK mutations and MYCN amplification have been associated with aggressive disease. In this study, we analyzed the efficacy of a Phase Ia-cleared, orally bioavailable dual ALK and focal adhesion kinase (FAK) inhibitor, ESK440, in multiple preclinical NB models. ESK440 potently inhibited proliferation of NB cell lines, with increased sensitivity in cell lines harboring ALK aberrations. ALK, FAK, and downstream target activation were rapidly decreased upon ESK440 treatment, and this was associated with impaired cellular migration and invasion. Importantly, ESK440 treatment also decreased MYCN levels. NB cell line and patient-derived xenograft studies showed significant reduction in tumor growth in ESK440-treated mice with no signs of toxicity. In certain NB models, ESK440 showed comparable or enhanced efficacy to lorlatinib, another clinical ALK inhibitor, and a lorlatinib-resistant cell line (COG-N-561 LR) retained sensitivity to ESK440. These preclinical results indicate that ESK440 is a promising targeted agent for ALK-driven NB and support future clinical studies to evaluate its efficacy in NB patients., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The University of Michigan has filed a provisional patent application based on the findings from this study (A.M.C. and S.C. are named as co-inventors). A.M.C. is a co-founder and serves on the scientific advisory board of Esanik Therapeutics, Inc. that licensed ESK440 from Teva Pharmaceuticals. Teva Pharmaceuticals or Esanik Therapeutics did not contribute to study design and funding. A.M.C. is also a co-founder and scientific advisor to LynxDx, Medsyn Bio, and Flamingo Therapeutics. He serves as an advisor to RAAPTA Therapeutics, Ascentage Pharma, Aurigene Oncology, and Tempus. The remaining authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: Neuroblastoma (NB) originates from differentiation arrest of sympathoadrenal progenitors in the neural crest. It is necessary to reveal the differentiation mechanism of NB. Previously, we reported that Purkinje cell protein 4 (PCP4) is a well-differentiated marker of NB tissues. Herein, we explored the underlying mechanism of PCP4 induced differentiation in order to find better treatment options for patients., Methods: We screened the interacting proteins of PCP4 by co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS/MS). Then we investigated the relevance between expression of calmodulin-dependent protein kinase II gamma (CAMK2G) and clinical features using R2 platform. We also explored the function of CAMK2G in NB cells by knockdown and RNA sequencing., Results: Here, we verified the binding of PCP4 and calmodulin (CaM) by Co-IP and identified a target kinase of CaM, CAMK2G, by LC-MS/MS. PCP4 overexpression activates the autophosphorylation of CAMK2G. Patients with high CAMK2G expression had better survival while low CAMK2G was associated with unfavorable clinical features including MYCN-amplification, unfavorable histology, progression and high INSS stage. CAMK2G knockdown inhibited neurite outgrowth and down-regulated neuronal differentiation markers (NF-H, MAP2), yet promoted migration, invasion and proliferation. Gene Ontology (GO) analysis showed that knockdown of CAMK2G downregulated the expression of neuronal differentiation-related genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that knockdown of CAMK2G upregulated the expression of migration-related genes., Conclusion: These findings indicate that CAMK2G activated by PCP4/CaM complex promotes differentiation and inhibits migration in NB cells., Level of Evidence: Not applicable., Competing Interests: Conflict of interest The authors declare no conflict of interest. There is not any competing financial interest in relation to the work described., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Neuroblastoma, a prevalent extracranial solid tumor in pediatric patients, demonstrates significant clinical heterogeneity, ranging from spontaneous regression to aggressive metastatic disease. Despite advances in treatment, high-risk neuroblastoma remains associated with poor survival. SLC1A5, a key glutamine transporter, plays a dual role in promoting tumor growth and immune modulation. However, its contributions to neuroblastoma biology remain largely unexplored., Methods: This study utilized clinical neuroblastoma samples from 20 patients and 1310 cases from four public datasets to investigate SLC1A5 expression, biological function, and prognostic significance. Differential expression, Kaplan-Meier survival analysis, gene set enrichment analysis, and weighted correlation network analysis were conducted. Functional validation included qPCR, immunohistochemistry, Western blotting, and cell proliferation assays using the SLC1A5 inhibitor V-9302. A prognostic signature, SRPS, was constructed and validated using machine-learning approaches. Immune infiltration analysis was performed to evaluate the tumor immune microenvironment., Results: SLC1A5 expression was significantly elevated in high-risk neuroblastoma and correlated with advanced stages and poor prognosis. GSEA revealed mTORC1 signaling enrichment in high SLC1A5 expression groups, validated by increased p-p70S6K levels in tumor samples and neuroblastoma cells. V-9302 treatment suppressed mTORC1 signaling and inhibited cell proliferation. Hub-genes were identified to form the SRPS model, which demonstrated superior prognostic performance compared to existing models. Immune infiltration analysis revealed a more immunosuppressive tumor microenvironment associated with high SLC1A5 expression. Additionally, SLC1A5 negatively regulated ST8SIA1, a gene crucial for GD2 biosynthesis, suggesting that SLC1A5 inhibition may enhance GD2-directed immunotherapies., Conclusion: SLC1A5 plays a pivotal role in neuroblastoma by promoting tumor progression and shaping an immunosuppressive microenvironment. The SRPS model, incorporating SLC1A5-associated genes, offers robust prognostic utility. Targeting SLC1A5 through advanced drug delivery systems and combined metabolic-immunotherapeutic strategies may enhance treatment specificity and efficacy. These findings provide a foundation for novel therapeutic approaches to improve outcomes in high-risk neuroblastoma patients., Competing Interests: Declarations. Ethics approval and consent to participate: The tissue samples and blood test results of patients utilized in this study were subjected to a comprehensive review and obtained approval from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (approval number: 2024-KY-0047). All research was conducted in accordance with the relevant guidelines and regulations of the Ethics Committee and research involving human research participants have been performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from the parents or guardians. Consent for publication: Written informed consent for publication was obtained from the parents or guardians. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The dynamic interplay between tumor cells and γδT cells within the tumor microenvironment significantly influences disease progression and immunotherapy outcome. In this study, we delved into the modulation of γδT-cell activation by tumor cell ligands CD112 and CD155, which interact with the activating receptor DNAM-1 on γδT cells. Spatial and single-cell RNA sequencing, as well as spatial metabolomic analysis, from neuroblastoma revealed that the expression levels and localization of CD112 and CD155 varied across and within tumors, correlating with differentiation status, metabolic pathways, and ultimately disease prognosis and patient survival. Both in vivo tumor xenograft experiments and in vitro coculture experiments demonstrated that a high CD112/CD155 expression ratio in tumors enhanced γδT cell-mediated cytotoxicity, whereas a low ratio fostered tumor resistance. Mechanistically, CD112 sustained DNAM-1-mediated γδT-cell activation, whereas CD155 downregulated DNAM-1 expression via E3 ubiquitin ligase tripartite motif-containing 21-mediated ubiquitin proteasomal degradation. By interacting with tumor cells differentially expressing CD112 and CD155, intratumoral γδT cells exhibited varying degrees of activation and DNAM-1 expression, representing three major functional subsets. This study underscores the complexity of tumor-immune cross-talk, offering insights into how tumor heterogeneity shapes the immune landscape. Significance: Tumor cells in different intratumoral neighborhoods display divergent patterns of ligands that regulate γδT-cell activation, highlighting multilevel regulation of antitumor immunity resulting from the heterogeneity of intercellular interactions in the tumor microenvironment., (©2024 The Authors; Published by the American Association for Cancer Research.)

Central nervous system neuroblastoma with forkhead box R2 (FOXR2) activation (NB-FOXR2) is a high-grade tumor of the brain hemispheres and a newly identified molecular entity. Tumors express dual neuronal and glial markers, leading to frequent misdiagnoses, and limited information exists on the role of FOXR2 in their genesis. To identify their cellular origins, we profiled the transcriptomes of NB-FOXR2 tumors at the bulk and single-cell levels and integrated these profiles with large single-cell references of the normal brain. NB-FOXR2 tumors mapped to LHX6+/DLX+ lineages derived from the medial ganglionic eminence, a progenitor domain in the ventral telencephalon. In vivo prenatal Foxr2 targeting to the ganglionic eminences in mice induced postnatal cortical tumors recapitulating human NB-FOXR2-specific molecular signatures. Profiling of FOXR2 binding on chromatin in murine models revealed an association with ETS transcriptional networks, as well as direct binding of FOXR2 at key transcription factors that coordinate initiation of gliogenesis. These data indicate that NB-FOXR2 tumors originate from LHX6+/DLX+ interneuron lineages, a lineage of origin distinct from that of other FOXR2-driven brain tumors, highlight the susceptibility of ventral telencephalon-derived interneurons to FOXR2-driven oncogenesis, and suggest that FOXR2-induced activation of glial programs may explain the mixed neuronal and oligodendroglial features in these tumors. More broadly, this work underscores systematic profiling of brain development as an efficient approach to orient oncogenic targeting for in vivo modeling, critical for the study of rare tumors and development of therapeutics. Significance: Profiling the developing brain enabled rationally guided modeling of FOXR2-activated CNS neuroblastoma, providing a strategy to overcome the heterogeneous origins of pediatric brain tumors that hamper tumor modeling and therapy development. See related commentary by Orr, p. 195., (©2024 The Authors; Published by the American Association for Cancer Research.)

Mouse models that faithfully represent the biology of human brain tumors are critical tools for unraveling the underlying tumor biology and screening for potential precision therapies. This is especially true of rare tumor types, many of which have correspondingly few xenograft or cell lines available. Although our understanding of the specific biological pathways driving cancer has improved significantly, identifying the appropriate progenitor populations to drive oncogenic processes represents a significant barrier to efficient mouse model production. In this issue of Cancer Research, Jessa and colleagues developed an innovative transcription factor fingerprinting method to map the cellular origin of central nervous system neuroblastoma, FOXR2-activated to medial ganglionic eminence-derived interneurons, which could then be efficiently targeted in the developing mouse brain using in utero electroporation. This approach serves as a blueprint for investigating other rare pediatric brain tumors, potentially accelerating progress toward the development of mouse models and identification of effective therapies. See related article by Jessa et al., p. 231., (©2024 American Association for Cancer Research.)

Translation initiation of most mammalian mRNAs is mediated by a 5' cap structure that binds eukaryotic initiation factor 4E (eIF4E). Notably, most mRNAs are still capped when eIF4E is inhibited, suggesting alternative mechanisms likely mediate cap-dependent mRNA translation without functional eIF4F. Here we found that, when eIF4E is inhibited, the ribosomal scaffold RACK1 recruits eIF3d on the 43S pre-initiation complex. Moreover, we found that it is just PKCBII in its active form that promotes the binding of RACK1 to eIF3d. These studies disclose a previously unknown role of ribosomal RACK1 for eIF3d specialized translation., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuroblastomas are the most common solid tumors outside of the brain that originate from immature neural crest cells, accounting for about 10% of all pediatric malignancies. The treatment for neuroblastomas involves a multimodal schedule, including surgery, radiation, chemotherapy, and immunotherapy. All these modalities are limited by side effects that might be severe, poor prognosis, and a high risk of recurrence. In the quest for additional therapeutic approaches, phytochemicals have attracted attention owing to their reported antitumor properties, safety, and multimechanistic mode of action. Several studies have used plant-derived bioactive compounds such as phenolics and flavonoids, suggesting modulation of biomolecules and signal transduction pathways involved in neuroblastoma. We reviewed the findings of recent preclinical and clinical studies demonstrating the effects of phytochemicals on neuroblastoma, shedding light on their molecular mechanism of action and potential therapeutic applications., (© 2024 International Union of Biochemistry and Molecular Biology.)

Background: RRS1 plays an important role in regulating ribosome biogenesis. Recently, RRS1 has emerged as an oncoprotein involved in tumorigenicity of some cancers. However its role in neuroblastoma remains unknown., Methods: RRS1 expression was detected in pediatric neuroblastoma patients' tissues and cell lines. The effects of RRS1 knockdown on proliferation, apoptosis, and cell cycle were evaluated in neuroblastoma cell lines. RRS1-related survival pathway was analyzed by co-immunoprecipitation (Co-IP), mass spectrometry, reverse transcription-quantitative real-time PCR (RT-qPCR), and western blot. Protein-protein interaction (PPI) network was constructed using Cytoscape software and the STRING databases., Results: Increased RRS1 level was found in neuroblastoma cases (35.6%) and cell lines. High RRS1 expression levels were associated with poor prognosis. RRS1 knockdown inhibited cell proliferation, induced apoptosis, and caused cell cycle arrest in SK-N-AS and SH-SY5Y cells. Co-IP and mass spectrometry analysis showed that RRS1 affects PI3K/Akt and nuclear factor κB (NF-κB) pathways. RT-qPCR and western blot results revealed that RRS1 knockdown inhibited the PI3K/Akt/NF-κB pathway through dephosphorylation of key proteins. In PPI network, AKT, PI3K, and P65 connected RRS1 with differentially expressed proteins more closely., Conclusions: This study suggests RRS1 knockdown may inhibit neuroblastoma cell proliferation by the PI3K/Akt/NF-κB pathway. Therefore, RRS1 may be a potential target for neuroblastoma treatment., Impact: RRS1 is involved in the progression of neuroblastoma. Knockdown of RRS1 contributes to inhibit the survival of neuroblastoma cells. RRS1 is associated with the PI3K/Akt/NF-κB signaling pathway in neuroblastoma cells. RRS1 may be a promising target for neuroblastoma therapy., Competing Interests: Competing interests: The authors declare no competing interests. Ethics statement: Informed consent was obtained from all patient’s parents and this study was approved by the ethics board of the Affiliated Hospital of Qingdao University., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

The SH-SY5Y human neuroblastoma cell line is a standard in vitro experimental model of neuronal-like cells used in neuroscience and toxicological research. These cells can be differentiated into mature neurons, most commonly using retinoic acid (RA). Despite differences in characteristics, both undifferentiated and differentiated SH-SY5Y cells are used in research. However, due to uncertainties regarding the expression of specific markers of neural function in each culture, there is no definite conclusion on which culture is better suited for (neuro)toxicological and/or neuroscience investigations. To address this dilemma, we investigated the basal expression/activity of the key elements of acetylcholine, dopamine, serotonin, and GABA neurotransmitter pathways, along with the elements involved in exocytosis of neurotransmitters, and neuron electrophysiological activity in undifferentiated and in RA-differentiated SH-SY5Y cells using a six-day differentiation protocol. Our findings revealed that both SH-SY5Y cell types are functionally active. While undifferentiated SH-SY5Y cells exhibited greater multipotency in the expression of tested markers, most of those markers expressed in both cell types showed higher expression levels in RA-differentiated SH-SY5Y cells. Our results suggest that the six-day differentiation protocol with RA induces maturation, but not differentiation of the cells into specific neuron phenotype. The greater multipotency of undifferentiated cells in neural markers expression, together with their higher sensitivity to xenobiotic exposure and more simple cultivation protocols, make them a better candidate for high throughput toxicological screenings. Differentiated neurons are better suited for neuroscience researches that require higher expression of more specific neural markers and the specific types of neural cells.

Background: Neuroblastoma (NB) is one of the most common pediatric solid tumors. Emerging evidence has indicated that ADGRL4 can act as a master regulator of tumor progression. In addition, it is well documented that the ERK/STAT3 signaling pathway can promote the proliferation, EMT, angiogenesis, and metastasis in tumors. The current study was formulated to elucidate the exact role of ADGRL4 in the malignant behaviors of NB cells and to investigate the intrinsic mechanism., Methods: In this work, expression differences of ADGRL4 in human NB cell lines and HUVECs were assessed via RT-qPCR and western blot analysis. For functional experiments, sh-ADGRL4 was transfected into SK-N-SH cells to generate ADGRL4 knockdown stable cell line. Moreover, ADGRL4 knockdown stable SK-N-SH cells were treated with LM22B-10 (an ERK activator) for rescue experiments. CCK-8, colony formation, would healing, and transwell assays determined NB cell growth, migration, and invasion. Meanwhile, proliferation-, metastasis- and EMT- associated proteins were also detected. Additionally, a tube formation assay was employed to evaluate in vitro angiogenesis. VM-cadherin, the marker of angiogenesis, was assessed using immunofluorescence staining., Results: Data showed notably upregulated ADGRL4 in NB cells, especially in SK-NSH cells. ADGRL4 knockdown inhibited NB cell growth, migration, invasion, EMT, and in vitro angiogenesis. ADGRL4 knockdown inactivated ERK/STAT3 signaling pathway. Activation of the ERK/STAT3 signaling pathway partially rescued the tumor suppression effects of ADGRL4 knockdown on NB cells., Conclusion: To conclude, the downregulation of ADGRL4 may inhibit cell growth, aggressiveness, EMT, and angiogenesis in NB by inactivating the ERK/STAT3 signaling pathway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Hematological and neurological expressed 1 (HN1), encoding a small protein, has been recently explored in different cancers owing to its higher expression in tumor samples as compared to adjacent normal. It was discovered and subsequently named because of its higher expression in hematological and neurological tissues in developing mice. Following discovery, it was considered a neuronal regeneration or dedifferentiation-related gene. However, since then, it has not been characterized in neuroblastoma or differentiated neurons. SH-SY5Y cell line presents a unique model of neuroblastoma often utilized in neurobiology research. In this study, first, we employed bioinformatics analysis along with in vitro evaluation using normal and retinoic acid (RA)-differentiated SH-SY5Y cells to determine the responses of HN1 and its function. The analysis revealed that HN1 expression is higher in neuroblastoma and lower in differentiated neurons and Parkinson's disease as compared to appropriate controls. Since HN1 coexpression network in neuroblastoma is found to be enriched in cell-cycle-related pathways, we have shown that HN1 expression increases in S-phase and remains lower in the rest of the cell cycle phases. Moreover, HN1 expression is also correlated with the microtubule stability in SH-SY5Y cells, which was investigated with nocodazole and taxol treatments. HN1 overexpression increased the ratio of S-type cells (undifferentiated), indicating that it acts as a dedifferentiating factor in neuroblastoma cells. Moreover, cell cycle dynamics also changed upon HN1 overexpression with alternating effects on SH-SY5Y and RA-differentiated (N-type) cells. Therefore, HN1 is a potential cell cycle regulatory element in the development of neuroblastoma or dedifferentiation of neurons, which requires further studies to decipher its mechanistic role., (© 2025 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

The clinical manifestations of Parkinson's disease (PD) are driven by aggregation of α-Synuclein (α-Syn) in the brain. However, there is increasing evidence that PD may be initiated in the gut and thence spread to the brain, eg, via the vagus nerve. Many studies link PD to changes in the gut microbiome, and bacterial amyloid has been shown to stimulate α-Syn aggregation. Yet, we are not aware of any studies reporting on a direct connection between microbiome components and α-Syn aggregation. Here, we report that soluble extract from the gut microbiome of the rats, particularly young rats transgenic for PD, shows a remarkably strong ability to inhibit in vitro α-Syn aggregation and keep it natively unfolded and monomeric. The active component(s) are heat-labile molecule(s) of around 30- to 100-kDa size, which are neither nucleic acid nor lipid. Proteomic analysis identified several proteins whose concentrations in different rat samples correlated with the samples' anti-inhibitory activity, while a subsequent pull-down assay linked the protein chaperone DnaK with the inhibitory activity of young rat's microbiome, confirmed in subsequent in vitro assays. Remarkably, the microbiome extracts also protected neuroblastoma SH-SY5Y cells and zebrafish embryos against α-Syn toxicity. Our study sheds new light on the gut microbiome as a potential source of protection against PD and opens up for new microbiome-based therapeutic strategies., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background/aim: Treatment with retinoic acid (RA) often promotes neuroblastoma differentiation and growth inhibition, including the suppression of the expression of the MYCN oncogene. However, RA also targets protumoral chemokines, such as CCL2, which may contribute to the development of resistance. The present study aimed to investigate the regulation and function of CCL2 and N-Myc in RA-treated neuroblastoma cells., Materials and Methods: In Kelly or SH-SY5Y cells, viability was quantified by cell fitness assays. Expression was analyzed using quantitative PCR and the regulation of proteins using enzyme-linked immunoabsorbent assays (ELISA) or western blots., Results: In MYCN-amplified Kelly cells, endogenous CCL2 levels were significantly lower compared to MYCN non-amplified SH-SY5Y cells. Treatment with 5 μM RA increased CCL2 release in both cell lines, but reduced N-Myc levels and cell numbers in Kelly cells. Over-expression of MYCN enhanced viability in SH-SY5Y cells, but did not affect RA-induced CCL2 release, while supplementation of CCL2 in Kelly cells did not prevent RA-mediated growth reduction. Impaired N-Myc or CCL2 signaling reduced the survival of all RA-treated cells and inhibition of N-Myc also decreased CCL2 levels. However, attenuated survival signaling was not generally associated with reduced levels of N-Myc or CCL2. Co-application of RA and the growth factor receptor inhibitors cediranib or crizotinib decreased N-Myc levels only in Kelly cells, while CCL2 release was dependent on the cell type and stimulus., Conclusion: CCL2 and N-Myc promote the viability of RA-treated cells, although the levels of these mediators were not consistently correlated with cellular outcomes, especially during apoptotic signaling., (Copyright © 2025, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: CDK4 is highly expressed and associated with poor prognosis and decreased survival in advanced neuroblastoma (NB). Targeting CDK4 degradation presents a potentially promising therapeutic strategy compared to conventional CDK4 inhibitors. However, the autophagic degradation of the CDK4 protein and its anti-proliferation effect in NB cells has not been mentioned., Results: We identified autophagy as a new pathway for the degradation of CDK4. Firstly, autophagic degradation of CDK4 is critical for NVP-BEZ235-induced G0/G1 arrest, as demonstrated by the overexpression of CDK4, autophagy inhibition, and blockade of autophagy-related genes. Secondly, we present the first evidence that p62 binds to CDK4 and then enters the autophagy-lysosome to degrade CDK4 in a CTSB-dependent manner in NVP-BEZ235 treated NB cells. Similar results regarding the interaction between p62 and CDK4 were observed in the NVP-BEZ235 treated NB xenograft mouse model., Conclusions: Autophagic degradation of CDK4 plays a pivotal role in G0/G1 cell cycle arrest in NB cells treated with NVP-BEZ235.

Background: Neuroblastoma (NB), the most prevalent solid tumor in children, arises from sympathetic nervous system and accounts for 15% of pediatric cancer mortality. This malignancy exhibits substantial genetic and clinical heterogeneity, thus complicating treatment strategies. Poly(ADP-ribose) polymerase 1 (PARP1), a key enzyme catalyzing polyADP-ribosylation (PARylation), plays critical roles in various cellular processes, and contributes to tumorigenesis and aggressiveness. However, the functions and regulatory mechanisms of PARP1 in NB progression still remain to be determined., Methods: The association of PARP1 expression with NB patients' survival was analyzed by mining of R2 database. Western blotting, reverse transcription-polymerase chain reaction, MTT colorimetric, soft agar, and matrigel invasion assays were utilized to assess PARP1 expression and its effects on aggressiveness of NB cell lines. Chromatin immunoprecipitation (ChIP) sequencing and ChIP assays were employed to investigate the binding of Yin Yang 1 (YY1) to PARP1 promoter. Protein interactions were explored by BioGRID database analysis, molecular docking, and co-immunoprecipitation assay. RNA sequencing and crosslinking-immunoprecipitation high throughput sequencing datasets were used to identify precursor mRNA splicing targets of non-POU domain containing octamer binding protein (NONO)., Results: High PARP1 expression was associated with poor survival of NB patients. PARP1 over-expression enhanced the proliferation and invasion of NB cell lines, confirming its oncogenic roles. YY1 was identified as a key transcriptional regulator facilitating PARP1 expression. Additionally, PARP1 interacted with NONO to induce its PARylation, resulting in stabilization of NONO protein via preventing ubiquitin-mediated degradation. NONO facilitated the splicing and mRNA maturation of target genes a disintegrin and metalloproteinase domain 8 (ADAM8) and testis-expressed gene 14 (TEX14) in a PARylation-dependent manner. Rescue experiments indicated that YY1 facilitated PARP1-mediated PARylation of NONO and subsequent mRNA maturation of ADAM8 and TEX14 in NB cells. In clinical NB cases, high expression of YY1, PARP1, NONO, ADAM8, or TEX14 was associated with poor survival of patients., Conclusions: These findings indicate that YY1 drives PARP1 expression essential for PARylation of NONO in mRNA maturation during NB progression., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: All authors have agreed to publish this manuscript. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Neuroblastoma is a common malignant tumor in childhood that seriously endangers the health and lives of children, making it essential to find effective prognostic markers to accurately predict their clinical outcomes. The development of high-throughput technology in the biomedical field has made it possible to obtain multi-omics data, whose integration can compensate for missing or unreliable information in a single data source. In this study, we integrated clinical data and two omics data, i.e., gene expression and DNA methylation data, to study the prognosis of neuroblastoma. Since the features in omics data are redundant, it is crucial to conduct feature selection on them. We proposed a two-step feature selection (TSFS) method to quickly and accurately select the optimal features, where the first step aims at selecting candidate features and the second step is to remove redundant features among them using our proposed maximal association coefficient (MAC). Our goal is to predict composite clinical outcomes for neuroblastoma patients, i.e., their survival time and vital status at the last follow-up, which was validated to be two inter-correlated tasks. We conducted a series of experiments and evaluated the experimental results using accuracy and AUC (area under the ROC curve) evaluation metrics, which indicated that by the combination of the integration of the three types of data, our proposed TSFS method and a multi-task learning method can synergistically improve the reliability and accuracy of the prediction models.

The zebrafish (Danio rerio) is a valuable model organism for studying human biology due to its easy genetic manipulation and small size. It is optically transparent and shares genetic similarities with humans, making it ideal for studying developmental processes, diseases, and drug screening via imaging-based approaches. Solid malignant tumors often contain hypoxic areas that stimulate the release of extracellular vesicles (EVs), lipid-bound structures released by cells into the extracellular space, that facilitate short- and long-range intercellular communication and metastatization. Here we investigate the effects of EVs derived from neuroblastoma (NB), a pediatric solid tumor, on metastatic niche formation using the zebrafish as an in vivo model. Intravascular injection in zebrafish embryos allows a non-invasive visualization of EVs dispersion, uptake, and interactions with host cells. To improve repeatability of our results and ease the injection steps, we used an agarose device replica molded from a custom designed micromilled aluminum mold. We first demonstrated that EVs released under hypoxic conditions promote angiogenesis and are more easily internalized by endothelial cells than those purified from normoxic cells. We also showed that injection of with hypoxic EVs increased macrophages mobilization. We then focused on the caudal hematopoietic tissue (CHT) region of the embryo as a potential metastatic site. After hypoxic EVs injection, we highlighted changes in the expression of mmp-9 and cxcl8b genes. Furthermore, we investigated the ability of NB-derived EVs to prime a metastatic niche by a two-step injection of EVs first, followed by NB cells. Interestingly, we found that embryos injected with hypoxic EVs had more proliferating NB cells than those injected with normoxic EVs. Our findings suggest that EVs released by hypoxic NB cells alter the behavior of recipient cells in the zebrafish embryo and promote metastatic outgrowth. In addition, we demonstrated the ability of the zebrafish embryo to be a suitable model for studying the interactions between EVs and recipient cells in the metastatic process., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fietta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

MYCN amplification is a hallmark of aggressive neuroblastoma, driving N-Myc overexpression and enhancing protein synthesis, making these processes potential therapeutic targets. Here, we present a protocol for quantifying nascent N-Myc and global protein translation in neuroblastoma cells. This protocol describes the steps for labeling nascent proteins and performing an optimized click chemistry reaction directly on the membrane after blotting, enabling high-sensitivity detection and analysis. Adaptable to other proteins of interest, this approach provides valuable insights into neuroblastoma protein synthesis. For complete details on the use and execution of this protocol, please refer to Chittavanich et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

It is little known that Acanthamoeba trophozoites and Toxocara canis eggs can reduce tumors in vitro and animal models. Although this has been known for many years, the mechanism that induces the antitumor effect in these parasites is still not known. We employed Western blot (WB) and immunofluorescence (IFC) by confocal microscopy to explore the potential protein binding between neuroblastoma (NB) SH-SY5Y cells and anti- Acanthamoeba and anti- Toxocara canis antibodies. Using WB, we detected two fragments of 70 kDa and 60 kDa recognized by the anti- Acanthamoeba antibodies, and two fragments of 115 kDa and 70 kDa recognized by the anti- Toxocara canis antibodies. In both cases, the IFC results were positive in the cell membrane of the SH-SY5Y cells. Our findings suggest a potential overlap of similar molecules between these parasites and tumor cells, which may contribute to tumor elimination. Investigating the relationship between anti- Acanthamoeba and anti- Toxocara canis antibodies in neoplastic cells could provide evidence for the future use of these anti-parasitic antibodies in targeting NB or other cancers.

The first of our aims in this study was to investigate the effects of 5-HT2AR, 5-HT7R, and A2AR blockades on miR-27b-3p expression in the short and long-term in neuroblastoma cells. Our second aim was to reduce the expression of pERK and suppress proliferation by blocking the 5-HT2AR with ketanserin. Our third aim was to reduce the expression of pAKT and induce apoptosis by blocking the A2AR and 5-HT7R with MSX3 and SB269970. Thus, we aimed to investigate the therapeutic efficacy of ketanserin, MSX3 and SB269970, individually or in combination, on neuroblastoma cells. We found that short and long-term blockades of A2A, 5-HT2A, and 5-HT7 receptors had different effects on miR-27b-3p expression. Blockade of A2AR and 5-HT7R with MSX3 and SB269970 decreased miR-27b-3p expression in the short term while increasing it in the long term. Ketanserin increased miR-27b-3p expression in both the short and long term. When 5-HT2AR was blocked with ketanserin, no significant difference was observed in pERK expression and proliferation in the short term. In contrast, a substantial decrease in pERK expression and proliferation was detected in the long term. Our findings show that the MSX3 + SB269970 dual combination and ketanserin + MSX3 + SB269970 triple combination are especially critical in suppressing pAKT expression in the long term. These findings showed that pAKT protein expression induced apoptosis due to decreased in neuroblastoma cells. Our study provides the first evidence for the relationships between ketanserin/miR-27b-3p/pERK, MSX3/miR-27b-3p/pAKT, and SB269970/miR-27b-3p/pAKT in neuroblastoma cells. Ketanserin, MSX3, and SB269970 drug combinations may be promising therapeutic agents in neuroblastoma cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Brain Research Organization (IBRO). Published by Elsevier Inc. All rights reserved.)

HuD plays a critical role in neurite outgrowth, neuronal plasticity, and survival. However, HuD autoantibodies from patients with paraneoplastic gut dysmotility can trigger the apoptotic cascade in human neuroblastoma cell line and myenteric neurons. The mechanism by which HuD regulates the apoptotic pathway is unclear. Apoptosis is one of the underlying causes of neurodegenerative diseases like Alzheimer's disease. In the current study, we found that HuD interacts with Msi2 transcript and positively regulates it in the mouse neuroblastoma (N2a) cells. MSI2 being an RNA binding protein has diverse mRNA targets and regulates the mitochondrial apoptotic pathway by interacting with and repressing APAF1 transcript. Conversely, the reduced levels of HuD leads to decreased Msi2 expression and increased APAF1 levels, which results in apoptosis in N2a cells. Overall, our research indicates that HuD and Msi2 possess an anti-apoptotic role in N2A cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gaikwad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Neuroblastoma (NB) is a severe pediatric tumor originating from the developing sympathetic nervous system, characterized by diverse clinical outcomes, including spontaneous regression and aggressive metastasis. This variability suggests the existence of different NB subtypes, necessitating accurate classification for effective targeted treatment. In this study, we employed the similarity network fusion (SNF) algorithm and identified three NB subtypes, including mesenchymal-like (MES), MYCN-like (MYCN), and neurogenic-like (Neurogenic). The MES subtype exhibited the highest activation of immune-related pathways. The MYCN subtype demonstrated the worst prognosis, with enrichment in cell growth and proliferation pathways. Conversely, the Neurogenic subtype showed the best prognosis, with enrichment in sympathetic nervous system development processes. Through single-cell RNA sequencing (scRNA-seq) analysis, we examined the tumor microenvironments of these distinct NB subtypes, revealing divergent differentiation trajectories for adrenergic cells within the MYCN and Neurogenic subtypes. We also identified a significant presence of naïve T cells in the MES subtype, as well as mesenchymal cell subtypes associated with the unique plasticity observed in both the MES and MYCN subtypes. Drug sensitivity prediction analysis suggested that the MES subtype may respond favorably to MEK inhibitors, while the MYCN subtype may be susceptible to Bcl-2 inhibitors. Our integrative multi-omics approach enabled precise stratification of NB into biologically distinct subtypes, potentially facilitating the development of subtype-specific therapeutic strategies for improved patient management and survival outcomes., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The cognitive dysfunction in the brain cause severe pathological consequences such as Alzheimer's disease (AD), Parkinson's disease. The current treatments are cost expensive and also cause negative side effects. Therefore it is inevitable to develop natural phyto-compounds as a drug like molecules to treat neurodegenerative diseases. In this context, we have assayed the neuroprotective effects of betanin, an indole derivative, in the neuroblastoma cell line SHSY-5Y cells. The neuroprotective effect was investigated in the β-amyloid (Aβ) - induced SHSY-5Y cells; betanin (25 µg) protected the SHSY-5Y cells from the toxic effects and maintained the cell viability. Moreover, the acridine orange and ethidum Bromide staining decipher that treatment of betanin in the Aβ-induced SHSY-5Y cells maintain the cell viablity sustainably. The Reactive Oxygen Species (ROS) assay infers that betanin quenches the generation of free radicals progressively in the Aβ-induced SHSY-5Y cells. In addition, the autophagy determination by flow cytometry revealed that betanin induces autophagy to remove the neurodegenerated cells. Further, we examined the docking and simulation patterns with the angiotensin-converting enzyme (ACE), TNF-α converting enzyme (TACE), glycogen synthase kinase 3 (GK3), and acetylcholinesterase enzymes (AChE) and amyloid precursor protein (APP). The insilico docking analysis denotes that betanin had a significant docking score with the target molecules. Thus, from the invitro and insilico studies, betanin strongly inhibit the toxic effects of Aβand protect the cells from degeneration., Competing Interests: Declarations. Ethical Approval: Not Applicable. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Selenocysteine (Sec) metabolism is crucial for cellular function and ferroptosis prevention and begins with the uptake of the Sec carrier, selenoprotein P (SELENOP). Following uptake, Sec released from SELENOP is metabolized via selenocysteine lyase (SCLY), producing selenide, a substrate for selenophosphate synthetase 2 (SEPHS2), which provides the essential selenium donor, selenophosphate (H 2 SePO 3 - ), for the biosynthesis of the Sec-tRNA. Here, we discovered an alternative pathway in Sec metabolism mediated by peroxiredoxin 6 (PRDX6), independent of SCLY. Mechanistically, we demonstrate that PRDX6 can readily react with selenide and interact with SEPHS2, potentially acting as a selenium delivery system. Moreover, we demonstrate the functional significance of this alternative route in human cancer cells, revealing a notable association between elevated expression of PRDX6 and human MYCN-amplified neuroblastoma subtype. Our study sheds light on a previously unrecognized aspect of Sec metabolism and its implications in ferroptosis, offering further possibilities for therapeutic exploitation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The function and mechanism of Cyclophilin A (CypA) in modulating gene expression associated with Alzheimer's disease (AD) remain unclear. This multifunctional protein is found to be elevated in the cerebrospinal fluid (CSF) of individuals at risk for AD. The cytotoxic effects of CypA, including both wild-type and the mutant R55A, were assessed using the MTT assay. Prior to this evaluation, the purified recombinant protein was validated through enzymatic activity assays and western blot analysis. Following treatment with CypA and transient transfection using the CypA construct, real-time PCR (qRT-PCR) and western blotting were conducted to analyze the expression of factors involved in various signaling pathways, with an emphasis on inflammation, cell death, and intercellular communication. The findings indicate that CypA has a significant impact on the gene expression of factors associated with inflammation and the progression of AD in SH-SY5Y cells. It can be concluded that CypA is capable of regulating gene expression in SH-SY5Y cells, either in a manner dependent on or independent of its enzymatic activity. Additionally, the influence of this multifunctional protein on gene expression is contingent upon the specific site of action, as well as the dosage and duration of exposure to the cells., Competing Interests: Declarations. Competing interests: The authors affirm that there are no competing interests to disclose., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Relapse rates in high-risk neuroblastoma remain exceedingly high. The malignant cells that are responsible for relapse have not been identified, and mechanisms of therapy resistance remain poorly understood. In this study, we used single-nucleus RNA sequencing and bulk whole-genome sequencing to identify and characterize the residual malignant persister cells that survive chemotherapy from a cohort of 20 matched diagnosis and definitive surgery tumor samples from patients treated with high-risk neuroblastoma induction chemotherapy. We show that persister cells share common mechanisms of chemotherapy escape, including suppression of MYC(N) activity and activation of NFκB signaling, and the latter is further enhanced by cell-cell communication between the malignant cells and the tumor microenvironment. Overall, our work dissects the transcriptional landscape of cellular persistence in high-risk neuroblastoma and paves the way to the development of new therapeutic strategies to prevent disease relapse. Significance: Approximately 50% of patients with high-risk neuroblastoma die of relapsed refractory disease. We identified the malignant cells that likely contribute to relapse and discovered key signaling pathways that mediate cellular persistence. Inhibition of these pathways and their downstream effectors is postulated to eliminate persister cells and prevent relapse. See related commentary by Wolf et al., p. 2308., (©2024 The Authors; Published by the American Association for Cancer Research.)

In our previous studies, we demonstrated that 20 h pre-exposure of SH-SY5Y human neuroblastoma cells to 1950 MHz, UMTS signal, at specific absorption rate of 0.3 and 1.25 W/kg, was able to reduce the oxidative DNA damage induced by a subsequent treatment with menadione in the alkaline comet assay while not inducing genotoxicity per se. In this study, the same cell model was used to test the same experimental conditions by setting different radiofrequency exposure duration and timing along the 72 h culture period. The results obtained in at least three independent experiments indicate that shorter exposure durations than 20 h, that is, 10, 3, and 1 h per day for 3 days, were still capable to exert the protective effect while not inducing DNA damage per se. In addition, to provide some hints into the mechanisms underpinning the observed phenomenon, thioredoxin-1, heat shock transcription factor 1, heat shock protein 70, and poly [ADP-ribose] polymerase 1, as key molecular players involved in the cellular stress response, were tested following 3 h of radiofrequency exposure in western blot and qRT-PCR experiments. No effect resulted from molecular analysis under the experimental conditions adopted., (© 2024 The Author(s). Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.)

The development of drug resistance reduces the efficacy of cancer therapy. Tumor cells can acquire resistance to MDM2 inhibitors, which are currently under clinical evaluation. We generated RG7388-resistant neuroblastoma cells, which became more proliferative and metabolically active and were less sensitive to DNA-damaging agents in vitro and in vivo, compared with wild-type cells. The resistance was associated with a mutation of the p53 protein (His193Arg). This mutation abated its transcriptional activity via destabilization of the tetrameric p53-DNA complex and was observed in many cancer types. Finally, we found that Cisplatin and various BH3-mimetics could enhance RG7388-mediated apoptosis in RG7388-resistant neuroblastoma cells, thereby partially overcoming resistance to MDM2 inhibition., (© 2024. The Author(s).)

The impact of exercise on pediatric tumor biology is essentially unknown. We explored the effects of regular exercise on tumor proteome profile (as assessed with liquid chromatography with tandem mass spectrometry) in a mouse model of one of the most aggressive childhood malignancies, high-risk neuroblastoma (HR-NB). Tumor samples of 14 male mice (aged 6-8 wk) that were randomly allocated into an exercise (5-wk combined aerobic and resistance training) or nonexercise control group (6 and 8 mice/group, respectively) were analyzed. The Search Tool for the Retrieval of Interacting Genes/Proteins database was used to generate a protein-protein interaction (PPI) network and enrichment analyses. The Systems Biology Triangle (SBT) algorithm was applied for analyses at the functional category level. Tumors of exercised mice showed a higher and lower abundance of 101 and 150 proteins, respectively, than controls [false discovery rate (FDR) < 0.05]. These proteins were enriched in metabolic pathways, amino acid metabolism, regulation of hormone levels, and peroxisome proliferator-activated receptor signaling (FDR < 0.05). The SBT algorithm indicated that 184 and 126 categories showed a lower and higher abundance, respectively, in the tumors of exercised mice (FDR < 0.01). Categories with lower abundance were involved in energy production, whereas those with higher abundance were related to transcription/translation, apoptosis, and tumor suppression. Regular exercise altered the abundance of hundreds of intratumoral proteins and molecular pathways, particularly those involved in energy metabolism, apoptosis, and tumor suppression. These findings provide preliminary evidence of the molecular mechanisms underlying the potential effects of exercise in HR-NB. NEW & NOTEWORTHY We used liquid chromatography with tandem mass spectrometry to explore the impact of a 5-wk exercise intervention on the tumor proteome profile in a mouse model of one of the most aggressive childhood malignancies, high-risk neuroblastoma. Exercise altered the abundance of hundreds of proteins and pathways, particularly those involved in energy metabolism and tumor suppression. These molecular changes could mediate, at least partly, the potential antitumorigenic effects of exercise.

Long noncoding RNAs (lncRNAs) participate in transcriptional, epigenetic, and post-transcriptional regulation of gene expression and may influence carcinogenesis. MALAT1 is a lncRNA that is expressed in endocrine and many other neoplasms and it has been shown to have oncogenic and/or tumor suppressor effects in tumor development. Olfactory neuroblastomas arise in the nasal cavity while sympathetic neuroblastomas are present mainly in the adrenal and periadrenal regions. These neoplasms have overlapping histopathological features. Rare cases of sympathetic neuroblastomas metastatic to the nasal cavity have been reported. PHOX2B has been shown to be relatively specific for sympathetic neuroblastomas, but only a limited number of cases of olfactory neuroblastomas have been examined for PHOX2B expression. This study aimed to explore the potential utilization of MALAT1 and PHOX2B in distinguishing these two entities. Tissue microarrays (TMA) were created for olfactory neuroblastomas (n = 26) and sympathetic neuroblastomas (n = 52). MALAT1 lncRNA expression was assessed by in situ hybridization using RNAScope technology. TMA slides were scanned by Vectra multispectral imaging system and image analysis and quantification were performed with inForm software. PHOX2B expression was analyzed by immunohistochemistry. MALAT1 showed predominantly nuclear expression in both tumor types and MALAT1 expression was 2-fold higher in olfactory neuroblastomas compared to sympathetic neuroblastomas (p < 0.0001). PHOX2B showed nuclear staining in most sympathetic neuroblastomas (51/52, 98 %) while only 1 olfactory neuroblastoma (3.8 %) was focally positive for this marker. These findings suggest immunostaining of PHOX2B could be an excellent marker in distinguishing between these two tumor types., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Neuronal differentiation and neurite growth are essential processes in nervous system development and are regulated by several factors. Although all-trans retinoic acid (ATRA) has been shown to mediate the differentiation of mouse neuroblastoma cells via the activation of several pathways, including Wnt/β-catenin signaling, the mechanism remains unclear. The pyruvate kinase, muscle (PKM) plays an important role in the glycolysis of neuroblastoma cells and regulates the Wnt signaling pathway in various cancer cells. In this study, we hypothesized that the Wnt/PKM axis regulates the differentiation of neuroblastoma cells (Neuro-2a and N1E-115). To test this hypothesis, we used inhibitors and activators of the Wnt/β-catenin and glycolytic pathways in ATRA-induced differentiated Neuro-2a and N1E-115 cells and established cell lines with silenced or a mutant replacement of Pkm. Western blot and qPCR showed that ATRA treatment activated the Wnt signaling pathway and inhibited PKM-mediated glycolysis. The oxygen consumption rate (indicating oxidative phosphorylation) significantly increased, whereas the extracellular acidification rate (indicating glycolysis) significantly decreased during differentiation; these effects were reversed upon PKM inhibition. The Wnt inhibitor ICG-001 and PKM activator ML-265 inhibited ATRA-induced Neuro-2a and N1E-115 differentiation, whereas RNA interference-mediated Pkm silencing promoted Neuro-2a and N1E-115 differentiation, which was reversed by PKM overexpression. Treatment with the Wnt activator kenpaullone promoted Neuro-2a and N1E-115 differentiation, which was reversed by ML-265 administration. These results indicate that Wnt/β-catenin signaling promotes Neuro-2a and N1E-115 differentiation by inhibiting PKM-mediated glycolysis during ATRA-induced differentiation. These findings may provide a new theoretical basis for the role of glycolysis in nerve differentiation., Competing Interests: Declaration of competing interest I have nothing to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Cytokine-induced killer (CIK) cell therapy has proven successful in clinical trials regarding glioblastoma. Equally important are the hints suggesting peroxisome proliferator-activated receptors (PPARs) ligands being co-expressed in the central nervous system (CNS). This provides a rationale about investigating the possible synergistic effect of CIK cells and PPARs., Methodology: We investigated neuroblastoma and glioblastoma cell lines with mature CIK cells and the PPARγ antagonist GW-9662 to assess the effects on cell viability, candidate gene expression (Wnt/β-catenin signalling, DNMT1) and global methylation levels (5-methylcytosine, LINE-1)., Results: Using a clinical applicable PPAR-γ inhibitor, we showed that (1) PPARγ-antagonist GW-9662 suppressed tumor cell growth in both neuroblastoma and glioblastoma cells, (2) PPARγ inhibition had restricted effect on the expression of Wnt/β-catenin associated genes, (3) inhibition of PPARγ led to downregulation of DNMT1 expression, supporting their hypothesized interaction in cancer, (4) a partial modulation of global LINE-1 methylation levels was observed, indicating their role in epigenetic processes, and (5) Combining PPARγ inhibition with CIK cell immunotherapy enhanced cell lysis significantly., Conclusion: We provide evidence that PPAR ligands in combination with CIK cell immunotherapy could be a valuable option for malignant CNS tumors., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Context: Lysiphyllum strychnifolium (Craib) A. Schmitz. (Fabaceae) is a Thai traditional medicine used to remove food and alcohol toxins from the body., Objective: This study investigates the molecular mechanism of L. strychnifolium extracts against paraoxon-ethyl-induced apoptosis in SH-SY5Y cells., Materials and Methods: The ethanol and water extracts of leaves and stems of L. strychnifolium were prepared at various concentrations (0-100 μg/mL) and co-treated to the cells with 0.375 mM paraoxon-ethyl for 24 and 48 h. Cell viability was performed using the PrestoBlue assay. ROS and caspase activity were detected using 2',7'-dichlorodihydrofluorecein diacetate and caspase-Glo® 3/7, 8, and 9 assay kits. Apoptotic and ER stress-related gene expression were determined by real-time PCR, and nuclear and mitochondrial morphology were observed using Hoechst 33342 and MitoTracker® Deep Red FM staining., Results: The most effective concentrations of each extract against paraoxon-ethyl-induced cell death were 25 μg/mL of leaf ethanol, 12.5 μg/mL of stem ethanol, 100 μg/mL of leaf water, and 25 μg/mL of stem water extracts. The leaf ethanol extract was the most effective at detoxifying, while stem extracts were highly toxic in high doses. The detoxifying L. strychnifolium extracts against paraoxon-ethyl-induced oxidative stress decreased p53, BiP/GRP78 , and CHOP gene expression and minimized caspase 9 and caspase 3, protecting cells from apoptosis. The extracts could also restore mitochondrial membrane potential and reduce the swollen globule mitochondrial shape., Discussion and Conclusion: These findings could potentially protect neuron cells from neurodegenerative issues due to oxidative damage, apoptosis, and other potential consequences.

An imbalance between reactive oxygen species (ROS) production and antioxidant defense driven by oxidative stress and inflammation is a critical factor in the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's. Coriander (Coriandrum sativum L.), a culinary plant in the Apiaceae family, displays various biological activities, including anticancer, antimicrobial, and antioxidant effects. Herein, neuroprotective properties of three major bioactive compounds derived from coriander (i.e., linalool, linalyl acetate, and geranyl acetate) were investigated on hydrogen peroxide-induced SH-SY5Y neuroblastoma cell death by examining cell viability, ROS production, mitochondrial membrane potential, and apoptotic profiles. Moreover, underlying mechanisms of the compounds were determined by measuring intracellular sirtuin 1 (SIRT1) enzyme activity incorporated with molecular docking. The results showed that linalool, linalyl acetate, and geranyl acetate elicited their neuroprotection against oxidative stress via protecting cell death, reducing ROS production, preventing cell apoptosis, and modulating SIRT1 longevity. Additionally, in silico pharmacokinetic predictions indicated that these three compounds are drug-like agents with a high probability of absorption and distribution, as well as minimal potential toxicities. These findings highlighted the potential neuroprotective linalool, linalyl acetate, and geranyl acetate for developing alternative natural compound-based neurodegenerative therapeutics and prevention., (© 2024. The Author(s).)

Neuroblastoma (NB) is the most common extracranial childhood cancer, caused by the improper differentiation of developing trunk neural crest cells (tNCC) in the sympathetic nervous system. The N 6 -methyladenosine (m 6 A) epitranscriptomic modification controls post-transcriptional gene expression but the mechanism by which the m 6 A methyltransferase complex METTL3/METTL14/WTAP is recruited to specific loci remains to be fully characterized. We explored whether the m 6 A epitranscriptome could fine-tune gene regulation in migrating/differentiating tNCC. We demonstrate that the m 6 A modification regulates the expression of HOX genes in tNCC, thereby contributing to their timely differentiation into sympathetic neurons. Furthermore, we show that posterior HOX genes are m 6 A modified in MYCN-amplified NB with reduced expression. In addition, we provide evidence that sustained overexpression of the MYCN oncogene in tNCC drives METTL3 recruitment to a specific subset of genes including posterior HOX genes creating an undifferentiated state. Moreover, METTL3 depletion/inhibition induces DNA damage and differentiation of MYCN overexpressing cells and increases vulnerability to chemotherapeutic drugs in MYCN-amplified patient-derived xenografts (PDX) in vivo, suggesting METTL3 inhibition could be a potential therapeutic approach for NB., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

During intracellular trafficking N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins catalyze the membrane fusion by assembling into a four-helix complex. In the mouse model, loss of the endosomal SNAREs vti1a and vti1b results in a perinatal lethal phenotype and neuronal defects including decreased neurite outgrowth in cultured primary neurons. We used a CRISPR/Cas9 system to generate a Vti1a Vti1b double knockout (DKO) in the neuroblastoma cell line N1E-115. Three different DKO cell lines were obtained and examined at genome and protein level. The double deficiency impaired proper differentiation based on lower levels of synaptic proteins as well as reduced neurite formation and elongation compared to wild type cells in differentiation medium. Neurite elongation can be induced by a variety of extracellular signals via different signaling cascades. Treatment with the Rho kinase inhibitor Y27632, which stimulates enlargeosome exocytosis, or with neurotrophic factors (BDNF, NGF and NT3) resulted in reduced stimulation of all DKO clones and in significantly shorter neurites compared to wild type cells. The loss of vti1a and vti1b disrupted Akt signaling during enlargeosome-mediated and Erk signaling during BDNF-induced neurite outgrowth., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Recent investigations indicate that tannic acid is associated with a decrease in oxidative damage. Growing evidence supports the protective effects of tannic acid on the central nervous system (CNS). However, uncertainties persist regarding its influence on hydrogen peroxide (H 2 O 2 )-triggered oxidative impairment in nerve cells and its interaction with apoptosis. Hence, the objective of this work was to examine the neuroprotective impact of tannic acid on SH-SY5Y cell impairment following H 2 O 2 -induced oxidative stress, particularly concerning apoptotic pathways. The control group received no treatment, while the H 2 O 2 group underwent treatment with 0.5 mM H 2 O 2 for a duration of 24 h. The tannic acid group received treatment with different concentrations of tannic acid for a duration of 24 h. Meanwhile, the tannic acid + H 2 O 2 group underwent pre-treatment with tannic acid for one hour and was subsequently subjected to 0.5 mM H 2 O 2 for one day. Within the tannic acid + H 2 O 2 group, the cell viability in SH-SY5Y cells was notably enhanced by tannic acid at concentrations of 2.5, 5, and 10 μM. It also resulted in a considerable rise in TAS (Total Antioxidant Status) levels and a concurrent decline in TOS (Total Oxidant Status) levels, serving as indicators of reduced oxidative stress. Additionally, tannic acid treatment resulted in decreased levels of apoptotic markers (Bax, cleaved PARP, and cleaved caspase 3) and oxidative DNA damage marker (8-oxo-dG), while increasing the anti-apoptotic marker Bcl-2. The findings from flow cytometry also revealed a significant reduction in the apoptosis rate following pretreatment with tannic acid. In summary, tannic acid demonstrates protective effects on SH-SY5Y cells in the face of H 2 O 2 -triggered oxidative damage by suppressing both oxidative stress and apoptosis. Nevertheless, additional research is warranted to assess the neuroprotective potential of tannic acid., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

miR-454-3p has been reported to be a tumor-suppressive microRNA (miRNA) in multiple cancer types. We identified the kinase STK33 mRNA, which is a high-risk factor for survival in neuroblastoma (NB) patients, as being a substrate of miR-454-3p in NB. Even though STK33 is an attractive target for several cancers, the development of inhibitors of STK33 has been challenging. For the various cell lines tested, we demonstrated reduced growth and viability with the miR-454-3p mimic. From among the candidate NB-associated miRNAs, miR-454-3p mimic and its antagonist had the most profound effect on STK33 mRNA and protein-level changes. Under various conditions of growth and external stress for the cells, the RNA levels for miR-454-3p and STK33 also negatively correlated. Luciferase reporter assays demonstrated STK33 as a substrate for miR-454-3p, and recombinant versions of STK33 resistant to miR-454-3p significantly blunted the suppressive effect of the miR-454-3p and established STK33 as the major functional substrate of miR-454-3p. Overexpression of miR-454-3p or knockdown of STK33 mRNA promoted autophagy and at the same time, increased the apoptotic markers in the tested NB cells, indicating a mechanism for the suppressive effect of the agents. Given the difficult-to-drug targets such as STK33 and the recent successes in RNA delivery methods for cancer treatment, it is thought that targeting cancer cells with a suppressive miRNA such as miR-454-3p for STK33-dependent cancer types may be an alternative means of NB therapy., Competing Interests: DECLARATION OF COMPETING INTERESTS There are no competing interests to declare., (Copyright © 2024. Published by Elsevier Inc.)

Despite the known therapeutic uses of dexamethasone (DEX), the specific mechanisms underlying its neurotoxic effects in neuronal cells, particularly in undifferentiated human neuroblastoma (SH-SY5Y) cells, remain inadequately understood. This study aims to elucidate these mechanisms, emphasizing bioenergetics, oxidative stress, and apoptosis, thereby providing novel insights into the cellular vulnerabilities induced by chronic DEX exposure. The findings revealed significant reductions in cell viability, altered membrane integrity with LDH leakage, decreased intracellular ATP production, and the electron transport chain complexes I and III activity inhibition. DEX significantly increased the release of the reactive species and peroxidation of lipids, as well as of Nrf2 expression. At the same time, it simultaneously led to a decline in the activities of the antioxidant catalase and superoxide dismutase enzymes, along with a depletion of glutathione reserves. The apoptosis process was exhibited by a significant elevation of caspases 3 and 8 activities with overexpression of mRNA BAX, inhibition of BCL-2, and a significant upregulation of the BAX/BCL-2 ratio. Assessment of neuronal development genes (GAP43, CAMK2A, CAMK2B, TUBB3, and Wnts) by quantitative PCR assay showed increased expression of CAMK2A, CAMK2B, and Wnt3a with a significant reduction in GAP43 mRNA levels. Collectively, this study proved that DEX was cytotoxic to SH-SY5Y via bioenergetic disruption, mitochondrial dysfunction, oxidative stress, and apoptosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Estramustine (EM), a clinically successful hormone-refractory anti-prostate cancer drug, exhibited potent anti-proliferative activity, depolymerized microtubules, blocked cells at mitosis, and induced cell death in different cancer cells. Altered iron metabolism is a feature of cancer cells. Using EM, we examined the plausible relationship between microtubule depolymerization and induction of ferroptosis in human neuroblastoma (SH-SY5Y and IMR-32) cells. EM reduced glutathione (GSH) levels and induced reactive oxygen species (ROS) generation. The pre-treatment of neuroblastoma cells with ROS scavengers (N-acetyl cysteine and dithiothreitol) reduced the anti-proliferative effects of EM. EM treatment increased labile iron pool (LIP), depleted glutathione peroxidase 4 (GPX4) levels, and lipid peroxidation, hallmark features of ferroptosis, highlighting ferroptosis induction. Ferroptosis inhibitors (deferoxamine mesylate and liproxstatin-1) abrogated the cytotoxic effects of EM, further confirming ferroptosis induction. Vinblastine and nocodazole also increased LIP and induced lipid peroxidation in neuroblastoma cells. This study provides evidence for the coupling of microtubule integrity to ferroptosis. The results also suggest that microtubule-depolymerizing agents may be considered for developing pro-ferroptosis chemotherapeutics., (© 2024 International Union of Biochemistry and Molecular Biology.)

Thirty selenium-containing coumarin derivatives were synthesized and evaluated for inhibitory activity against 17 malignant tumor cell lines. Among these, compound 11i demonstrated the most potent inhibition of neuroblastoma SK-N-SH cells, with an IC 50 of 2.5 ± 0.1 μM. Compound 11i notably inhibited SK-N-SH cell proliferation, migration, and invasion. Western blot and immunofluorescence analyses indicated that 11i increased the Bax/Bcl-2 protein expression ratio, promoted Cytochrome C release from mitochondria, and activated caspases 9 and 3, triggering the mitochondria-mediated apoptotic pathway and inducing endogenous tumor cell apoptosis. The compounds localized in the cytoplasm and co-localized with mitochondria, suggesting mitochondrial interaction and dysfunction. Computational docking studies revealed a strong binding affinity of 11i with Bcl-2 and mitochondrial G-quadruplexes. In a subcutaneous neuroblastoma-bearing mouse model, 11i showed notable anti-tumor efficacy with tumor inhibition rates of 79 % (10 mg/kg) and 93 % (20 mg/kg), exceeding that of cyclophosphamide. This study represents a novel finding on the anti-tumor activity of selenium-containing coumarin derivatives and provides a theoretical basis for developing coumarin-based therapeutics for neuroblastoma., Competing Interests: Declaration of competing interest The work described has not been submitted elsewhere for publication, in whole or in part, and it is not being submitted to any other journal. All the authors listed have reviewed the final version of the manuscript and approve it for publication., (Copyright © 2025 Elsevier Masson SAS. All rights reserved.)