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1.
Oncol Rep ; 49(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36416312

RESUMO

7­Methoxyheptaphylline (7­MH) is a carbazole extracted from Clausena harmandiana, a medicinal plant that is used to treat headaches and stomachaches. The aim of the present study was to examine the neuroprotective effects and anticancer activity of 7­MH. Cell death was assessed using an MTT assay and flow cytometry. The expression of apoptosis­related proteins was determined by western blot analysis. An animal model was used to test anti­metastasis. The interactions between 7­MH and the molecular target were observed using molecular docking. The results revealed that 7­MH provided protection against hydrogen peroxide (H2O2)­induced neuronal cell death. In cancer cells, 7­MH induced SH­SY5Y, 4T1, HT29, HepG2, and LNCaP cell death. 7­MH inhibited metastasis of HT29 cells in vitro and 4T1­Luc cells in vitro and in vivo. 7­MH inhibited proteins, including P­glycogen synthase kinase (GSK)­3, and cleaved caspase­3, but it activated anti­apoptotic proteins in H2O2­induced SH­SY5Y cell death. By contrast, 7­MH activated the cleaving of caspase­3 and GSK­3, but it suppressed anti­apoptotic proteins in SH­SY5Y cells. 7­MH reduced the levels of NF­κB and STAT3 in 4T1 cells; phospho­p65, Erk, and MAPK13 in LNCaP cells; and phospho­Erk and matrix metalloproteinase­9 in HT29 cells. Molecular docking analysis showed that 7­MH targets TAK1 kinase. The present study indicated that 7­MH induced apoptosis of cancer cells and provided protection against H2O2­induced neuron cell death via TAK1 kinase.


Assuntos
Peróxido de Hidrogênio , Neuroblastoma , Animais , Humanos , Caspase 3/metabolismo , Peróxido de Hidrogênio/farmacologia , Quinase 3 da Glicogênio Sintase , Simulação de Acoplamento Molecular , Linhagem Celular Tumoral , Neuroblastoma/metabolismo , Carbazóis/farmacologia
2.
Acta Histochem ; 124(2): 151856, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35077998

RESUMO

Neuroblastoma is a metastatic brain tumor particularly common in children. The cure rate is below 50% for patients of high-risk condition. Novel therapeutic agents and approaches are needed to improve the cure rate. Tumor necrosis factor-related and apoptosis-inducing ligand (TRAIL) is a promising proapoptotic factor that rapidly induces apoptosis preferentially in transformed and cancerous cells. Unfortunately, the common TRAIL resistance in cancers has hampered the clinical application of the ligand. Previously we prepared a novel TRAIL-armed ER derived nanosomal agent (ERN-T) that overcomes TRAIL resistance in some cancer lines when combined with a synthetic antagonist of inhibitors of apoptosis proteins (IAPs), AZD5582. However, how AZD5582 sensitizes cancer cells to ERN-T remains not well understood. In this study we continued to test the therapeutic efficacy of the combinatory therapy of ERN-T and AZD5582 on neuroblastoma, aiming to reveal the molecular mechanism underlying the synergism between AZD5582 and ERN-T. The obtained data revealed that ERN-Ts overcame TRAIL resistance and showed significant cytotoxicity on the resistant neuroblastoma line SH-SH5Y when combined with AZD5582 whilst sparing normal cells. The combination of low doses of ERN-Ts and AZD5582 induced intensive apoptosis in SH-SY5Y but not in normal skin fibroblasts (NSFs). Importantly we discovered that TRAIL sensitization in SH-SY5Y was associated with the concomitant downregulation of antiapoptotic factors cFLIP, MCL-1 and IAPs and upregulation of proapoptotic protein BAX and the death receptor 5 (DR5) by the cotreatment of ERN-T and AZD5582. In vivo study demonstrated that the combination of ERN-T and AZD5582 constituted a highly effective and safe therapy for subcutaneous SH-SY5Y xenograft neuroblastoma in nude mice. In conclusion, we identified that the concomitant regulation of both antiapoptotic and proapoptotic factors and DR5 is an essential molecular mechanism for overcoming TRAIL resistance in SH-SY5Y and the combination of ERN-T and AZD5582 potentially constitutes a novel therapeutic strategy, which is highly effective and safe for neuroblastoma.


Assuntos
Neuroblastoma , Ligante Indutor de Apoptose Relacionado a TNF , Alcinos , Animais , Apoptose , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oligopeptídeos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico
3.
J Tradit Chin Med ; 42(6): 877-884, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36378044

RESUMO

OBJECTIVE: To investigate the in-depth pharma-cological mechanisms of celastrol in children neuro-blastoma treatment. METHODS: In the current study, we examined the effects of celastrol on children neuroblastoma cells viability and proliferation by cell counting kit-8 assay and colony formation assay. Annexin V-FTIC and PI staining were applied to determine cell apoptosis after celastrol treatment. ROS generation levels were examined by 2', 7'-dichloroflfluorescin diacetate. RESULTS: We found that celastrol could suppress the proliferation of children neuroblastoma cells with few effects on normal cell lines . Further mechanisms studies have shown that celastrol inhibited cell cycle progression and induced cell apoptosis in QDDQ-NM and SH-SY5Y cells. In addition, ROS production might involve in celastrol-mediated apoptotic cell death in children neuroblastoma cells by activating caspase death pathway. CONCLUSIONS: Our findings demonstrated that celastrol could promote ROS generation-induced apoptosis in neuroblastoma cell by activating caspase death pathway. These findings suggested that celastrol might be a potential novel anti-neuroblastoma agent with minor cytotoxicity.


Assuntos
Neuroblastoma , Triterpenos , Criança , Humanos , Caspases/genética , Espécies Reativas de Oxigênio/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Triterpenos/farmacologia , Linhagem Celular Tumoral , Apoptose , Sobrevivência Celular , Proliferação de Células , Caspase 3/metabolismo
4.
Oncogene ; 41(46): 4994-5007, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36319669

RESUMO

Neuroblastomas require novel therapies that are based on the exploitation of their biological mechanism. To address this need, we analyzed the DNA methylation and expression datasets of neuroblastomas, extracted a candidate gene characterizing the aggressive features, and conducted functional studies. Based on the DNA methylation data, we identified a subgroup of neuroblastoma cases with 11q loss of heterozygosity with extremely poor prognosis. PHGDH, a serine metabolism-related gene, was extracted as a candidate with strong expression and characteristic methylation in this subgroup as well as in cases with MYCN amplification. PHGDH inhibition suppressed neuroblastoma cell proliferation in vitro and in vivo, indicating that the inhibition of serine metabolism by PHGDH inhibitors is a therapeutic alternative for neuroblastoma. Inhibiting the arginine metabolism, which is closely related to serine metabolism using arginine deiminase, had a combination effect both in vitro and in vivo, especially on extracellular arginine-dependent neuroblastoma cells with ASS1 deficiency. Expression and metabolome analyses of post-dose cells confirmed the synergistic effects of treatments targeting serine and arginine indicated that xCT inhibitors that inhibit cystine uptake could be candidates for further combinatorial treatment. Our results highlight the rational therapeutic strategy of targeting serine/arginine metabolism for intractable neuroblastoma.


Assuntos
Metilação de DNA , Neuroblastoma , Humanos , Metilação de DNA/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proliferação de Células/genética , Serina/metabolismo , Arginina/genética , Arginina/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica
5.
Int J Mol Sci ; 23(21)2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36361524

RESUMO

Hyperphosphorylation and aggregation of the microtubule binding protein tau is a neuropathological hallmark of Alzheimer's disease/tauopathies. Tau neurotoxicity provokes alterations in brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB)/cAMP-response-element binding protein (CREB) signaling to contribute to neurodegeneration. Compounds activating TRKB may therefore provide beneficial effects in tauopathies. LM-031, a coumarin derivative, has demonstrated the potential to improve BDNF signaling in neuronal cells expressing pro-aggregated ΔK280 tau mutant. In this study, we investigated if LM-031 analogous compounds provide neuroprotection effects through interaction with TRKB in SH-SY5Y cells expressing ΔK280 tauRD-DsRed folding reporter. All four LMDS compounds reduced tau aggregation and reactive oxygen species. Among them, LMDS-1 and -2 reduced caspase-1, caspase-6 and caspase-3 activities and promoted neurite outgrowth, and the effect was significantly reversed by knockdown of TRKB. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in these cells, implying that the neuroprotective effects of LMDS-1/2 are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. Furthermore, LMDS-1/2 demonstrated their ability to quench the intrinsic fluorescence of tryptophan residues within the extracellular domain of TRKB, thereby consolidating their interaction with TRKB. Our results suggest that LMDS-1/2 exert neuroprotection through activating TRKB signaling, and shed light on their potential application in therapeutics of Alzheimer's disease/tauopathies.


Assuntos
Doença de Alzheimer , Neuroblastoma , Fármacos Neuroprotetores , Tauopatias , Humanos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas tau/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neuroproteção , Doença de Alzheimer/tratamento farmacológico , Cumarínicos/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Neuroblastoma/metabolismo , Receptor trkB/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Caspases
6.
Int J Mol Sci ; 23(21)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36361771

RESUMO

More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors. To date, there is no approved therapy for dry AMD, including late-stage geographic atrophy. We present a treatment option for dry AMD that transfers the brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial (RPE) cells by electroporation using the plasmid-based Sleeping Beauty (SB) transposon system. ARPE-19 cells and primary human RPE cells were co-transfected with two plasmids encoding the SB100X transposase and the transposon carrying a BDNF transcription cassette. We demonstrated efficient expression and secretion of BDNF in both RPE cell types, which were further increased in ARPE-19 cell cultures exposed to hydrogen peroxide. BDNF-transfected cells exhibited lower apoptosis rates and stimulated neurite outgrowth in human SH-SY5Y cells. This study is an important step in the development of a cell-based BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat dry AMD or other degenerative retinal diseases.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Neuroblastoma , Humanos , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Neuroblastoma/metabolismo , Terapia Genética , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo
7.
PLoS One ; 17(11): e0277134, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36331951

RESUMO

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins ßIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.


Assuntos
Acetilcolinesterase , Neuroblastoma , Humanos , Acetilcolinesterase/metabolismo , Polpa Dentária/metabolismo , Neuroblastoma/metabolismo , Diferenciação Celular , Tretinoína/farmacologia , Células-Tronco , Colinérgicos , Células Cultivadas , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo
8.
Biomed Pharmacother ; 156: 113966, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36411643

RESUMO

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor arising from neural crest cells of the developing sympathetic nervous system. Despite marked advances in cancer treatment, the survival rate of high-risk NB remains unsatisfactory. As a key pro-inflammatory mediator regulating tumor microenvironment, prostaglandin E2 (PGE2) promotes NB proliferation, angiogenesis, and immune evasion via acting on four G protein-coupled receptors, particularly the EP2 subtype. Recent studies have been vigorously focused on developing and evaluating compounds targeting PGE2-regulated tumor inflammation in animal models of NB. In this review, we revisit these translational efforts and examine the feasibility of pharmacological inhibition of enzymes responsible for PGE2 biosynthesis or its signaling receptors as emerging therapeutic strategies for NB. We also explore the potential downstream oncogenic pathways upon the activation of PGE2 receptors, aiming to bridge the knowledge gap between tumorigenesis and the role of elevated PGE2/EP2 signaling, which is widely observed in high-risk NBs.


Assuntos
Dinoprostona , Neuroblastoma , Animais , Dinoprostona/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Receptores de Prostaglandina E , Transdução de Sinais , Microambiente Tumoral
9.
J Cell Sci ; 135(22)2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36321463

RESUMO

Notch signaling and its downstream gene target HES1 play a critical role in regulating and maintaining cancer stem cells (CSCs), similar to as they do during embryonic development. Here, we report a unique subclass of Notch-independent Hes-1 (NIHes-1)-expressing CSCs in neuroblastoma. These CSCs maintain sustained HES1 expression by activation of HES1 promoter region upstream of classical CBF-1 binding sites, thereby completely bypassing Notch receptor-mediated activation. These stem cells have self-renewal ability and potential to generate tumors. Interestingly, we observed that NIHes-1 CSCs could transition to Notch-dependent Hes-1-expressing (NDHes-1) CSCs where HES1 is expressed by Notch receptor-mediated promoter activation. We observed that NDHes-1-expressing CSCs also had the potential to transition to NIHes-1 CSCs and during this coordinated bidirectional transition, both CSCs gave rise to the majority of the bulk cancer cells, which had an inactive HES1 promoter (PIHes-1). A few of these PIHes-1 cells were capable of reverting into a CSC state. These findings explain the existence of a heterogenic mode of HES1 promoter activation within the IMR-32 neuroblastoma cell line and the potential to switch between them. This article has an associated First Person interview with the first authors of the paper.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neuroblastoma , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores Notch/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Regiões Promotoras Genéticas/genética , Linhagem Celular , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo
10.
PLoS One ; 17(11): e0275600, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36378656

RESUMO

Cell therapies using human induced pluripotent stem cell (hiPSC)-derived nephron progenitor cells (NPCs) are expected to ameliorate acute kidney injury (AKI). However, using hiPSC-derived NPCs clinically is a challenge because hiPSCs themselves are tumorigenic. LIN28A, ESRG, CNMD and SFRP2 transcripts have been used as a marker of residual hiPSCs for a variety of cell types undergoing clinical trials. In this study, by reanalyzing public databases, we found a baseline expression of LIN28A, ESRG, CNMD and SFRP2 in hiPSC-derived NPCs and several other cell types, suggesting LIN28A, ESRG, CNMD and SFRP2 are not always reliable markers for iPSC detection. As an alternative, we discovered a lncRNA marker gene, MIR302CHG, among many known and unknown iPSC markers, as highly differentially expressed between hiPSCs and NPCs, by RNA sequencing and quantitative RT-PCR (qRT-PCR) analyses. Using MIR302CHG as an hiPSC marker, we constructed two assay methods, a combination of magnetic bead-based enrichment and qRT-PCR and digital droplet PCR alone, to detect a small number of residual hiPSCs in NPC populations. The use of these in vitro assays could contribute to patient safety in treatments using hiPSC-derived cells.


Assuntos
Células-Tronco Pluripotentes Induzidas , Neuroblastoma , RNA Longo não Codificante , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Técnicas In Vitro , Néfrons , RNA Longo não Codificante/metabolismo , Neuroblastoma/metabolismo
11.
Hum Exp Toxicol ; 41: 9603271221136206, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36411272

RESUMO

In this study, we aimed to evaluate possible toxic effects of thimerosal, aluminum and combination of thimerosal and aluminum in SH-SY5Y cells. Inhibitory concentrations were determined by MTT assay; reactive oxygen species (ROS) were determined by a fluorometric kit and antioxidant/oxidant parameters were measured by spectrophotometric kits. Nuclear factor erythroid 2-associated factor 2 (Nrf2), norepinephrine (NE), dopamine transporter (DAT) and dopamine beta ß-hydroxylase (DBH) levels were measured by sandwich ELISA kits while 8-hydroxy deoxyguanosine (8-OHdG) and dopamine levels were determined by competitive ELISA kits. Thimerosal (1.15 µM) and aluminum (362 µM) were applied to cells at inhibitory concentrations 20 (IC20s) for 24 h. ROS increased significantly in cells aluminum- and aluminum+thimerosal-treated cells. Glutathione levels decreased in aluminum group while total antioxidant capacity and protein oxidation levels increased significantly in aluminum and aluminum+thimerosal groups. Lipid peroxidation increased significantly in groups treated with aluminum and aluminum+thimerosal. Nrf2 levels and DNA damage were significantly higher in all groups while dopamine levels significantly increased in cells treated with thimerosal and aluminum+thimerosal, DAT levels were found to be higher in all experimental groups compared to the control. These findings showed that both thimerosal and aluminum can change oxidant/antioxidant status, cause DNA damage, alter dopamine and DAT levels. Changes seen in cells treated with combined exposure to aluminum and thimerosal are more pronounced. Special care should be taken while vaccinating sensitive populations and safer alternatives for aluminum and thimerosal should used.


Assuntos
Neuroblastoma , Timerosal , Humanos , Timerosal/toxicidade , Hidróxido de Alumínio , Alumínio/toxicidade , Fator 2 Relacionado a NF-E2 , Dopamina , Antioxidantes/farmacologia , Espécies Reativas de Oxigênio , Neuroblastoma/metabolismo , Linhagem Celular , Oxidantes
12.
Bioorg Med Chem ; 74: 117069, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36283251

RESUMO

C-Abl is involved in various biological processes and plays an important role in neurodegenerative diseases, especially Parkinson's disease (PD). Previous studies have found that nilotinib shows a neuroprotective effect in cell and animal models of PD by inhibiting the activation of c-Abl. But the low blood-brain barrier permeability and potential toxicity limit the further use of nilotinib in PD. Based on molecular modeling studies, a series of 4-methyl-3-(pyridin-2-ylamino)benzamide derivatives were designed and synthesized. In particular, compound 9a exhibited significant inhibitory activity against c-Abl and a potent neuroprotective effect against MPP+-induced SH-SY5Y cell death. Moreover, 9a not only displayed lower cell toxicity compared with nilotinib, but also showed higher oral bioavailability and proper permeability of the blood-brain barrier. This paper provides 4-methyl-3-(pyridin-2-ylamino)benzamide derivatives as a new scaffold for c-Abl inhibitor with potential neuroprotective effect.


Assuntos
Neuroblastoma , Fármacos Neuroprotetores , Doença de Parkinson , Animais , Humanos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/metabolismo , Neuroblastoma/metabolismo , Barreira Hematoencefálica/metabolismo , Doença de Parkinson/metabolismo , Benzamidas/farmacologia , Benzamidas/metabolismo , Linhagem Celular Tumoral
13.
Int J Mol Sci ; 23(19)2022 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-36232980

RESUMO

Oxygen glucose deprivation (OGD) can produce hypoxia-induced neurotoxicity and is a mature in vitro model of hypoxic cell damage. Activated AMP-activated protein kinase (AMPK) regulates a downstream pathway that substantially increases bioenergy production, which may be a key player in physiological energy and has also been shown to play a role in regulating neuroprotective processes. Resveratrol is an effective activator of AMPK, indicating that it may have therapeutic potential as a neuroprotective agent. However, the mechanism by which resveratrol achieves these beneficial effects in SH-SY5Y cells exposed to OGD-induced inflammation and oxidative stress in a 3D gelatin scaffold remains unclear. Therefore, in the present study, we investigated the effect of resveratrol in 3D gelatin scaffold cells to understand its neuroprotective effects on NF-κB signaling, NLRP3 inflammasome, and oxidative stress under OGD conditions. Here, we show that resveratrol improves the expression levels of cell viability, inflammatory cytokines (TNF-α, IL-1ß, and IL-18), NF-κB signaling, and NLRP3 inflammasome, that OGD increases. In addition, resveratrol rescued oxidative stress, nuclear factor-erythroid 2 related factor 2 (Nrf2), and Nrf2 downstream antioxidant target genes (e.g., SOD, Gpx GSH, catalase, and HO-1). Treatment with resveratrol can significantly normalize OGD-induced changes in SH-SY5Y cell inflammation, oxidative stress, and oxidative defense gene expression; however, these resveratrol protective effects are affected by AMPK antagonists (Compounds C) blocking. These findings improve our understanding of the mechanism of the AMPK-dependent protective effect of resveratrol under 3D OGD-induced inflammation and oxidative stress-mediated cerebral ischemic stroke conditions.


Assuntos
Neuroblastoma , Fármacos Neuroprotetores , Proteínas Quinases Ativadas por AMP/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Catalase/metabolismo , Gelatina/farmacologia , Glucose/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/metabolismo , Interleucina-18/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroblastoma/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo , Oxigênio/metabolismo , Resveratrol/metabolismo , Resveratrol/farmacologia , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
14.
Int J Mol Sci ; 23(19)2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36233079

RESUMO

Peptides have revealed a large range of biological activities with high selectivity and efficiency for the development of new drugs, including neuroprotective agents. Therefore, this work investigates the neuroprotective properties of naturally occurring peptides, endomorphin-1 (EM-1), endomorphin-2 (EM-2), rubiscolin-5 (R-5), and rubiscolin-6 (R-6). We aimed at answering the question of whether well-known opioid peptides can counteract cell injury in a common in vitro model of Parkinson's disease (PD). Antioxidant activity of these four peptides was evaluated by the 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity, oxygen radical absorbance capacity (ORAC), and ferric-reducing antioxidant power (FRAP) assays, while neuroprotective effects were assessed in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y). The mechanisms associated with neuroprotection were investigated by the determination of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, and Caspase-3 activity. Among the tested peptides, endomorphins significantly prevented neuronal death induced by 6-OHDA treatment, decreasing MMP (EM-1) or Caspase-3 activity (EM-2). Meanwhile, R-6 showed antioxidant potential by FRAP assay and exhibited the highest capacity to recover the neurotoxicity induced by 6-OHDA via attenuation of ROS levels and mitochondrial dysfunction. Generally, we hypothesize that peptides' ability to suppress the toxic effect induced by 6-OHDA may be mediated by different cellular mechanisms. The protective effect caused by endomorphins results in an antiapoptotic effect (mitochondrial protection and decrease in Caspase-3 activity), while R-6 potency to increase a cell's viability seems to be mediated by reducing oxidative stress. Our results may provide new insight into neurodegeneration and support the short peptides as a potent drug candidate to treat PD. However, further studies should be conducted on the detailed mechanisms of how tested peptides could suppress neuronal injuries.


Assuntos
Neuroblastoma , Fármacos Neuroprotetores , Síndromes Neurotóxicas , Doença de Parkinson , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Neuroblastoma/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Peptídeos Opioides/farmacologia , Oxidopamina/farmacologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
Eur J Med Chem ; 244: 114818, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36223680

RESUMO

The epigenetic regulator lysine specific demethylase 1 (LSD1), a MYCN cofactor, cooperatively silences MYCN suppressor genes. Furthermore, LSD1 has been correlated with adverse effects in neuroblastic tumors by propagating an undifferentiated, malignant phenotype. We observed that high LSD1 mRNA expression in MYCN-expressing neuroblastoma (NB) correlated with poor prognosis, implicating LSD1 as an oncogenic accomplice in high-grade NB. Thus, LSD1 inhibition is a potential strategy for targeting treatment-resistant MYCN-expressing NB. Tranylcypromine-based covalent LSD1 inhibitors have demonstrated anti-tumor activity but are associated with undesirable off-target effects, such that only 2 non-covalent LSD1 inhibitors are in clinical trials. We now report 3 novel scaffolds for reversible LSD1 inhibition: 2-(arylsulfonamido)benzoic acid, N-(2-(1H-tetrazol-5-yl)phenyl)benzenesulfonamide and 2-(arylcarboxamido)benzoic acid analogues. The most active of these analogues, compound 48, exhibited potent and selective mixed reversible inhibition of LSD1 (IC50 = 0.58 µM) and significantly increased global H3K4me2 in NB cells. In addition, combination treatment with 48 and bortezomib in NB cells results in a synergistic effect.


Assuntos
Histona Desmetilases , Neuroblastoma , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Linhagem Celular Tumoral , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Ácido Benzoico
16.
Cells ; 11(20)2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36291086

RESUMO

Pannexin1 (Panx1) is expressed in both neurons and glia where it forms ATP-permeable channels that are activated under pathological conditions such as epilepsy, migraine, inflammation, and ischemia. Membrane lipid composition affects proper distribution and function of receptors and ion channels, and defects in cholesterol metabolism are associated with neurological diseases. In order to understand the impact of membrane cholesterol on the distribution and function of Panx1 in neural cells, we used fluorescence recovery after photobleaching (FRAP) to evaluate its mobility and electrophysiology and dye uptake to assess channel function. We observed that cholesterol extraction (using methyl-ß-cyclodextrin) and inhibition of its synthesis (lovastatin) decreased the lateral diffusion of Panx1 in the plasma membrane. Panx1 channel activity (dye uptake, ATP release and ionic current) was enhanced in cholesterol-depleted Panx1 transfected cells and in wild-type astrocytes compared to non-depleted or Panx1 null cells. Manipulation of cholesterol levels may, therefore, offer a novel strategy by which Panx1 channel activation might modulate various pathological conditions.


Assuntos
Astrócitos , Colesterol , Conexinas , Proteínas do Tecido Nervoso , Neuroblastoma , Humanos , Trifosfato de Adenosina/metabolismo , Anticolesterolemiantes/farmacologia , Astrócitos/metabolismo , Colesterol/metabolismo , Conexinas/metabolismo , Canais Iônicos/metabolismo , Lovastatina/farmacologia , Lipídeos de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/metabolismo , Estabilidade Proteica
17.
Front Immunol ; 13: 1016683, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36311753

RESUMO

Background: The prognosis of MYCN positive NB is poor, and there is no targeted drug for N-myc at present. This study aims to screen out hub genes closely related to MYCN, analyze the relationship between hub genes and NB microenvironment, and provide basis for molecular targeted therapy of MYCN positive NB. Methods: We combined the microarray data of GSE45547 (n=649) and GSE49710 (n=498), screened the DEGs between MYCN positive (n=185) and MYCN negative NB (n=951), performed WGCNA, Lasso regression and Roc analyses on the merged matrix, and obtained the hub genes related to MYCN in the training group. We performed ssGSEA on the experimental group to calculate the infiltration level of 28 kinds of immune cells in each sample, compared the differences of immune cell infiltration between MYCN positive and MYCN negative group. The influences of hub genes on the distribution of each immune cell were also analyzed by ssGSEA. The expression differences of the three hub genes were verified in the E-MTAB-8248 cohort (n=223), and the correlation between hub genes and prognosis of NB was calculated by Kaplan-Meier method in GSE62564 (n=498) and the validation group. We also verified the expression differences of hub genes by qRT-PCR in SK-N-BE(2), SKNDZ, Kelly and SH-SY5Y cell lines. Results: Here were 880 DEGs including 420 upregulated and 460 downregulated genes in MYCN positive NB in the training group. Overlap of the DEGs and WGCNA networks identified four shared genes, namely, ZNF695, CHEK1, C15ORF42 and EXO1, as candidate hub genes in MYCN positive NB. Three core genes, ZNF695, CHEK1 and C15ORF42, were finally identified by Lasso regression and Roc analyses. ZNF695, CHEK1 and C15ORF42 were highly expressed in MYCN positive NB tissues and cell lines. These three genes were closely related to the prognosis of children with NB. Except that Activated CD4 T cell and Type2 T helper cell increased, the infiltration levels of the other 26 cells decreased significantly in MYCN positive NB tissues. The infiltration levels of Type2 T helper cell and Activated CD4 T cell were also significantly positively correlated with the expression levels of the three hub genes. Conclusion: ZNF695, CHEK1 and C15ORF42 are highly expressed in MYCN positive NB, and their expression levels are negatively correlated with the prognosis of children with NB. The infiltration levels of Activated CD4 T cell and Type2 T helper cell increased in the microenvironment of MYCN positive NB and were significantly positively correlated with the expression levels of the three hub genes. The results of this study provide that ZNF695, CHEK1 and C15ORF42 may be potential prognostic markers and immunotherapy targets for MYCN positive NB.


Assuntos
Neuroblastoma , Criança , Humanos , Algoritmos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Microambiente Tumoral
18.
J Exp Clin Cancer Res ; 41(1): 314, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36284313

RESUMO

BACKGROUND: Beta-1,3-galactosyltransferase-4 (B3GALT4) plays a critical regulatory role in tumor biology. However, the role of B3GALT4 in modulating the tumor microenvironment (TME) of neuroblastoma (NB) remains unknown. METHODS: Public datasets and clinical NB samples were collected to evaluate the expression and clinical significance of GD2 and B3GALT4 in NB patients. CCK-8, colony formation, and transwell assays and experiments in tumor-bearing mouse models were conducted to investigate the function of B3GALT4. Flow cytometry, ELISA, immunohistochemistry, immunofluorescence, western blotting, and chemotaxis assays were conducted to ascertain the immunomodulatory mechanism of B3GALT4. The combined therapeutic effect of the lipid raft inhibitor MßCD and anti-GD2 mAb was validated in a murine model of NB. RESULTS: GD2 was overexpressed in NB tissues and high expression of GD2 was associated with poor prognosis in NB patients. B3GALT4 was downregulated in NB tissues, and low expression of B3GALT4 indicated poor prognosis in NB patients. Silencing B3GALT4 significantly enhanced tumor progression both in vitro and in vivo. Meanwhile, the overexpression of B3GALT4 increased the recruitment of CD8+ T lymphocytes via the chemokines CXCL9 and CXCL10. Additionally, B3GALT4 regulated NB-cell GD2 expression and lipid raft formation. Mechanistically, B3GALT4 regulated the expression of CXCL9 and CXCL10 via the c-Met signaling in the lipid rafts and the downstream AKT/mTOR/IRF-1 pathway. The lipid raft inhibitor, MßCD, attenuated B3GALT4 deficiency-induced tumor progression and immune evasion. Last, MßCD combined with anti-GD2 mAb treatment significantly enhanced the antitumor effect and the infiltration of CD8+ T cells. CONCLUSIONS: Upregulation of B3GALT4 promotes the secretion of CXCL9 and CXCL10 to recruit CD8+ T lymphocytes via the GD2-mediated lipid rafts and the c-Met/AKT/mTOR/IRF-1 pathway. Moreover, lipid raft inhibitors may enhance the efficacy of anti-GD2 immunotherapy for NB.


Assuntos
Neuroblastoma , Proteínas Proto-Oncogênicas c-akt , Animais , Camundongos , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Quimiocinas/uso terapêutico , Galactosiltransferases/uso terapêutico , Gangliosídeos/metabolismo , Microdomínios da Membrana , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Sincalida/uso terapêutico , Serina-Treonina Quinases TOR , Microambiente Tumoral , Proteínas Proto-Oncogênicas c-met/metabolismo
19.
Cells ; 11(17)2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36078115

RESUMO

Neuronal oxidative stress caused by mitochondrial dysfunction plays a crucial role in the development of Parkinson's disease (PD). Growing evidence shows that autophagy confers neuroprotection in oxidative-stress-associated PD. This work aims to investigate the involvement of TMEM166, an endoplasmic-reticulum-localized autophagy-regulating protein, in the process of PD-associated oxidative stress through the classic cellular PD model of neuroblastoma SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP+). Reactive oxygen species (ROS) production and mitochondrial membrane potential were checked to assess the oxidative stress induced by MPP+ and the cellular ATP generated was determined to evaluate mitochondrial function. The effect on autophagy induction was evaluated by analyzing p62 and LC3-II/I expression and by observing the LC3 puncta and the colocalization of LC3 with LAMP1/ LAMP2. The colocalization of mitochondria with LC3, the colocalization of Tom20 with LAMP1 and Tom20 expression were analyzed to evaluate mitophagy. We found that TMEM166 is up-regulated in transcript levels, but up-regulated first and then down-regulated by autophagic degradation in protein levels upon MPP+-treatment. Overexpression of TMEM166 induces mitochondria fragmentation and dysfunction and exacerbates MPP+-induced oxidative stress and cell viability reduction. Overexpression of TMEM166 is sufficient to induce autophagy and mitophagy and promotes autophagy and mitophagy under MPP+ treatment, while knockdown of TMEM166 inhibits basal autophagic degradation. In addition, overexpressed TMEM166 suppresses AMPK activation, while TMEM166 knockdown enhances AMPK activation. Pharmacological activation of AMPK alleviates the exacerbation of oxidative stress induced by TMEM166 overexpression and increases cell viability, while pharmacological inhibition mitophagy aggravates the oxidative stress induced by MPP+ treatment combined with TMEM166 overexpression. Finally, we find that overexpressed TMEM166 partially localizes to mitochondria and, simultaneously, the active AMPK in mitochondria is decreased. Collectively, these findings suggest that TMEM166 can translocate from ER to mitochondria and inhibit AMPK activation and, in response to mitochondrial oxidative stress, neuronal cells choose to up-regulate TMEM166 to promote autophagy/mitophagy; then, the enhancing autophagy/mitophagy degrades the TMEM166 to activate AMPK, by the two means to maintain cell survival. The continuous synthesis and degradation of TMEM166 in autophagy/mitochondria flux suggest that TMEM166 may act as an autophagy/mitochondria adaptor.


Assuntos
Proteínas Quinases Ativadas por AMP , Proteínas de Membrana , Doença de Parkinson , 1-Metil-4-fenilpiridínio/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Humanos , Proteínas de Membrana/metabolismo , Mitofagia , Neuroblastoma/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/metabolismo
20.
Pflugers Arch ; 474(11): 1133-1145, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36048287

RESUMO

Guanosine (GUO), widely considered a key signaling mediator, is implicated in the regulation of several cellular processes. While its interaction with neural membranes has been described, GUO still is an orphan neuromodulator. It has been postulated that GUO may eventually interact with potassium channels and adenosine (ADO) receptors (ARs), both particularly important for the control of cellular excitability. Accordingly, here, we investigated the effects of GUO on the bioelectric activity of human neuroblastoma SH-SY5Y cells by whole-cell patch-clamp recordings. We first explored the contribution of voltage-dependent K+ channels and, besides this, the role of ARs in the regulation of GUO-dependent cellular electrophysiology. Our data support that GUO is able to specifically modulate K+-dependent outward currents over cell membranes. Importantly, administering ADO along with GUO potentiates its effects. Overall, these results suggested that K+ outward membrane channels may be targeted by GUO with an implication of  ADO receptors in SH-SY5Y cells, but also support the hypothesis of a functional interaction of the two ligands. The present research runs through the leitmotif of the deorphanization of GUO, adding insight on the interplay with adenosinergic signaling and suggesting GUO as a powerful modulator of SH-SY5Y excitability.


Assuntos
Guanosina , Neuroblastoma , Adenosina , Guanosina/farmacologia , Humanos , Ligantes , Neuroblastoma/metabolismo , Canais de Potássio , Receptores Purinérgicos P1/metabolismo
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