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Fragile X syndrome (FXS) is a leading genetic cause of autism intellectual disorder and autism spectrum disorder (ASD), with either limited treatment options or incurable. Fragile X-related gene 1 (FXR1) is a homolog of the Fragile X mental retardation gene 1 (FMR1), the causative gene of FXS, and both are highly homologous and functionally identical. In FXS, both PI3K (AKT/mTOR signaling pathway) and ERK1/2 (MAPK signaling pathway) expression levels were abnormal. Dual speci?city phosphatase 6 (DUSP6) is a member of the mitogen-activated protein kinases (MAPKs) that participates in the crosstalk between the two signaling systems of MEK/ERK and mTOR. By interacting with multiple nodes of MAPK and PI3K/AKT signaling pathways (including the mTOR complex), DUSP6 regulates cellular growth, proliferation, metabolism and participates in pathological processes of cancer and cognitive impairment. However, whether there is an interaction between FXR1P and DUSP6 and the effects of DUSP6 on the growth of SK-N-SH cells remains elusive. As demonstrated by our results, FXR1P was identified in the cytoplasm and nucleus of SK-N-SH cells co-localized with DUSP6, which might have regulated ERK1/2 signaling pathways in SK-N-SH cells. To a certain extent, FXR1P may reverse the negative regulation of ERK1/2 by DUSP6. Moreover, we discovered that not only does DUSP6 inhibit proliferation, but it also promotes the apoptosis of SK-N-SH cells.
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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)
Subject(s)
Animals , Peptides , Bothrops , Crotalid Venoms/isolation & purification , Lectins, C-Type/isolation & purification , Neuroblastoma , Neutrophils , In Vitro TechniquesABSTRACT
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.
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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.
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Objective To observe the effect of Sirt1 on the phosphorylation of Tau protein in neuroblastoma SK-N-SH cell line.Methods We cultured SK-N-SH cells in vitro with adenovirus packaging of Sirt1 and SirtM (Sirt mutant),and then observed the expression of Sirt1 under an inverted fluorescence microscope.The expressions of Sirt1and SirtM were detected by Western blot;t-Tau protein and phosphorylation of Tau protein were detected by Western blot,Real-time PCR,immunohistochemistry and immunofluorescence;and the effect of Sirt1 on SK-N-SH apoptosis was investigated by flow cytometry.Results The t-Tau protein level and its phosphorylation were significantly decreased in Sirt1 and SirtM groups compared with those in control group,and Sirt1 group showed more significantly decreased ser404,thr231 phosphorylation of tau protein and the mRNA level of Tau.Flow cytometry showed that Sirt1 could significantly reduce the apoptosis of SK-N-SH cells compared with the control group.Conclusion Sirt1 can decrease the phosphorylation of Tau protein and reduce the apoptosis of SK-N-SH,which provides an important laboratory basis for studies on Tau protein disease and other neurodegenerative diseases.
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Objective@#To investigate the effect of manganese chloride (MnCl2) or 1-methyl-4-phenylpyridinium (MPP +) on oxidative stress and autophagy in human neuroblastomaSK-N-SH cells and the mechanism of the neurotoxicity of manganese.@*Methods@#SK-N-SH cells were treated with MnCl2 or MPP+ at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, and 2.0 mmol/L for 24 hours, and MTT assay was used to measure cell viability. The cells weretreated with MnCl2 or MPP+ at doses of 0.125, 0.25, and 0.5 mmol/L for 24 hours, and flow cytometry was used to measure the content of reactive oxygen species (ROS) in cells, a laser scanning confocal microscope was used to observe autophagosome in cells, and Western blot was used to measure the expression of autophagy-related proteins P62 and LC3-II/LC3-I.@*Results@#Compared with the control group, the 0.0625-2.0 mmol/L MnCl2 and 0.125-2.0 mmol/L MPP + treatment groups had significant reductions in the viability of SK-N-SH cells, and the 0.25-2.0 mmol/L MnCl2 treatment groups had significantly lower viability than the groups treated with the same doses of MPP+ (all P<0.05) . Compared with the control group, the 0.125-0.25 mmol/L MnCl2 and 0.125-0.5 mmol/L MPP+ treatment groups had significant increases in the content of ROS, and the 0.25-0.5 mmol/L MPP+ treatment groups had significantly higher content of ROS than the groups treated with the same doses of MnCl2 (all P<0.05) . Compared with the control group, the 0.25-0.5 mmol/L MnCl2 andMPP+ treatment groups had significant increases in autophagy-related proteins LC3-II/LC3-I and significant reductions in P62 expression; the 0.125-0.5 mmol/L MPP+ treatment groups had significantly higher LC3-II/LC3-I than the groups treated with the same doses of MnCl2, and the 0.125 and 0.25 mmol/L MPP + treatment groups had significantly lower P62 expression than the groups treated with the same doses of MnCl2 (all P<0.05) .@*Conclusion@#Both MnCl2 and MPP+ can induce oxidative stress and autophagy in SK-N-SH cells, and MPP+ has a significantly greater inductive effect on autophagy of SK-N-SH cells than MnCl2.
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OBJECTIVE: To evaluate the cytotoxicity of cucurbitacin B solid lipid nanoparticles (SLN) on human neuroblastoma SK-N-SH cell line in vitro. METHODS: The SK-N-SH cell line was treated with cucurbitacin B SLN at various concentrations. Growth suppression was evaluated by MTT method; apoptosis related alterations in morphology were ascertained under light microscopy. Flow cytometry (FCM) was used to investigate the distribution of cell life. RESULTS: Free cucurbitacin B and cucurbitacin B SLN inhibited the growth of SK-N-SH cells after 48 h treatment in a dose dependent manner, with IC50 values of 0.508 and 12.6 μmol · L-1, respectively. The apoptotic rates at concentrations of 0.143 and 0.716 μmol · L-1 were (34.9 ± 4.6)% and(53.6 ± 6.3)%, respectively, all of which were significantly higher than that of free cucurbitacin B (13.2 ± 2.3)% and(43.4 ± 5.5)% (P < 0.05), respectively. Under microscopy, the cells treated with free cucurbitacin B and cucurbitacin B SLN exhibited characteristics of apoptosis including decreased cell density of groups, reduced cell volume, and changed form of majority cell. Flow cytometry analysis suggested that free cucurbitacin B retarded the progression of cell cycle at G2/M and S phase, and cucurbitacin B SLN retarded the progression of cell cycle at G2/M phase. CONCLUSION: Cucurbitacin B can not only inhibit the proliferation but also induce apoptosis of human neuroblastoma SK-N-SH cell line, demonsting strong cytotoxicity. Cytotoxicity of cucurbitacin B SLN is higher than that of free cucurbitacin B.
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Ceramide induces cell death in a dose- and time-dependent manner in neuroblastoma SK-N-SH cells. To investigate the mechanism of SK-N-SH cell death by C2-ceramide, morphological features and Hoechst 33258 staining were analyzed. In these morphlogic study the cell death by ceramide showed typical apoptotic features, nuclear condensation, fragmentation, and membrane blebbing. Ceramide-induced apoptosis was accompanied by nuclear accumulation of p53. Inhibition of p53 expression with p53 antisense oligonucleotides inhibited apoptosis evoked by ceramide. Also, ceramide induced mitochondrial event, collapse of mitochondrial membrane potential (delta psi m) and interestingly, inhibition of p53 attenuated collapse of mitochondrial membrane potential, suggests that ceramide induces mitochondrial dysfunction through upregulation of p53 expression. These results suggest that ceramide-induced apoptosis is dependent upon increase in cellular p53 levels which play a critical role in the regulation of apoptotic cell death and p53 modulates mitochondrial function such as mitochondrial membrane potential level.
Subject(s)
Apoptosis , Bisbenzimidazole , Blister , Cell Death , Membrane Potential, Mitochondrial , Membranes , Neuroblastoma , Neurons , Oligonucleotides, Antisense , Up-RegulationABSTRACT
Magnolol, isolated from the stem bark of Magnolia officnalis, is typical Oriental herbs. It has been known to have many biological activities such as anti-platelet aggregation, hydroxyl radical scavenging, Ca2+ -channel blocking, a tonic, anti-rheumatic, anti-stress, anti-inflammatory, anti-cancer action and ischemic heart disease. But, it is still unclear how they effectively regulate their various biological properties. Ceramide is emerging as a second messenger of apoptotic cell death and there is increasing evidence that ceramide is involved in neurodegenerative disease and the process of senescence. The present study investigated the effect of Magnolol on ceramide-induced apoptosis in human neuroblastoma SK-N-SH cells. We showed that ceramide induced apoptosis through the mediation of reactive oxygen species (ROS) production and Magnolol, as an effective antioxidant, significantly inhibited the increase of ROS generation, thereby preventing apoptosis. Furthermore, an increase of caspase activity (apoptosis executors) resulted from ceramide reduced by Magnolol. These results implicate that ROS play on important roles in ceramide-induced apoptosis, also Magnolol protects via effectively inhibition of ROS generation by ceramide through selective pathway.
Subject(s)
Humans , Aging , Apoptosis , Cell Death , Hydroxyl Radical , Magnolia , Myocardial Ischemia , Negotiating , Neuroblastoma , Neurodegenerative Diseases , Oxidative Stress , Reactive Oxygen Species , Second Messenger SystemsABSTRACT
Aim To explore the effects and mechanism of ginsenoside-Rg1 on SK-N-SH cells treated with chronic morphine and naloxone-precipitated withdrawal. Methods Cells were pretreated with ginsenoside-Rg1 1,2,4,8,16,32 ?mol?L-1 for 24 h,then incubated for 24 h with morphine ( 100 ?mol?L-1 ) . MTT colorimetr was used to study the effects of ginsenoside-Rg1 on the multiplication of the cells treated with chro-nic morphine. After stimulated by the same concentra-tion of morphine,cells were added with different concentrations of Rg1 1,2,4 ?mol?L -1 for 24 h before stimulated with 10 ?mol?L -1 NAL. Fuorospectrophotometry RT-PCR and Western blot techniques were used to detect the effects of ginsenoside-Rg1 on the [Ca2+ ]i,CaMKⅡ ? mRNA and protein expression of the SK-N-SH cells treated with chronic morphine and naloxone-precipitated withdrawal. Results ① Compared with control group,morphine significantly inhibited cell multiplication and resulted in calcium overload,and the expression of CaMKⅡ-? mRNA and protein noticeably increased ( P
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Both Acanthopanax senticosus and Acorus gramineus Soland are typical Oriental herbs. They have been used as a tonic, anti-rheumatic, anti-inflammatory, anti-stress, anti-cancer agent. But, it is still unclear how they effectively regulate their various biological properties. Ceramide is emerging as a second messenger of apoptotic cell death and there is increasing evidence that ceramide is involved in neurodegenerative disease and the process of senescence. The present study investigated the different effects of A. senticosus and A. gramineus on ceramide-induced apoptosis in human neuroblastoma SK-N-SH cells. We showed that ceramide induced apoptosis through the mediation of reactive oxygen species(ROS) production and A. senticosus, as an effective antioxidant, significantly inhibited the increase of ROS generation, thereby preventing apoptosis. Furthermore, an increase of caspase activity (apoptosis executors) resulted from ceramide reduced by A. senticosus. But A. gramineus had almost no protective effects. These results implicate that ROS play on important roles in ceramide-induced apoptosis, also A. senticosus protects effectively via inhibition of ROS generation by ceramide through selective pathway.
Subject(s)
Humans , NeuroblastomaABSTRACT
AIM: To investigate the inhibitory effect s of a synthetic CRE-transcription factor decoy oligodeoxynucleotide (CRE-decoy ODN) on the upregulation of the expression of cholecystokinin (CCK) and fosB mRN A induced by chronic morphine administration in SK-N-SH cells. METHODS: The CRE cis-element, TGACGTCA, was palindromic, a sy nthetic single-stranded phosphorothioate oligodeoxynucleotide composed of the CR E sequence self-hybridizes to form a duplex/hairpin. The CRE-palindromic decoy a nd control oligodeoxynucleotides were added to the medium (1 h before exposure t o morphine) at 150 nmol/L in the presence of cationic lipid DOTAP. After the cel ls were treated with 100 ?mol/L morphine for 48 h, 10 ?mol/L naloxone was use d for 15 min. The effects of CRE-decoy ODN on the DNA-binding activity of CREB, the expression of CCK and fosB mRNA were detected by electrophoresis mobi lity shift assay (EMSA) and RT-PCR, respectively. The stability of cell-incorpo rated [ 32P]-labeled CRE-decoy ODN was extracted with phenol:chloroform a nd then subjected to 20% nondenaturing polyacrylamide gel electrophoresis and au toradiography. RESULTS: Chronic morphine administration and acute naloxone-prec ipitated withdrawal significantly activated the DNA-binding activity of CREB and the expression of CCK and fosB mRNA in SK-N-SH cells. The CRE-decoy ODN pen etrated into the cells, specifically downregulated these indexes. CONCLUSIONS: CRE-decoy ODN can significantly downregulates the e xpre ssion of CCK and fosB mRNA through specifically suppressing the DNA-binding activity of CREB activated by chronic morphine administration in SK-N-SH cells.