Human Neuroblastoma Cell Line Research Articles

Discovery of a novel DYRK1A inhibitor with neuroprotective activity by virtual screening and in vitro biological evaluation.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is implicated in accumulation of amyloid β-protein (Aβ) and phosphorylation of Tau proteins, and thus represents an important therapeutic target for neurodegenerative diseases. Though many DYRK1A inhibitors have been discovered, there is still no marketed drug targeting DYRK1A. This is partly due to the lack of effective and safe chemotypes. Therefore, it is still necessary to identify new classes of DYRK1A inhibitors. By performing virtual screening with the workflow mainly composed of pharmacophore modeling and molecular docking as well as the following DYRK1A inhibition assay, we identified compound L9, ((Z)-1-(((5-phenyl-1H-pyrazol-4-yl)methylene)-amino)-1H-tetrazol-5-amine), as a moderately active DYRK1A inhibitor (IC50: 1.67μM). This compound was structurally different from the known DYRK1A inhibitors, showed a unique binding mode to DYRK1A. Furthermore, compound L9 showed neuroprotective activity against okadaic acid (OA)-induced injury in the human neuroblastoma cell line SH-SY5Y by regulating the expression of Aβ and phosphorylation of Tau protein. This compound was neither toxic to the SH-SY5Y cells nor to the human normal liver cell line HL-7702 (IC50: >100μM). In conclusion, we have identified a novel DYRK1A inhibitor with neuroprotective activity through virtual screening and in vitro biological evaluation, which holds the promise for further study.

Platinum (II) Schiff Base Complexes and their Effects on the Inhibition of Amyloid β1–42 Aggregation

For synthesizing metal–organic molecules with pro‐drug properties to prevent the accumulation of amyloid beta (Aβ1–42), which is a feature of neurodegenerative Alzheimer's disease, two new Schiff bases with imine/amine donors 2‐([3,3‐diphenylalidene]amino)‐N,N‐dimethylethane‐1‐amine and N,N‐dimethyl‐2‐([quinolin‐4‐ylmethylene]amino)ethane‐1‐amine, as well as their novel Pt (II) complexes (I and II), were synthesized and characterized via Fourier transform infrared spectroscopy, 1H‐ and 13C‐nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and elemental analyses. The spectroscopic data has shown that the ligands are coordinated to the central atom in a chelating manner.Furthermore, the interaction between these complexes and Aβ1–42 was investigated using the electrochemical methods, square wave voltammetry and pencil graphite electrode, while monitoring the changes in the oxidation signal of the Try residue in Aβ1–42.The ability of the compounds to inhibit Aβ1–42 aggregation was investigated using the human neuroblastoma cell line (SH‐SY5Y). Complex II could actively inhibit the aggregation of Aβ1–42 at a molar ratio of 1.0/1.0, and its Aβ1–42 aggregation kinetics were fluorometrically determined using thioflavin T.These results were supported by scanning electron microscopy and transmission electron microscopy analyses. Moreover, the interaction of complex II with Aβ1–16 was investigated via 1H‐NMR spectroscopy.As a result, it was observed that complex II was effective in inhibiting the aggregation of Aβ1–42 at a molar ratio of 1.0/1.0. This result shows that studies can be conducted on the effects of Schiff base‐Pt complexes as potential pro‐drugs on Alzheimer's disease.

Dickopff 1 inhibits cancer stem cell properties and promotes neuronal differentiation of human neuroblastoma cell line SH-SY5Y

Neuroblastomas are pediatric tumors arising from undifferentiated cells of neural crest origin with stem cell-like characteristics. Dysregulation of Wnt/β-catenin signaling has been shown to be linked to the development of various tumors. Activated Wnt signaling results in β-catenin accumulation in the nucleus to support pro-neoplastic traits. DKK1, a secreted glycoprotein, is an inhibitor of Wnt signaling, and the addition of DKKI to the culture medium has been used to suppress the Wnt pathway. This study aimed to analyze the role of Dickopff-1 as a potential differentiating agent for the neuroblastoma cell line SH-SY5Y and neurospheres derived from it. The treatment of SH-5Y5Y derived neurospheres by DKK1 resulted in their disintegration and reduced proliferation markers like Ki67, PCNA. DKK1 treatment to the neurospheres also resulted in the loss of cancer stem cell markers like CD133, KIT and pluripotency markers like SOX2, OCT4, NANOG. DKK1 treatment caused reduction in mRNA expression of β-catenin and TCF genes like TCF4, TCF12. When the SH-SY5Y cancer cells were grown under differentiating conditions, DKKI caused neuronal differentiation by itself, and in synergy with retinoic acid. This was verified by the expression of markers like MAPT, DCX, GAP43, ENO2 and also with changes in neurite length. We concluded that Wnt inhibition, as exemplified by DKK1 treatment, is therefore a possible differentiating condition and also suppresses the proliferative and cancer stemness related properties of SH-SY5Y neuroblastoma cells.

Lysophosphatidic Acid Stimulates Mitogenic Activity and Signaling in Human Neuroblastoma Cells through a Crosstalk with Anaplastic Lymphoma Kinase.

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK.

Nuclear factor-κB activation by transforming growth factor-β1 drives tumour microenvironment-mediated drug resistance in neuroblastoma.

Intrinsic and extrinsic factors in the tumour microenvironment (TME) contribute to therapeutic resistance. Here we demonstrate that transforming growth factor (TGF)-β1 produced in the TME increased drug resistance of neuroblastoma (NB) cells. Human NB cell lines were tested in vitro for their sensitivity to Doxorubicin (DOX) and Etoposide (ETOP) in the presence of tumour-associated macrophages (TAM) and mesenchymal stromal cells/cancer-associated fibroblasts (MSC/CAF). These experiments were validated in xenotransplanted and primary tumour samples. Drug resistance was associated with an increased expression of efflux transporter and anti-apoptotic proteins. Upregulation was dependent on activation of nuclear factor (NF)-κB by TGF-β-activated kinase (TAK1) and SMAD2. Resistance was reversed upon pharmacologic and genetic inhibitions of NF-κB, and TAK1/SMAD2. Interleukin-6, leukaemia inhibitory factor and oncostatin M were upregulated by this TGF-β/TAK1/NF-κB/SMAD2 signalling pathway contributing to drug resistance via an autocrine loop activating STAT3. An analysis of xenotransplanted NB tumours revealed an increased presence of phospho (p)-NF-κB in tumours co-injected with MSC/CAF and TAM, and these tumours failed to respond to Etoposide but responded if treated with a TGF-βR1/ALK5 inhibitor. Nuclear p-NF-κB was increased in patient-derived tumours rich in TME cells. The data provides a novel insight into a targetable mechanism of environment-mediated drug resistance.

Fluoride Induces Neurocytotoxicity by Disrupting Lysosomal Iron Metabolism and Membrane Permeability.

The detrimental effects of fluoride on neurotoxicity have been widely recorded, yet the detailed mechanisms underlying these effects remain unclear. This study explores lysosomal iron metabolism in fluoride-related neurotoxicity, with a focus on the Steap3/TRPML1 axis. Utilizing sodium fluoride (NaF)-treated human neuroblastoma (SH-SY5Y) and mouse hippocampal neuron (HT22) cell lines, our research demonstrates that NaF enhances the accumulation of ferrous ions (Fe2+) in these cells, disrupting lysosomal iron metabolism through the Steap3/TRPML1 axis. Notably, NaF exposure upregulated ACSL4 and downregulated GPX4, accompanied by reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity and increased malondialdehyde (MDA) levels. These changes indicate increased vulnerability to ferroptosis within neuronal cells. The iron chelator deferoxamine (DFO) mitigates this disruption. DFO binds to lysosomal Fe2+ and inhibits the Steap3/TRPML1 axis, restoring normal lysosomal iron metabolism, preventing lysosomal membrane permeabilization (LMP), and reducing neuronal cell ferroptosis. Our findings suggest that interference in lysosomal iron metabolism may mitigate fluoride-induced neurotoxicity, underscoring the critical role of the Steap3/TRPML1 axis in this pathological process.

Discovery of a novel chemotype as DYRK1A inhibitors against Alzheimer's disease: Computational modeling and biological evaluation

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) plays an essential role in Tau and Aβ pathology closely related to Alzheimer's disease (AD). Accumulative evidence has demonstrated DYRK1A inhibition is able to reduce the pathological features of AD. Nevertheless, there is no approved DYRK1A inhibitor for clinical use as anti-AD therapy. This is somewhat due to the lack of effective and safe chemotypes of DYRK1A inhibitors. To address this issue, we carried out in silico screening, in vitro assays and in vivo efficacy evaluation with the aim to discover a new class of DYRK1A inhibitors for potential treatment of AD. By in silico screening, we selected and purchased 16 potential DYRK1A inhibitors from the Specs chemical library. Among them, compound Q17 (Specs ID: AO-476/40829177) potently inhibited DYRK1A. The hydrogen bonds between compound Q17 and two amino acid residues named GLU239 and LYS188, were uncovered by molecular docking and molecular dynamics simulation. The cell-based assays showed that compound Q17 could protect the SH-SY5Y human neuroblastoma cell line from okadaic acid (OA)-induced injury by targeting DYRK1A. More importantly, compound Q17 significantly improved cognitive dysfunction of 3 × Tg-AD mice, ameliorated pathological changes, and attenuated Tau hyperphosphorylation as well as Aβ deposition. In summary, our computational modeling strategy is effective to identify novel chemotypes of DYRK1A inhibitors with great potential to treat AD, and the identified compound Q17 in this study is worthy of further study.

Comparison of 177Lu-octreotate and 177Lu-octreotide for treatment in human neuroblastoma-bearing mice

BackgroundPatients with high-risk neuroblastoma (NB) have a 5-year event-free survival of less than 50 %, and novel and improved treatment options are needed. Radiolabeled somatostatin analogs (SSTAs) could be a treatment option. The aims of this work were to compare the biodistribution and the therapeutic effects of 177Lu-octreotate and 177Lu-octreotide in mice bearing the human CLB-BAR NB cell line, and to evaluate their regulatory effects on apoptosis-related genes. MethodsThe biodistribution of 177Lu-octreotide in mice bearing CLB-BAR tumors was studied at 1, 24, and 168 h after administration, and the absorbed dose was estimated to tumor and normal tissues. Further, animals were administered different amounts of 177Lu-octreotate or 177Lu-octreotide. Tumor volume was measured over time and compared to a control group given saline. RNA was extracted from tumors, and the expression of 84 selected genes involved in apoptosis was quantified with qPCR. ResultsThe activity concentration was generally lower in most tissues for 177Lu-octreotide compared to 177Lu-octreotate. Mean absorbed dose per administered activity to tumor after injection of 1.5 MBq and 15 MBq was 0.74 and 0.03 Gy/MBq for 177Lu-octreotide and 2.9 and 0.45 Gy/MBq for 177Lu-octreotate, respectively. 177Lu-octreotide treatment resulted in statistically significant differences compared to controls. Fractionated administration led to a higher survival fraction than after a single administration. The pro-apoptotic genes TNSFS8, TNSFS10, and TRADD were regulated after administration with 177Lu-octreotate. Treatment with 177Lu-octreotide yielded regulation of the pro-apoptotic genes CASP5 and TRADD, and of the anti-apoptotic gene IL10 as well as the apoptosis-related gene TNF. Conclusion177Lu-octreotide gave somewhat better anti-tumor effects than 177Lu-octreotate. The similar effect observed in the treated groups with 177Lu-octreotate suggests saturation of the somatostatin receptors. Pronounced anti-tumor effects following fractionated administration merited receptor saturation as an explanation. The gene expression analyses suggest apoptosis activation through the extrinsic pathway for both radiopharmaceuticals.

Effect of Gallic Acid on PTZ-induced Neurotoxıcıty, Oxidative Stress and Inflammation in SH-SY5Y Neuroblastoma Cells

Aim: Human neuroblastoma cell lines are widely used to elucidate the cellular and molecular mechanisms of neurotoxicants and to facilitate the prioritization of in vivo testing. Pentylenetetrazole (PTZ) is a tetrazole derivative. Although PTZ is the most commonly used chemical to create an in vivo and in vitro epilepsy (EP) model, its mechanism of action in neuronal cells has not been fully elucidated. Gallic acid (GA) has broad biological properties such as antioxidant, anti-microbial, and anti-inflammatory activities. This study aimed to investigate the effect of GA on PTZ-induced neurotoxicity in neuroblastoma cells. Methods: For the study, four groups were formed from SH-SY5Y neuroblastoma cells as control (C), GA (100 μM), PTZ (30 μM), and PTZ+GA. In the study, total antioxidant and oxidant status (TAS and TOS), inflammatory cytokines (TNF α, IL 1β, and IL 6), lipid peroxidation levels as malondialdehyde (MDA), glutathione peroxidase (GSHPx), and glutathione (GSH) levels in the SH-SY5Y neuroblastoma cells were determined. Results: The results showed that PTZ treatment caused neurotoxicity in the neuroblastoma cell line and increased TOS, TNF α, IL 1β, IL 6, and MDA levels while decreasing TAS, GSH, and GSHPx levels. This situation improved with GA treatment. Conclusion: As a result, it was determined that GA treatment showed a protective effect in the PTZ-induced neural toxicity model in SH-SY5Y human neuroblastoma cell lines.

Unsaturated Fatty Acid Synthesis Is Associated with Worse Survival and Is Differentially Regulated by MYCN and Tumor Suppressor microRNAs in Neuroblastoma.

MYCN amplification (MNA) and disruption of tumor suppressor microRNA (TSmiR) function are key drivers of poor outcomes in neuroblastoma (NB). While MYCN and TSmiRs regulate glucose metabolism, their role in de novo fatty acid synthesis (FAS) and unsaturated FAS (UFAS) remains poorly understood. Here, we show that FAS and UFAS (U/FAS) genes FASN, ELOVL6, SCD, FADS2, and FADS1 are upregulated in high-risk (HR) NB and that their expression is associated with lower overall survival. RNA-Seq analysis of human NB cell lines revealed parallel U/FAS gene expression patterns. Consistent with this, we found that NB-related TSmiRs were predicted to target these genes extensively. We further observed that both MYC and MYCN upregulated U/FAS pathway genes while suppressing TSmiR host gene expression, suggesting a possible U/FAS regulatory network between MYCN and TSmiRs in NB. NB cells are high in de novo synthesized omega 9 (ω9) unsaturated fatty acids and low in both ω6 and ω3, suggesting a means for NB to limit cell-autonomous immune stimulation and reactive oxygen species (ROS)-driven apoptosis from ω6 and ω3 unsaturated fatty acid derivatives, respectively. We propose a model in which MYCN and TSmiRs regulate U/FAS and play an important role in NB pathology, with implications for other MYC family-driven cancers.

Novel multifunctional tacrine-donepezil hybrids against Alzheimer's disease: Design synthesis and bioactivity studies.

A series of tacrine-donepezil hybrids were synthesized as potential multifunctional anti-Alzheimer's disease (AD) compounds. For this purpose, tacrine and the benzylpiperidine moiety of donepezil were fused with a hydrazone group to achieve a small library of tacrine-donepezil hybrids. In agreement with the design, all compounds showed inhibitory activity toward both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC50 values in the low micromolar range. Kinetic studies on the most potent cholinesterase (ChE) inhibitors within the series showed a mixed-type inhibition mechanism on both enzymes. Also, the docking studies indicated that the compounds inhibit ChEs by dual binding site (DBS) interactions. Notably, tacrine-donepezil hybrids also exhibited significant neuroprotection against H2O2-induced cell death in a differentiated human neuroblastoma (SH-SY5Y) cell line at concentrations close to their IC50 values on ChEs and showed high to medium blood-brain barrier (BBB) permeability on human cerebral microvascular endothelial cells (HBEC-5i). Besides, the compounds do not cause remarkable toxicity in a human hepatocellular carcinoma cell line (HepG2) and SH-SY5Y cells. Additionally, the compounds were predicted to also have good bioavailability. Among the tested compounds, H4, H16, H17, and H24 stand out with their biological profile. Taken together, the proposed novel tacrine-donepezil scaffold represents a promising starting point for the development of novel anti-ChE multifunctional agents against AD.

Zinc Deficiency Decreases Neurite Extension via CRMP2 Signal Pathway.

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3β (pGSK3β) relative to GSK3β in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3β inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3β. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

Exploring neuroadaptive cellular pathways in chronic morphine exposure: An in-vitro analysis of cabergoline and Mdivi-1 co-treatment effects on the autophagy-apoptosis axis.

The complex impacts of prolonged morphine exposure continue to be a significant focus in the expanding area of addiction studies. This research investigates the effectiveness of a combined treatment using Cabergoline and Mdivi-1 to counteract the neuroadaptive changes caused by in vitro morphine treatment. The impact of Methadone, Cabergoline, and a combination of Cabergoline and Mdivi-1 on the cellular and molecular responses associated with Morphine-induced changes was studied in human Neuroblastoma (SK-N-MC) and Glioblastoma (U87-MG) cell lines that were exposed to prolong Morphine treatment. Cabergoline and Mdivi-1 combined treatment effectively influenced the molecular alterations associated with neuroadaptation in chronic morphine-exposed neural cells. This combination therapy normalized autophagy and reduced oxidative stress by enhancing total-antioxidant capacity, mitigating apoptosis, restoring BDNF expression, and balancing apoptotic elements. Our research outlines morphine's dual role in modulating mitochondrial dynamics via the dysregulation of the autophagy-apoptosis axis. This emphasizes the significant involvement of DRP1 activity in neurological adaptation processes, as well as disturbances in the dopaminergic pathway during in vitro chronic exposure to morphine in neural cells. This study proposes a novel approach by recommending the potential effectiveness of combining Cabergoline and Mdivi-1 to modulate the neuroadaptations caused by morphine. Additionally, we identified BDNF and PCNA in neural cells as potential neuroprotective markers for assessing the effectiveness of drugs against opioid toxicity, emphasizing the need for further validation. The study uncovers diverse effects observed in pretreated morphine glioblastoma cells under treatment with Cabergoline and methadone. This highlights the potential for new treatments in the DRD2 pathway and underscores the importance of investigating the interplay between autophagy and apoptosis to advance research in managing cancer-related pain. The study necessitates an in-depth investigation into the relationship between autophagy and apoptosis, with a specific emphasis on protein interactions and the dynamics of cell signaling.

Abstract LB295: Unsaturated fatty acid synthesis is associated with poor prognosis and is differentially regulated by MYCN and tumor suppressor microRNAs in neuroblastoma

Abstract Dysregulated metabolism is a hallmark of cancer. While the role of glucose metabolism in cancer has been extensively studied, relatively little has been done to understand the role of poly-unsaturated fatty acids in cancer biology. To gain insight into their contribution to cancer progression we determined the expression patterns and outcome associations of both saturated and unsaturated fatty acid synthesis (UFAS) genes in pediatric neuroblastoma (NB), and fatty acid levels in NB cell lines derived from high-risk tumors. MYCN amplification and disruption of tumor suppressor miRNAs (TSmiR) function are central drivers of poor outcomes in NB. MYC, MYCN, and TSmiRs regulate glucose metabolism; however, their role in UFAS remains poorly understood. Using complementary analysis of publicly available genomic datasets and cultured NB cell measurements we examined the potential regulation of UFAS genes through MYCN and MYC activity and posttranscriptional regulation by TSmiRs. Here we show that de novo and UFAS pathway genes FASN, ELOVL6, SCD, FADS2, and FADS1 are upregulated in high-risk NB and are associated with poor prognosis. RNA-Seq analysis of eight human NB cell lines revealed parallel UFAS gene expression patterns. Consistent with this, we found that NB-related TSmiRs were predicted to extensively target these genes. Additionally, we observed that both MYC and MYCN upregulated UFAS pathway genes while suppressing TSmiR host gene expression, thereby creating a possible UFAS regulatory network between MYCN and TSmiRs in NB. Furthermore, using mass spectrometry analysis we found NB cells to be high in de novo synthesized omega 9 (ω9) unsaturated fatty acids and low in both ω6 and ω3. This finding provides a plausible means for NB to limit cell-autonomous immune stimulation and reactive oxygen species (ROS)-driven apoptosis from ω6 and ω3 unsaturated fatty acid derivatives, respectively. We propose a model in which the UFAS pathway, through novel regulation by MYCN and TSmiRs, plays a key role in neuroblastoma pathology with implications for other MYC-driven cancers. Citation Format: Dennis A. Sheeter, Secilia Garza, Hui Gyu Park, Lorraine-Rana Benhamou, Nikki R. Badi, Erika C. Espinosa, Kumar Kothapalli, J. Thomas Brenna, John T. Powers. Unsaturated fatty acid synthesis is associated with poor prognosis and is differentially regulated by MYCN and tumor suppressor microRNAs in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB295.

Synthesis of Novel Plant-Derived Encapsulated Radiolabeled Compounds for the Diagnosis of Parkinson's Disease and the Evaluation of Biological Effects with In Vitro/In Vivo Methods.

Parkinson's disease (PD) is a neurodegenerative disorder that affects millions of individuals globally. It is characterized by the loss of dopaminergic neurons in Substantia Nigra pars compacta (SNc) and striatum. Neuroimaging techniques such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) help diagnosing PD. In this study, the focus was on developing technetium-99m ([99mTc]Tc) radiolabeled drug delivery systems using plant-derived compounds for the diagnosis of PD. Madecassoside (MA), a plant-derived compound, was conjugated with Levodopa (L-DOPA) to form MA-L-DOPA, which was then encapsulated using Poly Lactic-co-Glycolic Acid (PLGA) to create MA-PLGA and MA-L-DOPA-PLGA nanocapsules. Extensive structural analysis was performed using various methods such as Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), dynamic light scattering (DLS), and scanning electron microscopy (SEM) to characterize the synthesized products. Radiochemical yields of radiolabeled compounds were determined using thin layer radio chromatography (TLRC) and high performance liquid radio chromatography (HPLRC) methods. In vitro cell culture studies were conducted on human neuroblastoma (SH-SY5Y) and rat pheochromocytoma (PC-12) cell lines to assess the incorporation of [99mTc]Tc radiolabeled compounds ([99mTc]Tc-MA, [99mTc]Tc-MA-L-DOPA, [99mTc]Tc-MA-PLGA and [99mTc]Tc-MA-L-DOPA-PLGA) and the cytotoxicity of inactive compounds (MA and MA-L-DOPA compounds and encapsulated compounds (MA-PLGA and MA-L-DOPA-PLGA). Additionally, the biodistribution studies were carried out on healthy male Sprague-Dawley rats and a Parkinson's disease experimental model to evaluate the compounds' bioactivity using the radiolabeled compounds. The radiochemical yields of all radiolabeled compounds except [99mTc]Tc-L-DOPA-PLGA were above 95% and had stability over 6h. The cytotoxic effects of all substances on SH-SY5Y and PC-12 cells increase with increasing concentration values. The uptake values of PLGA-encapsulated compounds are statistically significant in SH-SY5Y and PC-12 cells. The biodistribution studies showed that [99mTc]Tc-MA is predominantly retained in specific organs and brain regions, with notable uptake in the prostate, muscle, and midbrain. PLGA-encapsulation led to higher uptake in certain organs, suggesting its biodegradable nature may enhance tissue retention, and surface modifications might further optimize brain penetration. Overall, the results indicate that radiolabeled plant-derived encapsulated drug delivery systems with [99mTc]Tc hold potential as diagnostic agents for PD symptoms. This study contributes to the advancement of drug delivery agents in the field of brain research.

Abstract 5466: In vivo characterization of neuroblastoma intratumoral heterogeneity

Abstract Background: Neuroblastoma is a pediatric solid tumor that arises from neural crest-derived progenitor cells of the peripheral sympathetic nervous system (PSNS). Neuroblastoma patients display a significant level of genetic and phenotypic heterogeneity, with amplification of the MYCN oncogene associated with poor clinical outcomes. To better understand underlying genetic mechanisms associated with aggressive neuroblastoma, our group incorporated patient-relevant loss-of-function mutations into the established MYCN-driven zebrafish model of neuroblastoma (Tg(dbh:EGFP-MYCN)), which resulted in increased tumor penetrance and a highly metastatic phenotype. Aims & Methods: We aimed to investigate metastases-associated intratumoral heterogeneity in vivo using our patient-relevant metastatic zebrafish models, with functional validations performed using human neuroblastoma cell lines. Results: Single-cell RNA sequencing of metastatic zebrafish neuroblastoma tumors revealed intra-tumor cell heterogeneity, with a subset of cells highly expressing markers of more differentiated adrenergic-like cell fates (th, dbh, phox2a) while others enriched for markers of less differentiated mesenchymal-like progenitor cells (prrx1a, vim, fn1a). Differential cell labeling in zebrafish neuroblastoma suggests distinct tumor cell populations for follow-up validation and characterization throughout metastatic progression. Conclusion: Single-cell RNA sequencing of zebrafish metastatic neuroblastoma showed evidence of intratumoral heterogeneity that warrants further investigation. We are characterizing neuroblastoma intratumoral heterogeneity in our in vivo metastatic zebrafish models to examine the association between neuroblastoma tumor composition and metastatic potential. Altogether, this work aims to improve our understanding of the evolution of tumor cell heterogeneity associated with neuroblastoma metastasis and uncover novel therapeutic opportunities to improve patient outcomes. Citation Format: Willow R. Squires, Alex Weiss, Sarah Cohen-Gogo, Adam Shlien, David Kaplan, Meredith S. Irwin, Madeline N. Hayes. In vivo characterization of neuroblastoma intratumoral heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5466.

Abstract 5472: Direct cytotoxicity of hu14.18K.322A in high-risk neuroblastoma

Abstract Background: Neuroblastoma is a childhood cancer of the sympathetic nervous system and is associated with disialoganglioside GD2 overexpression. The main goal of this study is to investigate the involvement of GD2 targeting humanized antidisialoganglioside monoclonal antibody hu14.18K.322A (hu14) in high-risk neuroblastoma direct cell cytotoxicity, assess for synergy with induction chemotherapy agents, and delineate the mechanisms involved in cell death. Methods: For this study, we used a panel of GD2 expressing cell lines [SK-N-BE (2), SK-N-BE (1), LAN5, CHLA15] and a GD2 non-expressing cell line LAN6. We measured surface expression of GD2 and performed cell cycle analysis using flow cytometry. The role of hu14 in neuroblastoma cell death was assessed using cell viability assays, proliferation assays, real time PCR and western blot analysis. We measured synergy between hu14 and induction phase chemotherapy drugs. Further, we employed next generation sequencing (NGS) to analyze the transcriptomic changes in human neuroblastoma cell lines in response to hu14 treatment to understand the pathways involved and the molecular mechanisms of hu14-mediated cellular response. Results: Using a cell viability assay, we found that CHLA15 and SK-N-BE (1) cell lines were sensitive to hu14 treatment whereas SK-N-BE (2), LAN5, LAN6 were resistant. Hu14 treatment resulted in a dose dependent reduction in cell proliferation in these cell lines. We also found that hu14 treatment resulted in increased expression of apoptotic and autophagy pathway proteins, but not ferroptosis or necroptosis pathways proteins. Hu14 induced cell death was partially inhibited by a pan-caspase inhibitor, confirming that caspase-dependent pathways are involved. Western blot analysis also showed decreased levels of p53 phosphorylation in both sensitive cell lines upon hu14 treatment suggesting an important control mechanism centered on the p53 pathway. Hu14 treatment reduced the surface expression of GD2 in the sensitive cells suggesting direct inhibition of the GD2 synthesis pathway. However, PCR analysis of GD2 synthesis genes did not show any significant difference upon treatment suggesting posttranscriptional control of these genes. Furthermore, NGS analysis revealed alterations in several pathways, but these changes were distinct between the cell lines reconfirming the complexity of therapy response in high-risk Neuroblastoma. Conclusion: Taken together, our findings suggest that hu14.18K322A induces direct cell cytotoxicity in a subset of high-risk Neuroblastoma and synergizes well with standard chemotherapy drugs. Furthermore, our research highlights the complex nature of signaling pathways involved in hu14-mediated cytotoxicity that can be harnessed towards targeted therapy. Citation Format: Manu Gnanamony, Maria Thomas, Thu Hien Nguyen, Amber M. D'Souza, Pedro de Alarcon. Direct cytotoxicity of hu14.18K.322A in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5472.

Imidazo[1,2‐α] azine's esters increase selectivity and anti‐inflammatory activity in microglial cells

AbstractChronic neuroinflammation is crucial in many neurodegenerative diseases, including AD. Neuroinflammation activates microglia, the CNS's central effector cells, and immune cells. Toxins and neuroinflammatory reactions can kill or damage neurons, activating microglial cells. Inflammatory mediators from activated microglia can kill neurons like cytokines, chemokines, reactive oxygen species, and reactive nitrogen species. Imidazo[1,2‐α]azines with different acid groups in position two of the heterocycle system were synthesized and tested as anti‐inflammatory and cell immunomodulators. We measured the ability of human neuroblastoma (SHSY‐5Y) and microglial (HMC3) cell lines to decrease PGE2 production under pro‐inflammatory conditions. All derivatives reduced PGE2 production. Inhibitory profiles indicate that compounds 8a and 8b are more effective and potent than 9a and 9b, indicating a steric effect of carboxyphenyl on imidazo[1,2‐α]azines, reducing their potency. However, imidazo[1,2α]pyrimidines (8b and 9b) with nitrogen at position 8 were less effective than those observed by other groups (8a, 9a, and 10a). imidazo[1,2‐α]pyrimidines are more polar due to nitrogen at position 8. The synthetically derivative imidazo[1,2‐α]pyridines decreased PGE2 formation in vitro on neural cells, microglia (HMC3), and neuroblastoma (SH‐SY5Y) cells, proving that they are more effective than imidazo[1,2‐α]pyrimidines. Two product esters selectively reduced HMC3 microglial cell PGE2 production. This finding may help develop neurodegenerative disease anti‐inflammatories.

Injectable Hydrogel To Deliver Bone Mesenchymal Stem Cells Preloaded with Azithromycin To Promote Spinal Cord Repair.

Spinal cord injury is a disease that causes severe damage to the central nervous system. Currently, there is no cure for spinal cord injury. Azithromycin is commonly used as an antibiotic, but it can also exert anti-inflammatory effects by down-regulating M1-type macrophage genes and up-regulating M2-type macrophage genes, which may make it effective for treating spinal cord injury. Bone mesenchymal stem cells possess tissue regenerative capabilities that may help promote the repair of the injured spinal cord. In this study, our objective was to explore the potential of promoting repair in the injured spinal cord by delivering bone mesenchymal stem cells that had internalized nanoparticles preloaded with azithromycin. To achieve this objective, we formulated azithromycin into nanoparticles along with a trans-activating transcriptional activator, which should enhance nanoparticle uptake by bone mesenchymal stem cells. These stem cells were then incorporated into an injectable hydrogel. The therapeutic effects of this formulation were analyzed in vitro using a mouse microglial cell line and a human neuroblastoma cell line, as well as in vivo using a rat model of spinal cord injury. The results showed that the formulation exhibited anti-inflammatory and neuroprotective effects in vitro as well as therapeutic effects in vivo. These results highlight the potential of a hydrogel containing bone mesenchymal stem cells preloaded with azithromycin and trans-activating transcriptional activator to mitigate spinal cord injury and promote tissue repair.

Discovery, Structure-Based Modification, In Vitro, In Vivo, and In Silico Exploration of m-Sulfamoyl Benzoamide Derivatives as Selective Butyrylcholinesterase Inhibitors for Treating Alzheimer's Disease.

For the potential therapy of Alzheimer's disease (AD), butyrylcholinesterase (BChE) has gradually gained worldwide interest in the progression of AD. This study used a pharmacophore-based virtual screening (VS) approach to identify Z32439948 as a new BChE inhibitor. Aiding by molecular docking and molecular dynamics, essential binding information was disclosed. Specifically, a subpocket was found and structure-guided design of a series of novel compounds was conducted. Derivatives were evaluated in vitro for cholinesterase inhibition and physicochemical properties (BBB, log P, and solubility). The investigation involved docking, molecular dynamics, enzyme kinetics, and surface plasmon resonance as well. The study highlighted compounds 27a (hBChE IC50 = 0.078 ± 0.03 μM) and (R)-37a (hBChE IC50 = 0.005 ± 0.001 μM) as the top-ranked BChE inhibitors. These compounds showed anti-inflammatory activity and no apparent cytotoxicity against the human neuroblastoma (SH-SY5Y) and mouse microglia (BV2) cell lines. The most active compounds exhibited the ability to improve cognition in both scopolamine- and Aβ1-42 peptide-induced cognitive deficit models. They can be promising lead compounds with potential implications for treating the late stage of AD.