Proliferation Of SH-SY5Y Cells Research Articles

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on micropillar substrates

In this study, poly-L-lactic acid micropillar substrates were fabricated to evaluate the influence of topographic substrates on cell morphological and functional characteristics, such as spreading area, voltage-gated calcium channels (VGCCs) and membrane potential. The proliferation, spreading area, perimeter and circularity of SH-SY5Y cells interfaced with different substrates were first investigated. In addition, the cytoskeleton and focal adhesion of a cell as important manifestations of cell morphology were analyzed by immunofluorescence. VGCC responsiveness was evaluated by measuring the dynamic changes in intracellular Ca2+ evoked by 50 mM extracellular K+. To determine study whether the differences in VGCC responsiveness were caused by the differences in VGCC gene expression, the expression of N/L- type VGCCs was determined by qPCR and fluorescence staining. Notably, improved measurement of the membrane potential with potentiometric fluorescent dye TMRM was applied to determine the membrane potential of SH-SY5Y cells. Results indicated that the SH-SY5Y cells were deformed significantly to adapt to the substrates; however, no distinct effect on the proliferative ability of SH-SY5Y cells was observed. The micropillar substrates markedly influenced VGCC responsiveness, which correlated strongly with cell spreading but not with VGCC expression. The resting membrane potential of SH-SY5Y cells cultured on different substrates also changed, but no effect on responsiveness of VGCC was observed. These results suggest that the effect of the micropillar substrates on cell VGCC responsiveness may be attributed to changes in the functionality of the ion channel itself. Thus, topographic substrates can be used to engineer cell functionality in cell-based drug screening.

Vitis labruscana leaf extract ameliorates scopolamine-induced impairments with activation of Akt, ERK and CREB in mice

BackgroundGrapes are among the most widely consumed plants and are used as a folk medicine. Vitis species have been traditionally used as anti-inflammatory, analgesic, and memory-enhancing agents, but, their biological activities of discarded grape leaves are not completely understood. PurposeWe investigated the effects of alcoholic aqueous leaf extract of Vitis labruscana (LEVL) in a mouse model of memory impairment and tried to ascertain its mechanism. We also evaluated its effects in SH-SY5Y cells. MethodsLEVL (50, 100, and 150 mg/kg) was administered to ICR mice once daily for 7 days. Memory impairment was induced with intraperitoneal scopolamine injections (1 mg/kg) and measured with the Y-maze test and a passive avoidance task. LEVL-induced signaling was evaluated in SH-SY5Y cells and mouse hippocampi. ResultsWe first identified quercetin-3-O-glucuronide as LEVL's major component. We then showed that LEVL promoted phosphorylation of Akt, extracellular regulated kinase (ERK), and cyclic AMP response element binding protein (CREB) and proliferation of SH-SY5Y cells. Oral LEVL administration (100 mg/kg) for 7 days significantly reversed scopolamine-induced reductions of spontaneous alternation in the Y-maze test and scopolamine-induced shortening of latency times in the passive avoidance task's retention trial. Consistent with the cell experiment results, LEVL restored scopolamine-decreased phosphorylation of Akt, ERK, and CREB and scopolamine-reduced expression of brain-derived neuroprotective factor expression in mouse hippocampi. ConclusionOur results suggest that LEVL promotes phosphorylation of Akt, ERK, and CREB in the hippocampus and ameliorates scopolamine-induced memory impairment in mice.

Silencing of Y-box binding protein-1 by RNA interference inhibits proliferation, invasion, and metastasis, and enhances sensitivity to cisplatin through NF-κB signaling pathway in human neuroblastoma SH-SY5Y cells.

Y-box binding protein-1 (YB-1), a member of Y-box protein family binding DNA and RNA, has been proposed as a novel marker in multiple malignant tumors and found to be associated with tumor malignancy. Neuroblastoma is an embryonal tumor arising from neuroblast cells of the autonomic nervous system, which is the most common cancer diagnosed in infants. It has been reported that YB-1 is highly expressing in various human tumors including nasopharynx, thyroid, lung, breast, colon, ovary, and prostate cancers. This study aimed to investigate the functional role of YB-1 in neuroblastoma by silencing YB-1 using RNA interference (shRNA) in neuroblastoma SH-SY5Y cells. We found that silencing of YB-1 decreased the proliferation, migration, and invasion of SH-SY5Y cells. At molecular level, inhibition of YB-1 decreased the expression level of PCNA as well as MMP-2 in neuroblastoma SH-SY5Y cells. Also, we discovered that YB-1 silencing sensitized SH-SY5Y cells to cisplatin and promoted the apoptosis induced by cisplatin due to down-regulation of multidrug resistance (MDR) 1 protein via NF-κB signaling pathway. Therefore, we consider that targeting YB-1 is promising for neuroblastoma treatment and for overcoming its cisplatin resistance in the development of new neuroblastoma therapeutic strategies.

Androgen Receptor Regulates the Growth of Neuroblastoma Cells in vitro and in vivo.

Background: Neuroblastoma is the most common extracranial tumors in children. At present about the true etiology of neuroblastoma is unclear and many studies have tried to find effective treatments for these primary malignant tumors. Although it has been illustrated that androgen receptor (AR) was expressed in neuroblastoma cells in some former reports, the biological role of androgen receptor in the development of neuroblastoma is not fully understood.Methods: Androgen (R1881) and the antagonists of androgen receptor (MDV3100 and ARN509) were used to study the role of the androgen receptor signaling pathway in vitro and in vivo on SH-SY5Y and Neuro-2a (N2a) cell lines.Results: We found that AR expression showed an R1881 dose-dependent manner in neuroblastoma cells in vitro and R1881was able to increase, while both antagonists of androgen receptor (MDV3100 and ARN509) significantly decrease, the proliferation, migration, invasion and sphere formation of SH-SY5Y and N2a cells. Moreover, androgen promoted the growth of N2a tumor in vivo. However, when androgen receptor (AR) was effectively knocked down in the two cell lines by siRNA, either promoting or inhibiting effect of the androgen or androgen receptor antagonists, respectively, was attenuated.Conclusion: Our results suggested that androgen receptor may involve in the progression of neuroblastoma as well as provided insight into a new target for the diagnosis and treatment of neuroblastoma patients.

Down-regulation of C-terminal binding protein 2 (CtBP2) inhibits proliferation, migration, and invasion of human SHSY5Y cells in vitro

Neuroblastoma is the most common extracranial solid tumor in children and is responsible for ∼15% of pediatric cancer deaths. CtBP2 is a member of the CtBP family of proteins that functions as a transcription regulator and has been demonstrated to interact with the C-terminus of the adenoviral E1A oncoprotein. In this study, the expression of CtBP2 in the human neuroblastoma cell line SHSY5Y was down-regulated using lentiviral-mediated RNA interference. Down-regulation of CtBP2 inhibited the expression of c-myc, MMP2, and MMP9 proteins. Moreover, low expression of CtBP2 resulted in inhibited cell growth, proliferation, migration, and invasion, and the cell cycle was arrested at G2/M-phase. These results indicate that CtBP2 may be a potential target to suppress tumorigenesis in neuroblastoma.

Low Dose of Bisphenol A Activates NF-κB/IL-6 Signals to Increase Malignancy of Neuroblastoma Cells

Bisphenol A (BPA) can accumulate in the human body and promote the progression of various cancers. However, its role in the development of neuroblastoma (NB) is largely unknown. Our present study revealed that nanomolar concentrations of BPA can significantly increase the proliferation, migration and invasion of NB SH-SY5Y and SiMa cells, further evidenced by the upregulation of human proliferating cell nuclear antigen, Bcl-2, vimentin and fibronectin. Real-time PCR and ELISA results suggested that nanomolar BPA can increase the expression of interleukin-6 (IL-6), but had no effect on the expression of IL-2, IL-8, IL-10 or IL-12. The neutralization antibody of IL-6 can abolish BPA-induced proliferation and invasion of NB cells. The inhibitor of NF-κB (BAY 11-7082), but not PD98059 (PD, ERK1/2 inhibitor) or LY294002 (LY, PI3K/Akt inhibitor), attenuated BPA-induced IL-6 expression and cell proliferation and invasion. In addition, BPA treatment also rapidly increased the phosphorylation of p65 since treatment for 5 min. Collectively, our data revealed that nanomolar BPA can trigger the malignancy of NB cells via activation of NF-κB/IL-6 signals, suggesting that more attention should be paid to the potential health risks of daily BPA intake.

273 - The Flavonoid Isoliquiritigenin Is Toxic to Neuroblastoma Cells and Promotes Necroptosis

Neuroblastoma is the most common childhood cancer diagnosed in the first year of life that shows high mortality. The flavonoid, isoliquiritigenin (4,2’,4’-trihydroxychalcone; ISL), exhibits significant anti-tumor activities, including inhibition of proliferation and cell cycle progression. In the present work we evaluated the capacity of ISL to inhibit cellular proliferation and migration, and examined the anti-tumor activity of ISL in the human neuroblastoma cell line SH-SY5Y. Neuroblastoma cells were exposed to 10-200 µM ISL or vehicle (control) for 24h. Incubation with ISL decreased the number of viable cells, cell confluence and intracellular ATP concentration. There were no significant differences in caspase 3/7 activation between control and ISL groups and ISL did not appear to induce release of extracellular TNF and IL-1β from SH-SY5Y cells indicating that ISL did not induce cell death via apoptosis. An inhibitor of necroptosis, necrostatin-1, induced significant increases in ATP concentration in ISL treated neuroblastoma cells indicating involvement of this programmed cell death pathway. ISL treatment inhibited migration and proliferation of SH-SY5Y cells and also affected the cell cycle by decreasing the proportion cells in G0/G1 phase and increasing cells in S and G2/M phases. These findings suggest that ISL can influence proliferation by arresting SH-SY5Y cell growth cycle. Using ELISA the ratio of phosphorylated/total p38 and ERK 1/2 in cells treated with ISL was higher than in the control cells. Western blot assay confirmed increased expression of pERK1/2 after ISL treatment. The ratio of phosphorylated/total Akt in neuroblastoma cells incubated with ISL also increased significantly. Together the results suggest that ISL can induce cell cycle arrest and neuroblastoma cell death by necroptosis.

Canine mesenchymal stem cells are neurotrophic and angiogenic: An in vitro assessment of their paracrine activity

Mesenchymal stem cells (MSCs) have been used in cell replacement therapies for connective tissue damage, but also can stimulate wound healing through paracrine activity. In order to further understand the potential use of MSCs to treat dogs with neurological disorders, this study examined the paracrine action of adipose-derived canine MSCs on neuronal and endothelial cell models. The culture-expanded MSCs exhibited a MSC phenotype according to plastic adherence, cell morphology, CD profiling and differentiation potential along mesenchymal lineages. Treating the SH-SY5Y neuronal cell line with serum-free MSC culture-conditioned medium (MSC CM) significantly increased SH-SY5Y cell proliferation (P < 0.01), neurite outgrowth (P = 0.0055) and immunopositivity for the neuronal marker βIII-tubulin (P = 0.0002). Treatment of the EA.hy926 endothelial cell line with MSC CM significantly increased the rate of wound closure in endothelial cell scratch wound assays (P = 0.0409), which was associated with significantly increased endothelial cell proliferation (P < 0.05) and migration (P = 0.0001). Furthermore, canine MSC CM induced endothelial tubule formation in EA.hy926 cells in a soluble basement membrane matrix. Hence, this study has demonstrated that adipose-derived canine MSC CM stimulated neuronal and endothelial cells probably through the paracrine activity of MSC-secreted factors. This supports the use of canine MSC transplants or their secreted products in the clinical treatment of dogs with neurological disorders and provides some insight into possible mechanisms of action.

White sesame seed water-soluble fraction enhances human neuroblast cell viability via an anti-apoptotic mechanism

Defatted sesame seed flour is recovered as a byproduct after oil extraction and is usually considered a waste product. Previously, we showed that water-soluble fractions purified from defatted white and gold sesame seed flour exhibited good antioxidant activity in vitro. We also identified ferulic acid and vanillic acid as the bioactive antioxidants in both white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively). In this study, we hypothesized that WS-wsf and GS-wsf may have neuroprotective effects due to their antioxidant potential. Treatment with WS-wsf for 24 hours enhanced human neuroblastoma SH-SY5Y cell viability and proliferation, while GS-wsf, ferulic acid, and vanillic acid did not show similar effects. In addition, WS-wsf (1-3 mg/mL) significantly and dose-dependently protected SH-SY5Y cells against camptothecin-induced apoptosis, suggesting the involvement of an anti-apoptosis mechanism in the neuroprotective effects of WS-wsf. In fact, treatment with WS-wsf significantly decreased the mRNA expression levels of pro-apoptotic Bax and p53 genes. WS-wsf also enhanced Bcl-2 protein level and Akt phosphorylation. Taken together, this study showed that WS-wsf has interesting neuroprotective potential via an anti-apoptotic mechanism, which is independent from its antioxidant capacity.

Smoothened-independent activation of hedgehog signaling by rearranged during transfection promotes neuroblastoma cell proliferation and tumor growth

BackgroundRearranged during transfection (RET) proto-oncogene encodes a receptor tyrosine kinase for glial cell line-derived neurotrophic factor (GDNF) signaling, and high RET expression is closely related to the tumorigenesis and malignancy of neuroblastoma(NB). MethodsWe have investigated whether RET signals through hedgehog (HH) pathway in NB cell proliferation and tumor growth by in vitro cell culture and in vivo xenograft approaches. ResultsThe key members of both GDNF/RET and HH/GLI pathways are expressed in NB cell lines to different extents. Knockdown of RET in NB cells significantly attenuates the activity of HH signaling, whereas overexpression of RET robustly enhances the output of transcriptional activation by HH. Likewise, activation of RET by GDNF induces HH signaling, whereas knockdown of RET attenuates both basal and GDNF-induced activities of HH signaling. Moreover, protein kinase B lies on the downstream of GDNF/RET signaling module to inhibit the GSK3β, resulting in activation of HH signaling. Furthermore, either knockdown of RET by shRNA or inhibition of HH pathway by cyclopamine attenuates not only basal but also GDNF-induced proliferation of SH-SY5Y cells, and knockdown of either RET or smoothened in SH-SY5Y cell xenografts significantly attenuated the tumor growth. Finally, inhibition of HH signaling by GLI1 and GLI2 inhibitor, Gant61, reduces not only basal but also RET-induced proliferation of SH-SY5Y cells and outgrowth of xenografts. ConclusionGDNF/RET/AKT/GSK3β signaling module activates HH pathway to stimulate NB cells proliferation and tumor outgrowth. General significanceTargeting HH pathway is a rational approach for therapeutic intervention of NB with high RET expression.

JWH-133, a Selective Cannabinoid CB₂ Receptor Agonist, Exerts Toxic Effects on Neuroblastoma SH-SY5Y Cells.

Endocannabinoid system plays an important role in the regulation of diverse physiological functions. Although cannabinoid type 2 receptors (CB2) are involved in the modulation of immune system in peripheral tissues, recent findings demonstrated that they are also expressed in the central nervous system and could constitute a new target for the treatment of neurodegenerative disorders. At present, very little is known about the potential effects of CB2-mimetic drugs on neuronal cells. This study aimed to examine whether JWH-133, a selective CB2 receptor agonist, affects the survival of SH-SY5Y neuroblastoma cell line, a widely used experimental in vitro model to study mechanisms of toxicity and protection in nigral dopaminergic neurons. Cell viability was assessed using two complementary methods: MTT test measuring mitochondrial activity and LDHe test indicating disruption of cell membrane integrity. In addition, cell proliferation was measured using BrdU incorporation assay. JWH-133 (10-40 μM) induced a concentration-dependent decrease of SH-SY5Y cell viability and proliferation rate. Using AM-630, a reverse agonist of CB2 receptors, as well as Z-VAD-FMK, a pan-caspase inhibitor, we demonstrated that the cytotoxic effect of JWH-133 presumably was not mediated by activation of CB2 receptors or by caspase pathway. Results of this work suggest that agonists of CB2 receptors when administered in multiple/high doses may induce neuronal damage.

Proteomic Profiling of Neuroblastoma Cells Adhesion on Hyaluronic Acid-Based Surface for Neural Tissue Engineering.

The microenvironment of neuron cells plays a crucial role in regulating neural development and regeneration. Hyaluronic acid (HA) biomaterial has been applied in a wide range of medical and biological fields and plays important roles in neural regeneration. PC12 cells have been reported to be capable of endogenous NGF synthesis and secretion. The purpose of this research was to assess the effect of HA biomaterial combining with PC12 cells conditioned media (PC12 CM) in neural regeneration. Using SH-SY5Y cells as an experimental model, we found that supporting with PC12 CM enhanced HA function in SH-SY5Y cell proliferation and adhesion. Through RP-nano-UPLC-ESI-MS/MS analyses, we identified increased expression of HSP60 and RanBP2 in SH-SY5Y cells grown on HA-modified surface with cotreatment of PC12 CM. Moreover, we also identified factors that were secreted from PC12 cells and may promote SH-SY5Y cell proliferation and adhesion. Here, we proposed a biomaterial surface enriched with neurotrophic factors for nerve regeneration application.

Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia pastoris

Soluble receptor for advanced glycation end products (sRAGE), a natural inhibitor of RAGE, is considered to be a putative therapeutic molecule for a variety of diseases and a biomarker for certain conditions. To further study the function of sRAGE, recombinant rat sRAGE (rrsRAGE) was expressed and produced in a eukaryotic system. The open reading frame of rat sRAGE was cloned downstream of the methanol-inducible alcohol oxidase promoter of pPICZαA vector, and Pichia pastoris strain X-33 was used as the host strain. The expression of rrsRAGE was achieved by fermentation in a 15-L bioreactor and the resulting fermentation broth was subjected to purification on a cation exchange chromatography column. The purification of rrsRAGE reached 95% after size exclusion chromatography(SEC). The bioactivity of the purified protein was confirmed in a SH-SY5Y cell proliferation assay. The biological function of the purified rrsRAGE protein rat CCl4-induced model was then examined. Treatment with rrsRAGE resulted in significantly lower liver fibrosis and lower serum level of ALT, suggesting that sRAGE prevent liver from injury and fibrosis. In conclusion, we achieved high-efficiency production of bioactive rrsRAGE in P. pastoris.

Amyloid Beta Peptide 1-42 Induces SH-SY5Y Cell Apoptosis via the Promotion of Meg3 Long Noncoding RNA Expression

Background/Aim: The mechanisms and processes of amyloid beta (Aβ)_1-42/Aβ_1-40 degeneration and deposition of neuron damage are still not clear. The long noncoding RNA (lncRNA) is one of the members of the noncoding RNA family. In this study, we aimed to investigate whether Aβ_1-42 inhibited SH-SY5Y cells in vitro through modulating Meg3 lncRNA. Methods: The Alzheimer's disease (AD) senile plaque cell model was generated using synthetic Aβ_1-42-treated SH-SY5Y cells. MTT assays were used to determine the proliferation of SH-SY5Y cells. Quantitative (q)RT-PCR and Western blot analyses were used to test the expression levels of mRNA and protein. Northern blot analysis was used to confirm Meg3 lncRNA expression. Results: The MTT assays showed that exogenous Aβ_1-42 suppressed SH-SY5Y cells. The qRT-PCR and Western blot analyses revealed that the expression of p53 mRNA and protein was significantly increased in the AD model group, with a marked decrease in MDM2 and Ki-67 expression on day 7. Moreover, the qRT-PCR and Northern blot analyses confirmed that exogenous Aβ_1-42 promoted the expression of Meg3 lncRNA. There was a downregulation of Meg3 lncRNA expression in SH-SY5Y cells by siRNA, which could promote of the ability of MDM2 to degrade p53 protein on the ubiquitin pathway and delay SH-SY5Y apoptosis. Conclusion: Meg3 lncRNA is implicated as an important factor in the formation of mature Aβ peptides.

HIF-1α contributes to proliferation and invasiveness of neuroblastoma cells via SHH signaling.

The aim of this study was to investigate the effects of hypoxia-inducible factor-1α (HIF-1α) on the proliferation, migration and invasion of neuroblastoma (NB) cells and the mechanisms involved. We here initially used the real-time polymerase chain reaction (real-time PCR), Western blotting and immunohistochemistry (IHC) to detect the expression of HIF-1α and components of the sonic hedgehog (SHH) signaling pathway in NB cells and human specimens. Subsequently, cell proliferation, migration and invasion were analyzed using the cell counting assay, wound healing assay and Transwell system in two types of human NB cell lines, SH-SY5Y and IMR32. In addition, the role of HIF-1α in NB cells growth was determined in a xenograft nude mouse model. We found that the level of HIF-1α was significantly upregulated during NB progression and was associated with the expression of two components of SHH signaling, SHH and GLI1. We next indicated that the proliferation, migration and invasiveness of SH-SY5Y and IMR32 cells were significantly inhibited by HIF-1α knockdown, which was mediated by small interfering RNAs (siRNAs) targeting against its mRNA. Furthermore, the growth of NB cells in vivo was also suppressed by HIF-1α inhibition. Finally, the pro-migration and proliferative effects of HIF-1α could be reversed by disrupting SHH signaling. In conclusion, our results demonstrated that upregulation of HIF-1α in NB promotes proliferation, migration and invasiveness via SHH signaling.

SLIT2/ROBO1-miR-218-1-RET/PLAG1: a new disease pathway involved in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. We investigated changes in expression of microRNAs (miRNAs) and the genes they regulate in tissues of patients with HSCR. Quantitative real-time PCR and immunoblot analyses were used to measure levels of miRNA, mRNAs, and proteins in colon tissues from 69 patients with HSCR and 49 individuals without HSCR (controls). Direct interactions between miRNAs and specific mRNAs were indentified in vitro, while the function role of miR-218-1 was investigated by using miR-218 transgenic mice. An increased level of miR-218-1 correlated with increased levels of SLIT2 and decreased levels of RET and PLAG1mRNA and protein. The reductions in RET and PLAG1 by miR-218-1 reduced proliferation and migration of SH-SY5Y cells. Overexpression of the secreted form of SLIT2 inhibited cell migration via binding to its receptor ROBO1. Bowel tissues from miR-218-1 transgenic mice had nerve fibre hyperplasia and reduced numbers of gangliocytes, compared with wild-type mice. Altered miR-218-1 regulation of SLIT2, RET and PLAG1 might be involved in the pathogenesis of HSCR.

Xingshentongqiao decoction mediates proliferation, apoptosis, orexin-A receptor and orexin-B receptor messenger ribonucleic acid expression and represses mitogen-activated protein kinase signaling.

Background:Hypocretin (HCRT) signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ) is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R]) expression. The signaling pathways involved in these processes were also assessed.Methods:The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK) pathway activation was performed to further assess the signaling mechanism of XSTQ.Results:XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk) 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK).Conclusions:XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.

Myristoylated Alanine-Rich C Kinase Substrate Accelerates TNF-&amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt;-Induced Apoptosis in SH-SY5Y Cells in a Caspases-6 and/or -7-Dependent Manner

Cell proliferation, differentiation, and the elimination of unnecessary cells by apoptosis occur in the development of the nervous system. It is reported that brain dysplasia appears as the results of myristoylated alanine-rich C kinase substrate (MARCKS) knockout or the mutant mouse. We therefore expect that MARCKS participates in the development of the nervous system. However, the mechanism underlying such participation has not been identified. In this study, we observed the effects of the overexpression of MARCKS or unphosphorylatable MARCKS on cell proliferation and TNF-α-induced apoptosis in neuroblastoma SH-SY5Y cells. Furthermore, we restrained MARCKS expression by the RNAi method. In the results, MARCKS-overexpressing cells and not unphosphorylatable MARCKS-overexpressing cells showed increased cell proliferation rates. On the other hand, the RNAi decreased the proliferation of MARCKS-knocked down SH-SY5Y cells. These results indicated that MARCKS-overexpressing cells were more sensitive to TNF-α than normal SH-SY5Y cells. Moreover, in MARCKS-overexpressing cells TNF-α-induced apoptosis was inhibited by caspase-6 and -7 inhibitors but not by caspase-3 inhibitor. These results suggested that MARCKS participated in TNF-α-induced apoptosis in a caspase-6 and/or -7-dependent manner.

Mitochondrial ferritin, a new target for inhibiting neuronal tumor cell proliferation.

Mitochondrial ferritin (FtMt) has a significant effect on the regulation of cytosolic and mitochondrial iron levels. However, because of the deficiency of iron regulatory elements (IRE) in FtMt’s gene sequence, the exact function of FtMt remains unclear. In the present study, we found that FtMt dramatically inhibited SH-SY5Y cell proliferation and tumor growth in nude mice. Interestingly, excess FtMt did not adversely affect the development of drosophila. Additionally, we found that the expression of FtMt in human normal brain tissue was significantly higher than that of neuroblastoma, but not higher than that of neurospongioma. However, the expression of transferrin receptor 1 is completely opposite. We therefore hypothesized that increased expression of FtMt may negatively affect the vitality of neuronal tumor cells. Therefore, we further investigated the underlying mechanisms of FtMt’s inhibitory effects on neuronal tumor cell proliferation. As expected, FtMt overexpression disturbed the iron homeostasis of tumor cells and significantly downregulated the expression of proliferating cell nuclear antigen. Moreover, FtMt affected cell cycle, causing G1/S arrest by modifying the expression of cyclinD1, cyclinE, Cdk2, Cdk4 and p21. Remarkably, FtMt strongly upregulated the expression of the tumor suppressors, p53 and N-myc downstream-regulated gene-1 (NDRG1), but dramatically decreased C-myc, N-myc and p-Rb levels. This study demonstrates for the first time a new role and mechanism for FtMt in the regulation of cell cycle. We thus propose FtMt as a new candidate target for inhibiting neuronal tumor cell proliferation. Appropriate regulation of FtMt expression may prevent tumor cell growth. Our study may provide a new strategy for neuronal cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-014-1730-0) contains supplementary material, which is available to authorized users.

Dominant negative effect of polyglutamine expansion perturbs normal function of ataxin-3 in neuronal cells

The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado-Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.