Human Neuroblastoma Cell Line Research Articles

Dioscin-Mediated Autophagy Alleviates MPP+-Induced Neuronal Degeneration: An In Vitro Parkinson's Disease Model.

Autophagy is a cellular homeostatic process by which cells degrade and recycle their malfunctioned contents, and impairment in this process could lead to Parkinson’s disease (PD) pathogenesis. Dioscin, a steroidal saponin, has induced autophagy in several cell lines and animal models. The role of dioscin-mediated autophagy in PD remains to be investigated. Therefore, this study aims to investigate the hypothesis that dioscin-regulated autophagy and autophagy-related (ATG) proteins could protect neuronal cells in PD via reducing apoptosis and enhancing neurogenesis. In this study, the 1-methyl-4-phenylpyridinium ion (MPP+) was used to induce neurotoxicity and impair autophagic flux in a human neuroblastoma cell line (SH-SY5Y). The result showed that dioscin pre-treatment counters MPP+-mediated autophagic flux impairment and alleviates MPP+-induced apoptosis by downregulating activated caspase-3 and BCL2 associated X, apoptosis regulator (Bax) expression while increasing B-cell lymphoma 2 (Bcl-2) expression. In addition, dioscin pre-treatment was found to increase neurotrophic factors and tyrosine hydroxylase expression, suggesting that dioscin could ameliorate MPP+-induced degeneration in dopaminergic neurons and benefit the PD model. To conclude, we showed dioscin’s neuroprotective activity in neuronal SH-SY5Y cells might be partly related to its autophagy induction and suppression of the mitochondrial apoptosis pathway.

CRISPR/Cas9-Mediated Disruption of ZNF543 Gene: An Approach Toward Discovering Its Relation to TRIM28 Gene in Parkinson's Disease.

Genetic studies of familial forms of Parkinson's disease (PD) have shown that the ZNF543 gene is a candidate gene that operates relevant to this disease. However, until now, there is no evidence for ZNF543 gene function in PD, and mechanisms resulting from its mutation have not been elucidated. Given the same genetic location of the ZNF543 gene with TRIM28 and their effects on PD pathogenesis, we surmised that ZNF543 might act as a transcription factor for TRIM28 gene expression. By knocking out the ZNF543 gene via the CRISPR/Cas9 editing platform, we assessed the functional effect of loss of expression of this gene on TRIM28 gene expression. Four sgRNAs with different PAM sequences were designed against two parts of the regulatory region of ZNF543 gene, and highly efficient disruption of ZNF543 expression in human neuroblastoma cell line was evaluated by polymerase chain reaction and T7 endonuclease assay. Moreover, evaluation of TRIM28 gene expression in ZNF543-knocked-out cells indicated a significant increase in TRIM28 gene expression, suggesting that ZNF543 probably regulates the expression of TRIM28. This approach offers a window into pinpointing the mechanism by which ZNF543 gene mutations mediate PD pathogenicity.

Involvement of NLRP3/Caspase-1/GSDMD-Dependent pyroptosis in BPA-Induced apoptosis of human neuroblastoma cells

Bisphenol A (BPA) induces neurotoxicity via enhancing cell apoptosis and inflammation potently (effective at nanomolar concentrations), but its mechanisms remain unidentified. In this study, human neuroblastoma cell lines, IMR-32 and SK-N-SH cells, isolated from a male and a female subject, respectively, were exposed to BPA at various concentrations, with epigallocatechin gallate (EGCG, an antioxidant from green tea), Z-YVAD-FMK (a caspase-1 inhibitor), and ICI182.780 [an estrogen receptor (ER) inhibitor] as modulators. The results showed that BPA increased the mRNA levels of IL-18, ASC, GSDMD and protein levels of NLRP3, caspase-1 and GSDMD in both cell lines in a nonlinear manner. Noticeably, the direction of changes in the mRNA levels of caspase-1 and IL-1β were opposite, so did each of them in different cell lines: caspase-1 was enhanced in IMR-32 cells but suppressed in SK-N-SH cells, while IL-1β was suppressed in IMR-32 cells but enhanced in SK-N-SH cells. The level of GSDMD in situ increased along with the leakage of IL-1β, IL-18, caspase-1 and lactate dehydrogenase (LDH). Moreover, all the above effects of BPA were reversed by Z-YVAD-FMK, ICI182.780, and EGCG. Besides, BPA significantly increased reactive oxygen species production, LDH leakage and apoptosis, with reduced cell viability and mitochondrial membrane potential, in both cell lines, whereas Z-YVAD-FMK and ICI182.780 significantly alleviated the induction of Bak1, Bax, Bcl-2 and caspase-3 proteins by BPA. In summary, BPA may induce pyroptosis in neuroblastoma cells through NLRP3/caspase-1/GSDMD pathway, as mediated by ER; caspase-1-dependent pyroptosis may also contribute to BPA-induced apoptosis, an effect alleviated by EGCG.

Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis.

Histone deacetylase (HDAC) inhibitors are novel chemotherapy agents with potential utility in the treatment of neuroblastoma, the most frequent solid tumor of childhood. Previous studies have shown that the exposure of human neuroblastoma cells to some HDAC inhibitors enhanced the expression of the common neurotrophin receptor p75NTR. In the present study we investigated whether the upregulation of p75NTR could be exploited to render neuroblastoma cells susceptible to the cytotoxic action of an anti-p75NTR antibody conjugated to the toxin saporin-S6 (p75IgG-Sap). We found that two well-characterized HDAC inhibitors, valproic acid (VPA) and entinostat, were able to induce a strong expression of p75NTR in different human neuroblastoma cell lines but not in other cells, with entinostat, displaying a greater efficacy than VPA. Cell pretreatment with entinostat enhanced p75NTR internalization and intracellular saporin-S6 delivery following p75IgG-Sap exposure. The addition of p75IgG-Sap had no effect on vehicle-pretreated cells but potentiated the apoptotic cell death that was induced by entinostat. In three-dimensional neuroblastoma cell cultures, the subsequent treatment with p75IgG-Sap enhanced the inhibition of spheroid growth and the impairment of cell viability that was produced by entinostat. In athymic mice bearing neuroblastoma xenografts, chronic treatment with entinostat increased the expression of p75NTR in tumors but not in liver, kidney, heart, and cerebellum. The administration of p75IgG-Sap induced apoptosis only in tumors of mice that were pretreated with entinostat. These findings define a novel experimental strategy to selectively eliminate neuroblastoma cells based on the sequential treatment with entinostat and a toxin-conjugated anti-p75NTR antibody.

LncRNA TUG1 compromised neuronal mitophagy in cerebral ischemia/reperfusion injury by targeting sirtuin 1.

Mitophagy protects against cerebral ischemia/reperfusion (CI/R)-induced neuronal apoptosis via mitochondrial clearance. Although taurine-upregulated gene 1 (lncRNA TUG1) has been proposed to be involved in the neuronal apoptosis evoked by CI/R, its specific role in mitophagy during the progression of CI/R injury remains unknown. The CI/R rat model was established using middle cerebral artery occlusion/reperfusion (MCAO/R). Human neuroblastoma cell line SH-SY5Y was subjected to oxygen-glucose deprivation and reoxygenation (OGD/R). Ubiquitination assay, co-immunoprecipitation assay, RNA pull-down, and RNA immunoprecipitation were used to determine the interplay among TUG1, sirtuin 1 (SIRT1), and F-box and WD repeat domain-containing 7 (FBXW7). The upregulation of the TUG1 level and downregulation of the mitophagy were observed in both MCAO/R-treated rats and OGD/R-treated cells. The administration of si-TUG1 (a siRNA directed against TUG1) potentiated mitophagy and suppressed neuronal apoptosis in OGD/R-treated cells. However, the neuroprotective effect of si-TUG1 was reversed by mitophagy inhibitor or SIRT1 knockdown in vitro. Functionally, TUG1 enhanced FBXW7-mediated SIRT1 ubiquitination by upregulating FBXW7 expression. The overexpression of FBXW7 abrogated the si-TUG1-reinforced mitophagy by decreasing SIRT1 expression, thus aggravating neuronal apoptosis in the OGD/R+si-TUG1-treated cells. In rats with MCAO/R, the interference of TUG1 clearly decreased neuronal apoptosis, lessened the infarct volume, and relieved the neurological deficits. TUG1 knockdown promotes SIRT1-induced mitophagy by suppressing FBXW7-mediated SIRT1 degradation, thus relieving the neuronal apoptosis induced by CI/R injury. LncRNA TUG1 promotes neuronal apoptosis through inhibition of mitophagy. TUG1 decreased SIRT1 expression by promoting FBXW7-mediated SIRT1 ubiquitination. FBXW7/SIRT1 axis mediated the effect of TUG1 on OGD/R-induced neuronal apoptosis by regulating mitophagy.

The abnormal phosphorylation of the Rac1, Lim-kinase 1, and Cofilin proteins in the pathogenesis of Hirschsprung’s disease

ABSTRACT Rac1 can affect the migration of neural crest cells by regulating the polymerization of actin and the membrane formation process. But the role of the Rac1 signaling pathway in the pathogenesis of Hirschsprung’s disease (HSCR) remains unclear. In order to investigate the mechanism of the abnormal protein phosphorylation of Rac1, Lim-kinase 1 (Limk1) and Cofilin involved in the pathogenesis of HSCR. The protein phosphorylation levels of these proteins were detected by Western blot in 30 samples of HSCR narrow segment, 30 samples of transitional segment tissues, and 14 samples of normal intestinal tissues. Subsequently, in the SH-SY5Y human neuroblastoma cell line, a Rac1, Limk1, and Cofilin inhibitor group, a Rac1 overexpression group (PDGF-BB group), a Rac1 overexpression group + a Limk1 inhibitor group (P-B group), a Rac1 overexpression group + a Cofilin inhibitor group (P-C group) were established. The results showed that the expressions of p-Rac1, p-Limk1, and p-Cofilin in HSCR narrow segment and transitional segment were lower than those in normal intestine (p < 0.05). The expression levels of p-Rac1, p-Limk1, and p-Cofilin in the relative inhibitor group were significantly lower than those in the control group (p < 0.05), and the proliferation and migration levels in the control group and Rac1 overexpression group were significantly higher than those in the Rac1, Limk1, and Cofilin inhibitor group (p < 0.05). In conclusion, the decreased phosphorylation of the Rac1/Limk1/Cofilin signaling pathway in HSCR could inhibit the proliferation and migration of SH-SY5Y cells, and this might be associated with the pathogenesis of HSCR.

Photodynamic therapy with 5-aminolevulinic acid: A new diagnostic, therapeutic, and surgical aid for neuroblastoma

Background5-Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) is widely used in cancer therapy because of the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX). We aimed to assess the susceptibility of human neuroblastoma cell lines to ALA-PDT and determine the mechanism of PDT. MethodsWe used four human neuroblastoma cell lines (GOTO, NB9, IMR32, and NB1) and a gastric cancer cell line (MKN45) as a positive control. Cells were treated with increasing concentrations of ALA, and the ALA-induced production of PpIX in tumor cells was quantified using fluorescence spectrophotometry. PDT photocytotoxicity was measured by exposing the cells to a 630-nm irradiation for 10 min, and apoptotic cells stained with phosphatidylserine (PS) and propidium iodide (PI) were detected through flow cytometry. ResultsALA cytotoxicity was not observed in any cell line. The intracellular concentration of PpIX increased in an ALA dose-dependent manner, and intracellular fluorescence of PpIX increased in a time-dependent manner. The viability of NB-1 cells treated with 250 μM 5-ALA rapidly decreased to 5%. Photocytotoxicity was observed in the following order: NB1, IMR32, NB-9, and GOTO. Photocytotoxicity was positively correlated with intracellular PpIX concentrations. PS+/PI- cells increased up to 21% after 12 h, and PS+/PI+ cells accounted for 35% of all cells after 24 h, which suggests that ALA-PDT induced apoptotic cell death. ConclusionThis study shows that neuroblastoma cell lines were susceptible to 5-ALA-PDT, resulting in persistent apoptotic cell death. Levels of evidenceN/A for basic study.

The Thermal Dose of Photothermal Therapy Generates Differential Immunogenicity in Human Neuroblastoma Cells.

Simple SummaryPhotothermal therapy (PTT) is an effective thermal therapy for treating tumors. PTT has been combined with immunotherapy in various preclinical cancer models showing promising treatment outcomes. However, in these studies, PTT has primarily been utilized for maximizing tumor cell death. Previously, we observed that based on the “thermal dose” applied, PTT can generate dramatically different responses from the immune system when tested in cellular and animal models. Here, we sought to provide a framework to systematically assess the effect of PTT-based thermal doses on the immunogenic correlates of treated tumors as a measure of the effectiveness of PTT in eliciting an antitumor immune response. In human neuroblastoma tumor cells in vitro, we determined specific phenotypic markers, which demonstrated that SH-SY5Y cells were more responsive to PTT-based thermal dose compared with LAN-1 cells, which possess a high-risk phenotype. Our findings suggest the importance of conducting tumor thermal dose-responsiveness studies in vitro as an early measure of PTT effectiveness against a specific tumor.Photothermal therapy (PTT) is an effective method for tumor eradication and has been successfully combined with immunotherapy. However, besides its cytotoxic effects, little is known about the effect of the PTT thermal dose on the immunogenicity of treated tumor cells. Therefore, we administered a range of thermal doses using Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT) and assessed their effects on tumor cell death and concomitant immunogenicity correlates in two human neuroblastoma cell lines: SH-SY5Y (MYCN-non-amplified) and LAN-1 (MYCN-amplified). PBNP-PTT generated thermal dose-dependent tumor cell killing and immunogenic cell death (ICD) in both tumor lines in vitro. However, the effect of the thermal dose on ICD and the expression of costimulatory molecules, immune checkpoint molecules, major histocompatibility complexes, an NK cell-activating ligand, and a neuroblastoma-associated antigen were significantly more pronounced in SH-SY5Y cells compared with LAN-1 cells, consistent with the high-risk phenotype of LAN-1 cells. In functional co-culture studies in vitro, T cells exhibited significantly higher cytotoxicity toward SH-SY5Y cells relative to LAN-1 cells at equivalent thermal doses. This preliminary report suggests the importance of moving past the traditional focus of using PTT solely for tumor eradication to one that considers the immunogenic effects of PTT thermal dose to facilitate its success in cancer immunotherapy.

Protective Effect of Amber Extract on Human Dopaminergic Cells against 6-Hydroxydopamine-Induced Neurotoxicity.

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disease, after Alzheimer’s disease. In our previous study, we found that amber—a fossilized plant resin—can protect cells from apoptosis by decreasing the generation of reactive oxygen species (ROS). In this study, we focused on the effect of amber on 6-hydroxydopamine-induced cell apoptosis in the human neuroblastoma cell line SHSY5Y (one model for PD). Initially, we determined the protective effect of amber on the PD model. We found that amber extract has a protective effect against 6-hydroxydopamine-induced cell apoptosis. The decrease in ROS, cleaved caspase-3, pERK, and extracellular signal-regulated kinase (ERK) protein levels confirmed that amber extract decreases apoptosis via the ROS-mediated ERK signaling pathway. Furthermore, we determined the effects of amber extract on autophagy. The results showed that amber extract increased the levels of LC3II and Beclin-1, suggesting that amber extract can protect neuronal cells against 6-hydroxydopamine-induced cell apoptosis by promoting autophagy.

Serine-Threonine Kinase Receptor Associate Protein (STRAP) confers an aggressive phenotype in neuroblastoma via regulation of Focal Adhesion Kinase (FAK)

BackgroundSerine-threonine kinase receptor associated protein (STRAP), a scaffolding protein, is upregulated in many solid tumors. As such, we hypothesized that STRAP may be overexpressed in neuroblastoma tumors and may play a role in neuroblastoma tumor progression. MethodsWe examined two publicly available neuroblastoma patient databases, GSE49710 (n = 498) and GSE49711 (n = 498), to investigate STRAP expression in human specimens. SK-N-AS and SK-N-BE(2) human neuroblastoma cell lines were stably transfected with STRAP overexpression (OE) plasmid, and their resulting phenotype studied. PamChip® kinomic peptide microarray evaluated the effects of STRAP overexpression on kinase activation. ResultsIn human specimens, higher STRAP expression correlated with high-risk disease, unfavorable histology, and decreased overall neuroblastoma patient survival. STRAP OE in neuroblastoma cell lines led to increased proliferation, growth, supported a stem-like phenotype and activated downstream FAK targets. When FAK was targeted with the small molecule FAK inhibitor, PF-573,228, STRAP OE neuroblastoma cells had significantly decreased growth compared to control empty vector cells. ConclusionIncreased STRAP expression in neuroblastoma was associated with unfavorable tumor characteristics. STRAP OE resulted in increased kinomic activity of FAK. These findings suggest that the poorer outcomes in neuroblastoma tumors associated with STRAP overexpression may be secondary to FAK activation.

Protective Effects of Probucol on Different Brain Cells Exposed to Manganese

Manganese (Mn) is an essential metal for many functions in the body. However, in excess, it can be neurotoxic and cause a Parkinson-like syndrome, known as manganism. Here, we aimed to identify a protective effect of probucol, a lipid-lowering agent with anti-inflammatory and antioxidant properties, against Mn-induced toxicity in human neuroblastoma (SH-SY5Y) and glioblastoma (C6) cell lines. The cells were incubated with increasing concentrations ofMnfollowed by probucol addition 1, 3, 6, and/or 24h toassess themetal toxic doses and measurethe protectiveeffectofprobucol against Mn-induced oxidative damage. Longer exposition to Mn showed decreased SH-SY5Y cellular viability inconcentrations higherthan100µM, andprobucolwas able to prevent this effect. The C6cells were more sensitive to the Mn deleterious actions, decreasing the cell viabilityafter 6h of 500µM Mn exposure. In addition,probucol prevents the complexI and II of the mitochondrial respiratory chain (MRC) inhibition caused by Mn and decreased the intracellular ROS production. Taken together, our results showed that Mn toxicity affects differently both cell lines and probucolhas a protective effect against the oxidative imbalance in the central nervous system.

Long read sequencing reveals novel isoforms and insights into splicing regulation during cell state changes

BackgroundAlternative splicing is a key mechanism underlying cellular differentiation and a driver of complexity in mammalian neuronal tissues. However, understanding of which isoforms are differentially used or expressed and how this affects cellular differentiation remains unclear. Long read sequencing allows full-length transcript recovery and quantification, enabling transcript-level analysis of alternative splicing processes and how these change with cell state. Here, we utilise Oxford Nanopore Technologies sequencing to produce a custom annotation of a well-studied human neuroblastoma cell line SH-SY5Y, and to characterise isoform expression and usage across differentiation.ResultsWe identify many previously unannotated features, including a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2. We show differential expression and usage of transcripts during differentiation identifying candidates for future research into state change regulation.ConclusionsOur work highlights the potential of long read sequencing to uncover previously unknown transcript diversity and mechanisms influencing alternative splicing.

Induction of serine hydroxymethyltransferase 2 promotes tumorigenesis and metastasis in neuroblastoma.

High-risk neuroblastoma (NB) remains an extremely difficult subgroup to cure and is associated with MYCN amplification. Serine hydroxymethyltransferase 2 (SHMT2) regulates serine metabolism in a myc-dependent manner; it is upregulated in several cancers and is associated with tumor aggressiveness. Akt-2, an important regulator of MYCN via the PI3K/Akt pathway, induces metastatic potential in NB. The association between SHMT2 and PI3K/Akt in hepatocyte regeneration has been well established but its mechanistic interaction in cancer has yet to be clearly elucidated. Herein, we evaluated the exact role of SHMT2 on the PI3K/Akt pathway, in addition to NB tumorigenesis and metastatic potential in vitro. SHMT2 gene expression and overall survival (OS) were assessed. Two human NB cell lines were examined. SHMT2 silencing and overexpression were performed. The downstream effects were analyzed with immunoblotting, RT-qPCR and functional assays were performed. We found SHMT2 gene expression is associated with decreased OS and MYCN amplification. SHMT2 protein and mRNA expression are increased in MYCN-amplified cells. SHMT2 expression has a direct interaction with Akt-2 and MYCN. Induction of SHMT2 increased cellular proliferation, colony formation and cellular migration and SHMT2 expression was increased in metastatic NB cells. We conclude that SHMT2 regulates N-Myc via phosphorylation of Akt-2 and plays an important role in NB tumorigenesis by contributing to cell growth, migration, colony formation and metastasis in vitro.

In vitro neuroprotective effects of allicin on Alzheimer’s disease model of neuroblastoma cell line

Background/Aim: Alzheimer's disease is a progressive disorder that causes atrophy and neuronal death in the brain. Currently, there is not any effective therapy for Alzheimer's disease. The current research was designed to investigate the beneficial effects of allicin on Alzheimer’s disease in SHSY-5Y cells in vitro and elucidating the neuroprotective mechanism of allicin. Methods: Human neuroblastoma cell line (SH-SY5Y) was differentiated with retinoic acid to conduct the in vitro Alzheimer’s Disease model. Amyloid β1-42 protein was applied to the cells for 24 hours (2.5 µM) to induce cytotoxicity. Allicin was applied to the cell cultures in a wide spectrum dose (10 μM, 50 μM, 100 μM) to investigate neuroprotective effect against amyloid β for 24 hours. MTT and LDH analyses were performed to assess the cell viability. MDA and ROS concentrations and SOD activity were analyzed to determine the oxidative stress. Moreover, the effects of allicin on the caspase-3 expression in amyloid β induced neurotoxicity were determined by the RT-PCR analysis. Results: Amyloid β markedly decreased cell viability of SH-SY5Y in MTT analysis and elevated LDH levels. In contrast, in MTT analysis, the allicin markedly increased cell viability, indicating that allicin induces cell proliferation. Moreover, in LDH analysis, allicin treatment markedly decreased LDH release. Exposure to amyloid β markedly increased MDA and ROS levels, in comparison with the control. Moreover, amyloid β decreased activity of SOD in SH-SY5Y cells. Allicin markedly balanced out the amyloid β-induced MDA and ROS generation. In the same pathway, allicin increased activity of SOD in amyloid β -exposed SH-SY5Y cells. The caspase 3 expression was increased in amyloid β group in comparison to the control group and allicin markedly lowered the expression of caspase-3 levels. Conclusion: The beneficial effects of allicin on amyloid β -induced neurotoxicity on SH-SY5Y cells were reported for the first time in terms of cell viability, oxidative stress and apoptosis.

In Vitro Cytotoxic activity of Rhinacanthin Enriched Extract from Leaves of Rhinacanthus nasutus (L.) Kurz., (Acanthaceae) against Neuroblastoma Cell Line

In the present study, the shade dried powdered leaves of Rhinacanthus nasutus (L.) Kurz., was subjected to maceration with ethyl acetate, concentrated and evaporated to dryness. The ethyl acetate extract was subjected to anion exchange column chromatography and the isolated rhinacanthin enriched extract was analyzed by thin layer chromatography and fourier-transform infrared studies. In the present study, the in vitro cytotoxic activity of the rhinacanthin rich extract of Rhinacanthus nasutus (L) Kurz., over SH-SY5Y human neuroblastoma cell line was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay and compared with the standard drug doxorubicin. The preliminary phytochemical screening revealed the presence of alkaloids, quinones, glycosides, carbohydrates, amino acids and tannins. The thin layer chromatography profiling showed the presence of secondary metabolites in various mobile phase systems. Fourier-Transform Infrared analysis of rhinacanthin enriched extract confirmed the presence of functional groups including 1, 4-quinone carbonyl component. The results of in vitro cell line studies revealed a dose dependent cytotoxic effect on human neuroblastoma cell lines with an IC50 value of 88.9 μg/ml. Thus the results validate the potential of rhinacanthin enriched extract against neuroblastoma cancer and based on this initial screening, further studies at elucidation of its molecular mechanism as cancer therapeutics can be undertaken.

The histone deacetylase inhibitor OBP-801 has invitro/invivo anti-neuroblastoma activity.

Patients with high-risk neuroblastoma have a poor prognosis; new therapeutic agents are therefore required. We investigated the antitumor effects of OBP-801, a novel histone deacetylase inhibitor, its underlying mechanism, and its potential as a therapeutic agent for patients with neuroblastoma. The study included five human neuroblastoma cell lines: IMR32, GOTO, KP-N-RTBM, SK-N-AS, and SH-SY5Y. We investigated cell proliferation, cell cycle status, protein expression patterns, and apoptosis in neuroblastoma cells after OBP-801 treatment invitro. Cell survival rate and cell cycle were analyzed using the WST-8 assay and flow cytometry, respectively. Apoptosis was detected using annexin V staining, and the expression of apoptosis-related proteins was investigated by western blotting. The antitumor activity of OBP-801 was examined in an invivo xenograft mouse model. Dose-effect curve analysis showed that the mean half-maximal inhibitory concentration value was 5.5 ± 5.9 nM for the MYCN-amplified cell lines (IMR32, GOTO, and KP-N-RTBM) and 3.1 ± 0.7 nM for the MYCN-nonamplified cell lines (SK-N-AS and SH-SY5Y). OBP-801 inhibited cell proliferation and growth in all the cell lines. It induced G2/M phase arrest through the p21 (CDKN1A) pathway, increasing histone H3 levels and, subsequently, apoptosis in human neuroblastoma cells. OBP-801 suppressed the growth of neuroblastoma cells in the mouse xenograft model. Overall, OBP-801 induces M-phase arrest and apoptosis in neuroblastoma cells via mitotic catastrophe. Our results indicate that OBP-801 is a promising therapeutic agent with fewer adverse effects for patients with neuroblastoma.

Potential Effects of Melatonin on TRPA1 Channels in the Prevention and Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia and is defined as a progressive neurodegenerative disease. Main clinical features of AD are progressive impairment in learning and memory loss. Several studies have indicated that mitochondria play a critical role in the pathogenesis of AD. In this study, we investigated the effect of melatonin on mitochondria-dependent TRPA1 ion channels in neuroblastoma cells by creating an in vitro model of Alzheimer's disease. Okadaic acid was applied to SH-SY5Y (human neuroblastoma cell line) cells to create an AD model. After cellular differentiation, the following 7 main groups were created: Group 1 (Control), Group 2 (Mel+AD), Group 3 (Mel+AD+AP18), Group 4 (AD), Group 5 (AD+AP18), Group 6 (AD+Mel), and Group 7 (AD+Mel+AP18), and Alzheimer's disease was determined in vitro by examining the effect of melatonin on calcium-dependent TRPA1 channels in neuroblastoma cells. The Ca2+concentration was greater in the melatonin+AD, AD and AD+melatonin groups than in the control (p<0.001). However, there was no statistically significant difference between Mel+AD+AP18, AD+Mel+AP18 and the control. We determined that Ca2+ levels were lower in the melatonin+AD and AD+melatonin groups than in the AD group (p<0.001 and p<0.05). Additionally, cytosolic Ca2+ concentrations were found to be lower in the melatonin+AD group than in the AD+melatonin group (p<0.05). In evaluating the apoptosis and oxidative stress levels, we found that the apoptosis and intracellular ROS values were higher in the melatonin+AD, AD and AD+melatonin groups than in the control (p<0.001). In this respect, the mitochondrial depolarization and caspase-3 and caspase-9 levels were higher in the melatonin+AD, AD and AD+melatonin groups than in the control group (p<0.001). Additionally, the mitochondrial depolarization, caspase-3 and caspase-9 values were higher in the AD group than in the melatonin+AD and AD+melatonin groups (p<0.001), while mitochondrial depolarization and caspase-3 levels were lower in the melatonin+AD group than in the AD+melatonin group (p<0.001). However, in the same groups, there was no statistically significant difference in caspase-9 results. Additionally, the caspase-9 values were lower in the melatonin+AD group, AD group and AD+melatonin groups than in the melatonin+AD+AP18, AD+AP18 and AD+melatonin+AP18 groups, respectively (p<0.001 and p<0.05). Our results suggest that melatonin may be an effective option in the treatment and prophylaxis of Alzheimer's disease by reducing cytosolic Ca2+ concentration, apoptosis and intracellular ROS through TRPA1 channels.

Activation of Atg7-dependent autophagy by a novel inhibitor of the Keap1-Nrf2 protein-protein interaction from Penthorum chinense Pursh. attenuates 6-hydroxydopamine-induced ferroptosis in zebrafish and dopaminergic neurons.

The death of dopaminergic neurons is a dominant factor during the occurrence and development of Parkinson's disease (PD). Previous studies demonstrated that ferroptosis is implicated in the death of dopaminergic neurons. Besides, polyphenols have been proven to be effective in preventing the death of dopaminergic neurons. This work aims to explore the neuroprotective effect and mechanism of thonningianin A (Th A), a polyphenolic compound in natural plant foods, against 6-hydroxydopamine (6-OHDA)-induced ferroptosis in dopaminergic cells. The results of molecular docking and other binding assays collectively demonstrated that Th A can strongly target the Kelch domain of Keap1. Th A treatment significantly facilitated the nuclear factor erythroid 2-like 2 (Nrf2) nuclear translocation and subsequently increased the heme oxygenase-1 (HO-1) protein level through inhibiting the protein-protein interaction (PPI) of Keap1 and Nrf2. Compared with the nomifensine (Nomi) treatment, Th A had a more potent protective effect on 6-OHDA-induced ferroptosis during PD pathology in zebrafish, which was associated with assuaging the reduction of the total swimming distance, glutathione (GSH) depletion, iron accumulation, lipid peroxidation, and aggregation of α-synuclein (α-syn). Furthermore, Th A also exhibited a strong protective effect against 6-OHDA-induced ferroptosis in vitro in the human neuroblastoma cell line SH-SY5Y. Th A degraded Keap1 protein through activating Atg7-dependent autophagy. Additionally, Th A treatment facilitated the degradation of Keap1 protein by promoting the interaction between p62/SQSTM1 (sequestosome 1, hereafter referred to as p62) and Keap1. Taken together, our findings indicated that Th A protects dopaminergic cells against 6-OHDA-induced ferroptosis through activating the Nrf2-based cytoprotective system, thus enabling a potential application of Keap1-Nrf2 PPI inhibitors in the restraint of ferroptosis and treatment of PD.

Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide induces ferroptosis in neuroblastoma cells through redox imbalance.

As a widespread environmental pollutant, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-induced neurotoxicity has received increasing attention. Studies have shown that BPDE-induced neurodegeneration is due partly to neuronal apoptosis. Unlike apoptosis, ferroptosis is a non-apoptotic form of programmed cell death, but its specific role in the neurotoxicity of BPDE remains unclear. In this work, we investigated the ferroptosis in BPDE-induced cell death in human neuroblastoma cell line SH-SY5Y using a specific pharmacological inhibitor. Lipid peroxides, malondialdehyde production, glutathione / glutathione peroxidase activity, superoxide dismutase activity, and iron content were evaluated. Consistent with previous studies, our data showed that 0.5 μM BPDE poisoning for 24 hr could induce cell apoptosis and that cell survival could be improved by using apoptosis inhibitors. But with prolonged exposure time (72 hr) or increased exposure dose (1.0 μM), we have elucidated and validated that BPDE triggered ferroptosis in human SH-SY5Y cells. We also revealed that suppression of ferroptosis by specific inhibitors, ferrostatin-1 and deferoxamine, significantly rescued the phenotypes of ferroptosis induced by BPDE. BPDE downregulated Nrf2 and its target genes related to redox regulation, GPX4 and SLC7A11, but upregulated HO-1. Our results first demonstrated that BPDE caused cytotoxic effects on cell death via apoptosis and ferroptosis. Most notably, long-term environmental exposure to BPDE becomes a concern due to ferroptosis. Redox imbalance is controlled by the Nrf2, SLC7A11, and HO-1, through which lipid peroxides and ferrous ion accumulation cause ferroptosis after BPDE treatment. These findings highlight that targeting ferroptosis could serve as an effective protective strategy for neurotoxicity of BPDE.

Itchy E3 ubiquitin-protein ligase promotes neuroblastoma progression in vitro and in vivo

[Objective] Neuroblastoma is the most common pediatric neuroendocrine tumor. Patients with high-risk neuroblastoma have poor clinical outcomes. Understanding the mechanisms underlying neuroblastoma progression could help identify potential therapeutic targets. This study aimed to explore the roles of itchy E3 ubiquitin-protein ligase (ITCH) in neuroblastoma progression using neuroblastoma cell lines and xenograft models of neuroblastoma. [Methods] ITCH-silencing or overexpressing neuroblastoma cells were established using two different human neuroblastoma cell lines, SK-N-AS and SH-SY5Y. In vitro and in vivo experiments were carried out to determine the effects of ITCH on neuroblastoma cell behaviors. The dual-luciferase reporter assay and co-transfection experiments were applied to determine the interaction of ITCH and miR-145-5p during neuroblastoma progression. [Results] In both cell lines, ITCH overexpression significantly promotes the proliferation, migration, and invasion capacities of neuroblastoma cells, while ITCH silencing with ShITCH suppressed neuroblastoma cell proliferation and induced apoptosis. Moreover, overexpression of ITCH decreased 51% and 54% the protein expressions of large tumor suppressor kinase 1 (LATS1), and inhibited 59% and 66% the phosphorylation of Yes-associated protein (YAP), concomitant with 2.02-fold and 2.56-fold increased expressions of cell proliferation marker Ki67 and 2.51-fold and 2.26-fold elevated levels of anti-apoptosis marker Bcl2 in SK-N-AS and SH-SY5Y cells, respectively. The dual-luciferase reporter assay demonstrated that ITCH interacted with miR-145-5p. Further in vitro and xenograft experiments showed that ITCH negatively affected the tumor-suppressive effect of miR-145-5p. [Conclusion] ITCH promotes neuroblastoma cell proliferation and metastasis by inhibiting LATS1 and promoting YAP nuclear translocation.