Low-intensity pulsed ultrasound enhances neurite growth in serum-starved human neuroblastoma cells.

Introduction: Low-intensity pulsed ultrasound (LIPUS) is a recognized tool for promoting nerve regeneration and repair; however, the intracellular mechanisms of LIPUS stimulation remain underexplored. Method: The present study delves into the effects of varying LIPUS parameters, namely duty cycle, spatial average-temporal average (SATA) intensity, and ultrasound amplitude, on the therapeutic efficacy using SK-N-SH cells cultured in serum-starved conditions. Four distinct LIPUS settings were employed: (A) 50 mW/cm2, 40%, (B) 25 mW/cm2, 10%, (C) 50 mW/cm2, 20%, and (D) 25 mW/cm2, 10%. Results: Immunochemistry analysis exhibited neurite outgrowth promotion in all LIPUS-treated groups except for Group D. Further, LIPUS treatment was found to successfully promote brain-derived neurotrophic factor (BDNF) expression and enhance the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, protein kinase B (Akt), and mammalian target of rapamycin (mTOR) signaling pathways, as evidenced by western blot analysis. Discussion: The study suggests that the parameter combination of LIPUS determines the therapeutic efficacy of LIPUS. Future investigations should aim to optimize these parameters for different cell types and settings and delve deeper into the cellular response mechanism to LIPUS treatment. Such advancements may aid in tailoring LIPUS treatment strategies to specific therapeutic needs.

LncRNA ENST00000440246.1 Promotes Alzheimer's Disease Progression by Targeting PP2A.

Alzheimer's disease (AD) is an extremely prevalent neurodegenerative disease. Long noncoding RNAs (lncRNAs) play pivotal roles in the regulation of AD. However, the function of most lncRNAs in AD remains to be elucidated. In this study, the effects of lncRNA ENST00000440246.1 on the biological characteristics of AD were explored. Differentially expressed lncRNAs in AD were identified through bioinformatics analysis and peripheral blood from thirty AD patients was collected to verify the expression of these lncRNAs by quantitative real-time polymerase chain reaction (RT-qPCR). The correlations between lncRNAs and the Mini-Mental State Examination (MMSE) or the Montreal Cognitive Assessment (MoCA) were assessed by Pearson's correlation analysis. Immunofluorescence (IF), Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted to evaluate the biological effect of ENST00000440246.1 and protein phosphatase 2 A (PP2A) in SK-N-SH cells. Gene expression at the protein and mRNA levels was analyzed by Western blotting and RT-qPCR. The interaction between PP2A and ENST00000440246.1 was confirmed by IntaRNA and RNA pulldown assays. ENST00000440246.1 was upregulated and significantly negatively correlated with the MMSE and MoCA scores and the overexpression of ENST00000440246.1 inhibited cell proliferation and facilitated apoptosis and Aß expression in SK-N-SH cells. Mechanistically, ENST00000440246.1 targeted PP2A and regulated AD-related gene expression. The silencing of ENST00000440246.1 had the opposite effect. Furthermore, PP2A overexpression reversed the influence of ENST00000440246.1 overexpression in SK-N-SH cells. In conclusion, ENST00000440246.1 could promote AD progression by targeting PP2A, which indicates that ENST00000440246.1 has the potential to be a diagnostic target in AD.

Targeting HNRNPA2B1 inhibits enterovirus 71 replication in SK-N-SH cells.

OBJECTIVE: To investigate the effect of heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) on the replication of enterovirus 71 (EV-71) in SK-N-SH cells. METHODS: The mRNA and protein expression of HNRNPA2B1 in SK-N-SH cells were detected by real-time quantitative PCR (qRT-PCR) and western blotting (WB), respectively. WB was used to detect HNRNPA2B1 protein expression in the nucleus and cytosol. The localization of HNRNPA2B1 protein in the nucleus and cytosol was detected by immunofluorescence (IF). The expression of HNRNPA2B1 was inhibited by small interfering RNA (si-HNRNPA2B1). Viral RNA, viral structural protein VP1, and viral titer were detected by qRT-PCR, WB, and viral dilution counting, respectively. RESULTS: EV-71 infection significantly upregulates the expression of HNRNPA2B1 in SK-N-SH cells. EV-71 infection promotes HNRNPA2B1 nucleus-cytoplasm redistribution. Down-regulation of HNRNPA2B1 expression significantly inhibited EV-71 replication. CONCLUSION: HNRNPA2B1 protein redistributed from nucleus to cytoplasm and is highly expressed in the cytoplasm during EV-71 infection. Inhibition of HNRNPA2B1 levels effectively inhibits EV-71 replication in SK-N-SH cells.

Enhancing the neuroprotective effect of squid outer skin astaxanthin against rotenone-induced neurotoxicity in in-vitro model for Parkinson's disease.

Rotenone is a widely used organic pesticide that induces neurotoxicity via inhibition of mitochondrial complex I and oxidative stress actions for the most of dopaminergic neurons as that occurring in Parkinsonism disease (PD). Astaxanthin (ASX) is a natural pigment (carotenoids) and a potent therapeutic compound due to its antioxidant and anti-inflammatory properties. The commercially important cephalopod Doryteuthis singhalensis is widely distributed in tropical and subtropical waters in World Ocean. D. singhalensis is an important source of astaxanthin that contains valuable biological active compounds with many valuable pharmacological effects. The present study evaluated the effect of astaxanthin in preventing rotenone-induced toxicity of SK-N-SH human neuroblastoma cells in an in vitro model of experimental Parkinsonism. The results revealed the strongly significant antioxidant capability of extracted squid astaxanthin in 1,1- diphenyl- 2- picrylhydrazyl (DPPH) radical scavenging activity. In addition, astaxanthin treatment based on dose dependent manner significantly attenuated rotenone induced cytotoxicity, mitochondrial dysfunction and oxidative stress in SKN- SH cells. It is concluded that the marine squid derived astaxanthin could be used as a potential neuroprotector against rotenone induced toxicity due to its antioxidant, and anti-apoptotic properties. Consequently, it could be a supportive remedy for neurodegenerative diseases like Parkinson's disease.

Neuroprotective Effect of Dexmedetomidine Pretreatment on Sevoflurane- Initiated Neurotoxicity Via the Mir-204-5p/SOX4 Axis.

BACKGROUND: Sevoflurane (Sev) is a type of volatile anesthetic commonly used in clinic practices and can initiate long-term neurotoxicity, while dexmedetomidine (Dex) possesses a neuroprotective function in multiple neurological disorders. OBJECTIVE: This work expounded on the function of Dex pretreatment in Sev-initiated neurotoxicity. METHODS: At first, human neuroblastoma cells (SK-N-SH cells) were treated with different concentrations of Sev or Dex, followed by the cell counting kit (CCK)-8 assay to decide the appropriate concentrations of Sev or Dex. Cell viability, lactate dehydrogenase (LDH) productions, and apoptotic rate of SK-N-SH cells were examined by the CCK-8 assay, LDH cytotoxicity kit, and flow cytometry assay in sequence. Further, reactive oxygen species (ROS) levels and proinflammatory cytokine contents were examined by the ROS assay kit and the enzyme-linked immunosorbent assay kits. The expression patterns of microRNA (miR)-204-5p and SRY-box transcription factor 4 (SOX4) in SK-N-SH cells were measured by real-time quantitative polymerase chain reaction or Western blotting. The binding relationship between miR-204-5p and SOX4 was confirmed by the dual-luciferase assay. After transfection of miR-204-5p mimics or SOX4 siRNA, the role of the miR-204-5p/SOX4 axis in Sev-initiated neurotoxicity was detected. RESULTS: Sev treatment reduced SK-N-SH cell viability in a concentration-dependent manner, and Dex pretreatment diminished Sev-initiated neurotoxicity. Mechanically, Dex pretreatment limited Sevinduced upregulation of miR-204-5p and further increased SOX4 expression levels. miR-204-5p upregulation or SOX4 knockdown averted the neuroprotection function of Dex pretreatment in Sevinitiated neurotoxicity. CONCLUSION: Dex pretreatment decreased miR-204-5p expression levels and upregulated SOX4 expression levels, palliating Sev-initiated neurotoxicity.

Biphasic Dose-Response of Mn-Induced Mitochondrial Damage, PINK1/Parkin Expression, and Mitophagy in SK-N-SH Cells.

Excessive manganese (Mn) exposure produces neurotoxicity with mitochondrial damage. Mitophagy is a protective mechanism to eliminate damaged mitochondria to protect cells. The aim of this study was to determine the dose-response of Mn-induced mitochondria damage, the expression of mitophagy-mediated protein PINK1/Parkin and mitophagy in dopamine-producing SK-N-SH cells. Cells were exposed to 0, 300, 900, and 1500 µM Mn2+ for 24 h, and ROS production, mitochondrial damage and mitophagy were examined. The levels of dopamine were detected by ELISA and neurotoxicity and mitophagy-related proteins (α-synuclein, PINK1, Parkin, Optineurin, and LC3II/I) were detected by western blot. Mn increased intracellular ROS and apoptosis and decreased mitochondrial membrane potential in a concentration-dependent manner. However, at the low dose of 300 µM Mn, autophagosome was increased 11-fold, but at the high dose of 1500 µM, autophagosome was attenuated to 4-fold, together with decreased mitophagy-mediated protein PINK1/Parkin and LC3II/I ratio and increased Optineurin expression, resulting in increased α-synuclein accumulation and decreased dopamine production. Thus, Mn-induced mitophagy exhibited a novel biphasic regulation: at the low dose, mitophagy is activated to eliminate damaged mitochondria, however, at the high dose, cells gradually loss the adaptive machinery, the PINK1/Parkin-mediated mitophagy weakened, resulting in neurotoxicity.

Investigating the adverse outcome pathways (AOP) of neurotoxicity induced by DBDPE with a combination of in vitro and in silico approaches.

Current studies have shown an association between DBDPE and neurotoxicity. In this study, the adverse outcome pathway (AOP) and mechanistic analysis of DBDPE-induced neurotoxicity were explored by a combination of in vitro and in silico approaches in SK-N-SH cells. DBDPE-induced oxidative stress caused DNA strand breaks, resulting in the activation of poly (ADP-ribose) (PAR) polymerase-1 (PARP-1). Activation of PARP1 could cause toxic damage in various organ systems, especially in the nervous system. DBDPE-induced apoptosis via the caspase-dependent intrinsic mitochondrial pathway and the PARP1-dependent pathway. Activation of PARP1 by DBDPE was deemed the initiating event, thereby affecting the key downstream biochemical events (e.g., ROS production, DNA damage, membrane potential changes, and ATP reduction), which induced apoptosis. Furthermore, excessive activation of PARP1 was accompanied by the translocation of the apoptosis-inducing factor (AIF), which was associated with PARP1-dependent cell death. The inhibition of PARP1 by PJ34 reduced DBDPE-induced apoptosis and maintained cellular ATP levels. PJ34 also prevented the translocation of AIF from the mitochondria to the nucleus. These findings improve the understanding of the mechanism of DBDPE-induced neurotoxic effects and provide a theoretical basis for the ecological risk of DBDPE.

DBDPE and ZnO NPs synergistically induce neurotoxicity of SK-N-SH cells and activate mitochondrial apoptosis signaling pathway and Nrf2-mediated antioxidant pathway.

Decabromodiphenyl ethane (DBDPE), a new brominated flame retardant, could negatively affect neurobehavior and pose health risks to humans. Humans are also exposed to widely used nanomaterials. This study investigated the combined toxic effects and action types of DBDPE and Zinc oxide nanoparticles (ZnO NPs) on human neuroblastoma SK-N-SH cells and the toxicity mechanisms. DBDPE inhibited the viability of SK-N-SH cells by 21.87% at 25 mg/L. ZnO NPs synergistically exacerbated the toxic effects of DBDPE. DBDPE and ZnO NPs caused excessive ROS production and inhibition of antioxidant enzyme (SOD and GSH) activity in cells, thus causing oxidative cellular damage. Moreover, DBDPE and ZnO NPs caused apoptosis by disrupting mitochondrial kinetic homeostasis, reducing mitochondrial membrane potential (MMP), increasing cytochrome C release and regulating Bax/Bcl-2 and Caspase-3 mRNA and protein expression. DBDPE and ZnO NPs increased the mRNA expression of nuclear factor erythroid 2- related factor (Nrf2) and its downstream genes. The molecular mechanisms revealed that oxidative stress, apoptosis and mitochondrial dysfunction were the critical factors in combined cytotoxicity. The bioinformatics analysis further indicated that co-exposure affected Nrf2 activation, apoptotic factors expression and mitochondrial fusion. The findings enrich the risk perception of neurotoxicity caused by DBDPE and ZnO NPs.

Transcriptional landscape of human neuroblastoma cells in response to SARS-CoV-2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and the neurological symptoms of SARS-CoV-2 infection have already been reported. However, the mechanisms underlying the effect of SARS-CoV-2 infection on patients with central nervous system injuries remain unclear. METHODS: The high-throughput RNA sequencing was applied to analyze the transcriptomic changes in SK-N-SH cells after SARS-CoV-2 infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to identify the functions of differentially expressed genes and related pathways. RESULTS: A total of 820 mRNAs were significantly altered, including 671 upregulated and 149 downregulated mRNAs (showing an increase of ≥ 2-fold or decrease to ≤ 0.5-fold, respectively; p ≤ 0.05). Moreover, we verified the significant induction of cytokines, chemokines, and their receptors, as well as the activation of NF-κB, p38, and Akt signaling pathways, in SK-N-SH by SARS-CoV-2. CONCLUSIONS: To our knowledge, this is the first time the transcriptional profiles of the host mRNAs involved in SARS-CoV-2 infection of SK-N-SH cells have been reported. These findings provide novel insight into the pathogenic mechanism of SARS-CoV-2 and might constitute a new approach for future prevention and treatment of SARS-CoV-2-induced central nervous system infection.

SNHG14 Upregulation Was a Molecular Mechanism Underlying MPP+ Neurotoxicity in Dopaminergic SK-N-SH Cells via SNHG14-miR-519a-3p-ATG10 ceRNA Pathway.

Long non-coding RNA small nuclear RNA host gene 14 (SNHG14) is a novel contributor of dopaminergic neuronal injury in Parkinson's disease. We further explored its role in 1-methyl-4-phenylpyridinium (MPP+)-damaged dopaminergic neurons (DAn) and the possible mechanism involving SNHG14, microRNA (miR)-519a-3p, and autophagy-related 10 (ATG10). MPP+ cytotoxicity was measured by MTS cell viability assay, flow cytometry, and a series of assay kits for detecting apoptosis and oxidative stress. Molecule expression was examined by qPCR and Western blotting, and RNA interaction was predicted by starBase2.0 of ENCORI platform and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. SNHG14 and ATG10 expression was increased, and miR-519a-3p was decreased in MPP+-treated SK-N-SH cells, and SNHG14 knockdown alleviated MPP+-induced SK-N-SH cell damage by regulating cell viability, cell cycle arrest, apoptosis, and oxidative stress. Additionally, antisense RNA of miR-519a-3p abated the suppressive role of SNHG14 knockdown, and ectopic expression of ATG10 counteracted the protective role of miR-519a-3p against MPP+ neurotoxicity. Mechanistically, SNHG14 and ATG10 were competitive endogenous RNAs (ceRNAs) for miR-519a-3p, and ATG10 expression could be positively modulated by SNHG14 via sponging miR-519a-3p. Target silencing SNHG14 and restoring miR-519a-3p could prevent DAn from MPP+ toxicity via regulation of ATG10.

BAX But Not BCL2 Is Necessary for Apoptosis in Neuroblastoma Cells Treated With Casiopeína® IIIia.

BACKGROUND/AIM: The emerging antineoplastics Casiopeínas® induce uncoupling of the respiratory chain, production of reactive oxygen species (ROS), entry of Bax into mitochondria, and exit of Ca2+ and Bcl-2 from them, leading to apoptosis. This study aimed to elucidate whether BAX and BCL2 are necessary for apoptosis. MATERIALS AND METHODS: We silenced BAX and BCL2 by CRISPR-Cas9, assessed ROS and calcium retention capacity (CRC) by spectrofluorometry, and caspase-3 with inmunoblotting in neuroblastoma (NB) cells and 3T3-L1 fibroblasts treated with cisplatin and Casiopeína IIIia (CasIIIia). RESULTS: We observed an increase in O2•- production only in BCL2KO NB cells treated with cisplatin (three-fold) and CasIIIia (five-fold), whereas the production of H2O2 in BCL2KO NB cells treated with cisplatin and CasIIIia increased five-fold and three-fold, respectively. The baseline calcium-retention capacity (CRC) was 1.7 relative fluorescence units (RFU) in both cell types. In BAXKO, cisplatin and CasIIIia increased CRC to ~2.3 RFU, and in BCL2KO, they decreased CRC to ~1.1 RFU. We did not detect caspase-3 in BAXKO NB cells. CONCLUSION: Only BAX is essential for CasIIIia-induced apoptosis.

[Direct Molecular Fishing of Zinc-Dependent Protein Partners of Amyloid-beta 1-16 with the Taiwan (D7H) Mutation and Phosphorylated Ser8 Residue].

We previously showed that the metal-binding domain 1-16 of intact amyloid-beta (Aß) is involved in interactions with a number of proteins from the cytosolic fraction of SK-N-SH human neuroblastoma cells in a zinc-dependent manner only. It is known that hereditary mutations in the Aß metal-binding domain (Aß(1-16)), which accelerate the development of Alzheimer's disease and post-translational modifications of amino acid residues, can significantly affect the domain's structure in the presence of zinc ions. In this work, using the molecular fishing methodology for Aß(l-16) isoforms with the Taiwanese mutation (D7H) and a phosphorylated Ser8 residue, proteins from the cytosol of SK-N-SH cells were found that are able to form zinc-dependent non-covalent complexes with these domains. The partner proteins identified for these isoforms differed from those for intact Aß(1-16). In contrast, the Aß(1-16) isoform with the English mutation (H6R) and the Aß(1-16) isoform containing both an isomerized Asp7 residue and phosphorylated Ser8 residue did not interact with cytosolic proteins. The results are useful for developing methods for rational modulation of protein-protein interactions involving natural isoforms of beta-amyloid, and also indicate the possible role of beta-amyloid with phosphorylated Ser8 as a molecule involved in normal physiological processes.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro.

BACKGROUND: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. METHODS: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. RESULTS: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). CONCLUSION: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

ABSTRACT Background: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p 0.001) or indirectly (p 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)

Cytokine-induced killer cells/natural killer cells combined with anti-GD2 monoclonal antibody increase cell death rate in neuroblastoma SK-N-SH cells.

Neuroblastoma (NB) is one of the most common extracranial, solid, pediatric malignancies. Despite improvements in conventional therapies, including surgery, chemotherapy and radiation therapy, the prognosis of stage IV NB remains poor, indicating that novel treatment strategies are required. Immunotherapies, such as anti-GD2 monoclonal antibodies, used alone or in combination with cytokines, and peripheral blood mononuclear cells or cord blood mononuclear cells (CBMNCs), have been indicated to cause NB cell death and to prolong patient survival in high-risk NB; however, they remain limited by severe cytotoxicity and side effects. In the present study, it was determined that anti-GD2 monoclonal antibody alone or CBMNC-isolated cytokine-induced killer (CIK)/natural killer (NK) cells alone significantly induced cell death of NB SK-N-SH cells, and the combination of anti-GD2 antibody and CIK/NK cells could significantly increase the cell death rate compared with either treatment alone. In addition, based on a method referred to our previous study, it was identified that a two-cytokine culture system, using interleukin IL-2 and IL-7, effectively stimulated the proliferation of CIK/NK cells. These results serve to suggest a novel treatment strategy for relapsed/refractory NB with high efficiency and few side effects.

A metabolomics study on effects of polyaromatic compounds in oil sand extracts on the respiratory, hepatic and nervous systems using three human cell lines.

Polyaromatic compounds (PACs) are by-products of combustion and are the major pollutants from the oil and gas industry. However, the mechanism of PACs induced toxicity still remains elusive. The aim of this study was to elucidate the effects of a typical mixture of PACs found in oil sand extract (OSE) on the respiratory, hepatic and nervous systems in humans using in vitro cell culture models followed by non-targeted metabolomics analysis. OSE collected from Alberta, Canada was fractionated into PAC and alkane fractions, and their effects after 24 h exposure on the cell viability measured by MTT assay in three human cell lines (A549, HepG2, and SK-N-SH) were studied. The PAC fractions showed significant dose-dependent cytotoxicity. A549 cells showed the highest sensitivity to OSE extracts, followed by SK-N-SH and HepG2. In contrast, the alkane fractions showed no effects on cell viability. The three human cell lines were further exposed with the PACs at 10% and 20% lethal concentration for 24 h. Metabolomics analysis of the cell extracts indicated that PACs treatments showed different disruptions on possible metabolic pathways on the three cell lines. PACs altered the sex steroid hormone metabolism and regulated the levels of leukotrienes metabolites in all three cell types. The amino acids L-cysteine, L-glutamine, L-tyrosine that are known to cause respiratory effects were significantly up-regulated in A549 cells. The PACs treated HepG2 cells showed down-regulation in metabolites responsible for the inflammatory mediation. Treatment of the differentiated SK-N-SH cells showed up-regulated metabolites involved with butanoate, fatty acid, and pyrimidine metabolism. Leukotriene metabolites were found to be significantly increased in all PACs treated cells. In conclusion, our results showed that PACs in OSE can alter the metabolism of the human lung, liver and neuronal cells and may induce toxicity in multiple target organs.

[The role of lysosomes in manganese-induced toxicity in SK-N-SH cells].

Objective: To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells. Methods: SK-N-SH cells were treated with MnCl(2) at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl(2) at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit. Results: Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl(2) treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl(2) treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl(2) treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl(2), while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn't observed significant difference of the activity of CTSD between different MnCl(2) treatment groups. Conclusion: MnCl(2) could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.

Status of topoisomerase-2ß protein in all-trans retinoic acid-treated human neuroblastoma (SK-N-SH) cells.

Of the mammalian topoisomerase (Topo)-2 isozymes (α and ß), Topo-2ß protein has been reported to regulate neuronal development and differentiation. However, the status of Topo-2ß in all-trans retinoic acid (ATRA)-treated human neuroblastoma (SK-N-SH) cells is not understood. More information about the effects of ATRA on SK-N-SH cells is needed to reveal the role of ATRA in the regulation of Topo-2ß levels and spontaneous regression of SK-N-SH cells to predict the clinical activity. This study was proposed to investigate the status and role of Topo-2ß protein in ATRA-induced survival and neuronal differentiation of SK-N-SH cells. Microscopic, sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitations and Western blot analysis were used to study and compare Topo-2ß protein among 10 µM ATRA-treated SK-N-SH cells and controls at different time points. The level of Topo-2ß protein increased in the initial days of treatment but markedly decreased upon induction of differentiation by ATRA in later stages. Upon ATRA treatment, SK-N-SH cells stretched, exhibited neurite extensions, and acquired a neuronal phenotype. Both treated and untreated SK-N-SH cells were able to migrate, occupy the scratched area, and completely recolonized 24 hours later. These results suggest an indirect role of Topo-2ß protein in regulation of genes involved in cell migration and differentiation of ATRA-treated SK-N-SH cells. This study suggests that Topo-2ß may be part of activation/repression of protein complexes activated by epigenetic modifying agents, differentiating signals, and inducible locus. However, detailed studies are needed to explore the ATRA-downstream genes leading to Topo-2ß regulation and regulatory proteins of neuronal differentiation.

The role of lysosomes in manganese-induced toxicity in SK-N-SH cells / 中华劳动卫生职业病杂志

Objective@#To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells.@*Methods@#SK-N-SH cells were treated with MnCl2 at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl2 at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit.@*Results@#Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl2 treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl2 treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl2 treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl2, while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn’t observed significant difference of the activity of CTSD between different MnCl2 treatment groups.@*Conclusion@#MnCl2 could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.