Etched-suppressed gold nanorods providing highly distinctive plasmonic patterns: Towards multiplex analysis of neuroblastoma biomarkers.

BACKGROUND: On-site monitoring of vanillylmandelic acid (VMA), homovanillic acid (HVA), and dopamine (DA) as key diagnostic biomarkers for a wide range of neurological disorders holds utmost significance in clinical settings. Numerous colorimetric sensors with mechanistic approaches based on aggregation or silver metallization have been introduced for this purpose. However, these mechanisms have drawbacks, such as sensitivity to environmental factors and probe toxicity. Therefore, there is a great demand for a robust yet non-toxic colorimetric sensor that employs a novel route to monitor these biomarkers effectively. RESULTS: Here, we present a single-component multi-colorimetric probe based on the controllable etching suppression of gold nanorods (AuNRs) upon exposure to the mild etchant N-bromosuccinimide (NBS), designed to accurately detect and discriminate VMA, HVA, DA, and their corresponding mixtures, i.e. , VMA: HVA, VMA:DA, HVA:DA, and VMA:HVA:DA. To enhance the sensitivity and automation capabilities of the designed multi-colorimetric sensor, two machine learning techniques were employed: linear discriminant analysis (LDA) for the qualitative classification and partial least-squares regression (PLSR) for the quantitative analysis of pure biomarkers and their mixtures. The outcomes revealed a high correlation between measured and predicted values, covering a linear range of 0.8-25, 1.2-25, and 2.7-100 µmol L-1, with remarkably low detection limits of 0.260, 0.397, and 0.913 µmol L-1 for VMA, HVA, and DA, respectively. Lastly, the performance of the probe was validated by successfully detecting the neuroblastoma biomarker VMA:HVA in human urine. SIGNIFICANCE: Our designed multi-colorimetric probe introduces a rapid, cost-effective, user-friendly, non-toxic, and non-invasive approach to detecting and discriminating not only the pure biomarkers but also their corresponding binary and ternary mixtures. The distinctive response profiles produced by the probe in the presence of different mixture ratios can indicate various disease states in patients, which is highly crucial in clinical diagnostics.

Phase I study of safety and efficacy of allogeneic natural killer cell therapy in relapsed/refractory neuroblastomas post autologous hematopoietic stem cell transplantation.

Despite low incidence, neuroblastoma, an immunologically cold tumor, is the most common extracranial solid neoplasm in pediatrics. In relapsed/refractory cases, the benefits of autologous hematopoietic stem cell transplantation (auto-HSCT) and other therapies are limited. Natural killer (NK) cells apply cytotoxicity against tumor cells independently of antigen-presenting cells and the adaptive immune system. The primary endpoint of this trial was to assess the safety of the injection of allogenic, ex vivo-expanded and primed NK cells in relapsed/refractory neuroblastoma patients after auto-HSCT. The secondary endpoint included the efficacy of this intervention in controlling tumors. NK cells were isolated and primed ex vivo (by adding interleukin [IL]-2, IL-15, and IL-21) in a GMP-compliant CliniMACS system and administered to four patients with relapsed/refractory MYCN-positive neuroblastoma. NK cell injections (1 and 5 × 107 cells/kg in the first and second injections, respectively) were safe, and no acute or sub-acute adverse events were observed. During the follow-up period, one complete response (CR) and one partial response (PR) were observed, while two cases exhibited progressive disease (PD). In follow-up evaluations, two died due to disease progression, including the case with a PR. The patient with CR had regular growth at the 31-month follow-up, and another patient with PD is still alive and receiving chemotherapies 20 months after therapy. This therapy is an appealing and feasible approach for managing refractory neuroblastomas post-HSCT. Further studies are needed to explore its efficacy with higher doses and more frequent administrations for high-risk neuroblastomas and other immunologically cold tumors.Trial registration number: irct.behdasht.gov.ir (Iranian Registry of Clinical Trials, No. IRCT20201202049568N1).

Oncolytic virotherapy augments self-maintaining natural killer cell line cytotoxicity against neuroblastoma.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer related deaths. Targeting neuroblastoma with immunotherapies has proven challenging due to a paucity of immune cells in the tumor microenvironment and the release of immunosuppressive cytokines by neuroblastoma tumor cells. We hypothesized that combining an oncolytic Herpes Simplex Virus (oHSV) with natural killer (NK) cells might overcome these barriers and incite tumor cell death. METHODS: We utilized MYCN amplified and non-amplified neuroblastoma cell lines, the IL-12 expressing oHSV, M002, and the human NK cell line, NK-92 MI. We assessed the cytotoxicity of NK cells against neuroblastoma with and without M002 infection, the effects of M002 on NK cell priming, and the impact of M002 and priming on the migratory capacity and CD107a expression of NK cells. To test clinical applicability, we then investigated the effects of M002 and NK cells on neuroblastoma in vivo. RESULTS: NK cells were more attracted to neuroblastoma cells that were infected with M002. There was an increase in neuroblastoma cell death with the combination treatment of M002 and NK cells both in vitro and in vivo. Priming the NK cells enhanced their cytotoxicity, migratory capacity and CD107a expression. CONCLUSIONS: To the best of our knowledge, these investigations are the first to demonstrate the effects of an oncolytic virus combined with self-maintaining NK cells in neuroblastoma and the priming effect of neuroblastoma on NK cells. The current studies provide a deeper understanding of the relation between NK cells and neuroblastoma and these data suggest that oHSV increases NK cell cytotoxicity towards neuroblastoma.

Decoding pediatric spinal tumors: a single-center retrospective case series on etiology, presentation, therapeutic strategies, and outcomes.

INTRODUCTION: Spinal tumors (ST) often result in dire prognosis, carrying risks such as permanent paralysis, sensory loss, and sphincter dysfunction. Data on their incidence and etiology in pediatric populations are markedly scant. Our study investigates the etiology, clinical manifestation, treatment, and outcomes of pediatric ST. METHODS: We conducted a retrospective review of our institutional pediatric oncology and neurosurgery database, examining 14 patients under 18 years admitted with ST due to oncological diseases since 2005. We analyzed the clinical presentations, evaluations, molecular diagnostics and treatments for these patients. RESULTS: The study spanned 15 years and included 14 pediatric patients, each diagnosed with distinct spinal tumor entity. The mean patient age was approximately 19.6 ± 10.1 months. Severe axial pain along the vertebral column was observed in 13 patients, while acute neurological deterioration manifested in 7 patients. As a first-line intervention, 13 patients underwent decompressive surgery through laminectomy and tumor resection, and only one patient received chemotherapy solely. Before surgery, seven patients were unable to walk; post-surgery, six of them regained their ability to ambulate. The diagnosis encompassed a range of neoplasms: two instances of Ewing sarcoma, 3 instances of teratoma, one case presenting an atypical teratoid Rhabdoid tumor, two instances each of low-grade astrocytoma and neuroblastoma, and single instances of ependymoma, meningioma, rhabdomyosarcoma, and embryonal tumors with multilayered rosettes (ETMRs). Three patients succumbed two years after initiating therapy. CONCLUSION: Despite their rarity, intraspinal tumors in pediatric patients pose substantial therapeutic challenges. The intertwined complexities of the disease entity and the patient's neurological status demand swift initiation of an individualized therapeutic strategy. This crucial step helps optimize outcomes for this patient cohort, who frequently grapple with debilitating health conditions. Inclusion of these patients within a registry is mandatory to optimize treatment outcomes due to their rarity in pediatric population.

Dose-Dependent Alterations of Lysosomal Activity and Alpha-Synuclein in Peripheral Blood Monocyte-Derived Macrophages and SH-SY5Y Neuroblastoma Cell Line by upon Inhibition of MTOR Protein Kinase - Assessment of the Prospects of Parkinson's Disease Therapy.

To date, the molecular mechanisms of the common neurodegenerative disorder Parkinson's disease (PD) are unknown and, as a result, there is no neuroprotective therapy that may stop or slow down the process of neuronal cell death. The aim of the current study was to evaluate the prospects of using the mTOR molecule as a potential target for PD therapy due to the dose-dependent effect of mTOR kinase activity inhibition on cellular parameters associated with, PD pathogenesis. The study used peripheral blood monocyte-derived macrophages and SH-SY5Y neuroblastoma cell line. As a result, we have for the first time showed that inhibition of mTOR by Torin1 only at a concentration of 100 nM affects the level of the lysosomal enzyme glucocerebrosidase (GCase), encoded by the GBA1 gene. Mutations in GBA1 are considered a high-risk factor for PD development. This concentration led a decrease in pathological phosphorylated alpha-synuclein (Ser129), an increase in its stable tetrameric form with no changes in the lysosomal enzyme activities and concentrations of lysosphingolipids. Our findings suggest that inhibition of the mTOR protein kinase could be a promising approach for developing therapies for PD, particularly for GBA1-associated PD.

Effectiveness of Nutrition Support Team Intervention in Pediatric Patients with Cancer.

Malnutrition in children with cancer is associated with poor prognosis. This study aimed to determine whether nutritional support team (NST) interventions prevent adverse events and improve the nutritional status in pediatric patients admitted for cancer treatment. This was a historical cohort study of pediatric patients with acute lymphocytic leukemia, acute myeloid leukemia, neuroblastoma, or brain tumor who received chemotherapy or underwent hematopoietic stem cell transplantation. Patients admitted between June 2013 and October 2014 were classified into the intervention group. Those admitted between January 2011 and December 2012 were classified into the control group. We created a homogeneous probability model using the inverse probability of treatment weighting method, and compared outcomes. A total of 75 patients were included in the study (38 and 37 in the intervention and control groups, respectively). The intervention group had significantly fewer incidents of nothing by mouth (nil per os [NPO]) (p=0.037) and days of NPO (p=0.046) than the control group. There was no significant difference between the intervention and control groups regarding the change in body mass index z-score between admission and discharge (p=0.376). NST interventions for children with cancer were associated with a reduction in the number of NPO occurrences and NPO days. These findings suggest that NST interventions contribute to continued oral intake.

GSTO2 ameliorates human neuroblastoma cell apoptosis, inflammation, ferroptosis, and oxidative stress by upregulating GPX4 expression in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a severe hemorrhagic stroke and induces severe secondary neurological injury. However, its pathogenesis remains to be explored. The present work investigates the role of glutathione S-transferase omega 2 (GSTO2) in ICH and the underlying mechanism. Human neuroblastoma cells (SK-N-SH) were stimulated using hemin to mimic ICH-like injury. Protein expression levels of GSTO2 and glutathione peroxidase 4 (GPX4) were detected by western blot analysis assay. Cell viability was assessed by cell counting kit-8 assay. Cell proliferation was investigated by 5-ethynyl-2'-deoxyuridine assay. Cell apoptosis was analyzed by flow cytometry. Interleukin-6 and tumor necrosis factor-α levels were quantified by enzyme-linked immunosorbent assays. Fe2+ colorimetric assay kit was used to detect Fe2+ level. A cellular reactive oxygen species (ROS) assay kit was used to detect ROS levels. Malondialdehyde (MDA) level was assessed using the MDA content assay kit. GSH level was quantified using the GSH assay kit. Co-immunoprecipitation assay was performed to identify the association between GSTO2 and GPX4. Hemin stimulation suppressed SK-N-SH cell proliferation and promoted cell apoptosis, cell inflammation, ferroptosis, and oxidative stress. GSTO2 expression was downregulated in hemin-treated SK-N-SH cells in comparison with the control group. In addition, ectopic GSTO2 expression counteracted hemin-induced inhibitory effect on cell proliferation and promoting effects on cell apoptosis, inflammation, ferroptosis, and oxidative stress. Moreover, GSTO2 was associated with GPX4 in SK-N-SH cells. GPX4 silencing attenuated GSTO2 overexpression-induced effects on hemin-stimulated SK-N-SH cell injury. GSTO2 ameliorated SK-N-SH cell apoptosis, inflammation, ferroptosis, and oxidative stress by upregulating GPX4 expression in ICH, providing a therapeutic strategy for ICH.

Autophagic degradation of CDK4 is responsible for G0/G1 cell cycle arrest in NVP-BEZ235-treated neuroblastoma.

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.

Tannic acid protects neuroblastoma cells against hydrogen peroxide - triggered oxidative stress by suppressing oxidative stress and apoptosis.

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 (H2O2)-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 H2O2-induced oxidative stress, particularly concerning apoptotic pathways. The control group received no treatment, while the H2O2 group underwent treatment with 0.5 mM H2O2 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 + H2O2 group underwent pre-treatment with tannic acid for one hour and was subsequently subjected to 0.5 mM H2O2 for one day. Within the tannic acid + H2O2 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 H2O2-triggered oxidative damage by suppressing both oxidative stress and apoptosis. Nevertheless, additional research is warranted to assess the neuroprotective potential of tannic acid.

Cytotoxic effects induced by combined exposure to the mycotoxins sterigmatocystin, ochratoxin A and patulin on human tumour and healthy 3D spheroids.

Humans are exposed to complex mixtures of mycotoxins through diet. Despite the serious threat they pose, mycotoxin risk assessment often overlooks co-exposure. With the aim of filling this gap, the present study investigates the combined cytotoxicity of sterigmatocystin (STE), ochratoxin A (OTA) and patulin (PAT) in human tumour Neuroblastoma and healthy Mesenchymal Stem Cells three-dimensional (3D) spheroids. The range of concentrations tested (1.56-50 µM for STE, 0.78-25 µM for OTA and 0.15-5 µM for PAT) was selected considering the IC50 values obtained in previous studies and the estimated dietary exposure of consumers. To ensure appropriate experimental conditions, assessments for single mycotoxins and their combinations were conducted simultaneously. The nature of the toxicological interactions among the mycotoxins was then defined using the isobologram analysis. Our results demonstrated increased cytotoxicity in mycotoxin mixtures compared to individual exposure, with abundance of synergistic interactions. These findings highlight that the co-occurrence of STE, OTA and PAT in food may increase their individual toxic effects and should not be underestimated. Moreover, the use of advanced culture models increased the reliability and physiological relevance of our results which can serve as a groundwork for formulating standardized regulatory approaches towards mycotoxin mixtures in food and feed.

Comprehensive analysis identifies ubiquitin ligase FBXO42 as a tumor-promoting factor in neuroblastoma.

Neuroblastoma, the deadliest solid tumor in children, exhibits alarming mortality rates, particularly among high-risk cases. To enhance survival rates, a more precise risk stratification for patients is imperative. Utilizing proteomic data from 34 cases with or without N-Myc amplification, we identified 28 differentially expressed ubiquitination-related proteins (URGs). From these, a prognostic signature comprising 6 URGs was constructed. A nomogram incorporating clinical-pathological parameters yielded impressive AUC values of 0.88, 0.93, and 0.95 at 1, 3, and 5 years, respectively. Functional experiments targeting the E3 ubiquitin ligase FBXO42, a component of the prognostic signature, revealed its TP53-dependent promotion of neuroblastoma cell proliferation. In conclusion, our ubiquitination-related prognostic model robustly predicts patient outcomes, guiding clinical decisions. Additionally, the newfound pro-proliferative role of FBXO42 offers a novel foundation for understanding the molecular mechanisms of neuroblastoma.

Neuroblastoma, Version 2.2024, NCCN Clinical Practice Guidelines in Oncology.

Neuroblastoma is the most common extracranial solid tumor diagnosed in children. This inaugural version of the NCCN Guidelines for Neuroblastoma provides recommendations for the diagnosis, risk classification, and treatment of neuroblastoma. The information in these guidelines was developed by the NCCN Neuroblastoma Panel, a multidisciplinary group of representatives with expertise in neuroblastoma, consisting of pediatric oncologists, radiologists, pathologists, surgeons, and radiation oncologists from NCCN Member Institutions. The evidence-based and consensus recommendations contained in the NCCN Guidelines are intended to guide clinicians in selecting the most appropriate treatments for their patients with this clinically heterogeneous disease.

CAR T-cell-mediated delivery of bispecific innate immune cell engagers for neuroblastoma.

Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.

Preclinical spheroid models identify BMX as a therapeutic target for metastatic MYCN nonamplified neuroblastoma.

The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest-derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients. To identify critical genes driving NB aggressiveness, we performed combined chromatin and transcriptome analyses on matched patient-derived xenografts (PDXs), spheroids, and differentiated adherent cultures derived from metastatic MYCN nonamplified tumors. Bone marrow kinase on chromosome X (BMX) was identified among the most differentially regulated genes in PDXs and spheroids versus adherent models. BMX expression correlated with high tumor stage and poor patient survival and was crucial to the maintenance of the self-renewal and tumorigenic potential of NB spheroids. Moreover, BMX expression positively correlated with the mesenchymal NB cell phenotype, previously associated with increased chemoresistance. Finally, BMX inhibitors readily reversed this cellular state, increased the sensitivity of NB spheroids toward chemotherapy, and partially reduced tumor growth in a preclinical NB model. Altogether, our study identifies BMX as a promising innovative therapeutic target for patients with high-risk MYCN nonamplified NB.

Parainfluenza Virus 5 V Protein Blocks Interferon Gamma-Mediated Upregulation of NK Cell Inhibitory Ligands and Improves NK Cell Killing of Neuroblastoma Cells.

Natural killer (NK) cells can be effective immunotherapeutic anti-cancer agents due to their ability to selectively target and kill tumor cells. This activity is modulated by the interaction of NK cell receptors with inhibitory ligands on the surface of target cells. NK cell inhibitory ligands can be upregulated on tumor cell surfaces in response to interferon-gamma (IFN-γ), a cytokine which is produced by activated NK cells. We hypothesized that the resistance of tumor cells to NK cell killing could be overcome by expression of the parainfluenza virus 5 (PIV5) V protein, which has known roles in blocking IFN-γ signaling. This was tested with human PM21-NK cells produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Infection of human SK-N-SH neuroblastoma cells with PIV5 blocked IFN-γ-mediated upregulation of three NK cell inhibitory ligands and enhanced in vitro killing of these tumor cells by PM21-NK cells. SK-N-SH cells transduced to constitutively express the V protein alone were resistant to IFN-γ-mediated increases in cell surface expression of NK cell inhibitory ligands. Real-time in vitro cell viability assays demonstrated that V protein expression in SK-N-SH cells was sufficient to increase PM21-NK cell-mediated killing. Toward a potential therapeutic application, transient lentiviral delivery of the V gene also enhanced PM21-NK cell killing in vitro. Our results provide the foundation for novel therapeutic applications of V protein expression in combination with ex vivo NK cell therapy to effectively increase the killing of tumor cells.

Joint metabolomics and transcriptomics analysis systematically reveal the impact of MYCN in neuroblastoma.

The limited understanding of the molecular mechanism underlying MYCN-amplified (MNA) neuroblastoma (NB) has hindered the identification of effective therapeutic targets for MNA NB, contributing to its higher mortality rate compared to MYCN non-amplified (non-MNA) NB. Therefore, a comprehensive analysis integrating metabolomics and transcriptomics was conducted to systematically investigate the MNA NB. Metabolomics analysis utilized plasma samples from 28 MNA NB patients and 68 non-MNA NB patients, while transcriptomics analysis employed tissue samples from 15 MNA NB patients and 37 non-MNA NB patients. Notably, joint metabolomics and transcriptomics analysis was performed. A total of 46 metabolites exhibited alterations, with 21 displaying elevated levels and 25 demonstrating reduced levels in MNA NB. In addition, 884 mRNAs in MNA NB showed significant changes, among which 766 mRNAs were higher and 118 mRNAs were lower. Joint-pathway analysis revealed three aberrant pathways involving glycerolipid metabolism, purine metabolism, and lysine degradation. This study highlights the substantial differences in metabolomics and transcriptomics between MNA NB and non-MNA NB, identifying three abnormal metabolic pathways that may serve as potential targets for understanding the molecular mechanisms underlying MNA NB.

Genipin promotes the apoptosis and autophagy of neuroblastoma cells by suppressing the PI3K/AKT/mTOR pathway.

This study investigated the underlying function and mechanism of genipin in neuroblastoma (NB). Using flow cytometry analysis and cytotoxicity tests, in vitro studies were conducted to assess the effects of genipin on the SK-N-SH cell line. The mechanism of action of genipin was explored through immunofluorescence staining, Western blotting, and caspase-3 activity assays. In addition, we also created a xenograft tumour model to investigate the effects of genipin in vivo. This research confirmed that genipin suppressed cell viability, induced apoptosis, and promoted autophagy, processes that are likely linked to the inhibition of the PI3K/AKT/mTOR signalling pathway. Autophagy inhibition increases the sensitivity of SK-N-SH cells to genipin. Furthermore, combination treatment with a PI3K inhibitor enhanced the therapeutic efficacy of genipin. These results highlight the potential of genipin as a candidate drug for the treatment of NB.

Encapsulation of 4-oxo-N-(4-hydroxyphenyl) retinamide in human serum albumin nanoparticles promotes EZH2 degradation in preclinical neuroblastoma models.

Neuroblastoma is the most prevalent and aggressive solid tumor that develops extracranially in children between the ages of 0-14 years, which accounts for 8-10% of all childhood malignancies and ∼15% of pediatric cancer-related mortality. The polycomb repressive complex 2 (PRC2) protein, EZH2, is overexpressed in neuroblastoma and mediates histone H3 methylation at lysine 27 (K27) positions through its methyl transferase activity and is a potential epigenetic silencer of many tumor suppressor genes in cancer. Phosphorylation of EZH2 decreases its stability and leads to proteasomal degradation. The 4-oxo-N-(4-hydroxyphenyl) retinamide (4O4HPR) promotes EZH2 degradation via activation of PKC-δ, but its limited solubility and physiological instability limit its application. In the current study, the encapsulation of 4O4HPR in Human Serum Albumin Nanoparticles (HSANPs) enhanced the solubility and physiological stability of the nanoformulation, leading to improved therapeutic efficacy through G2-M cell cycle arrest, depolarization of mitochondrial membrane potential, generation of reactive oxygen species and caspase 3 mediated apoptosis activation. The molecular mechanistic approach of 4O4HPR loaded HSANPs has activated caspase 3, which further cleaves PKC-δ into two fragments wherein the cleaved fragment of PKC-δ possesses the kinase activity that phosphorylates EZH2 and decreases the protein stability leading to its further ubiquitination in SH-SY5Y cells. Co-immunoprecipitation experiments revealed the direct interaction between PKC-δ and EZH2 phosphorylation, followed by ubiquitination. Moreover, 4O4HPR loaded HSANPs demonstrated improved in vivo biodistribution, greater dispersibility, and biocompatibility and exhibited enhanced protein instability and degradation of EZH2 in the neuroblastoma xenograft mouse model.

Prenyl Pterocarpans from Algerian Bituminaria bituminosa and Their Effects on Neuroblastoma.

The pterocarpan fraction from aerial parts of Bituminaria bituminosa was investigated for both chemical characterization and biological evaluation. Chemical studies were in accordance with the literature data on Bituminaria genus resulting in the identification of typical 4,8-prenyl pterocarpans. Three new members, bituminarins A-C (1-3), were isolated along with main bitucarpin A (4), erybraedin C (5) and erybraedin D (6) already reported from this plant. Further, biological studies evidenced antiproliferative properties of the most abundant pterocarpans 4 and 5 on neuroblastoma SH-SY5Y cell line, in agreement with previously described antiproliferative activity of these compounds against cancer cell lines other than neuroblastoma. The structure and the stereochemistry of the new molecules was determined by extensive spectroscopic analysis and chemical derivatization methods. The biological investigation was carried out by using an assay platform based on a live-cell imaging system revealing an apoptotic cell death induction.