Involvement of mitogen-activated protein kinase in peroxynitrite-induced cell death of human neuroblastoma SH-SY5Y cells

We have investigated the activation of mitogen-activated protein kinase (MAP kinase) in relation to cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Exposure of the cells to peroxynitrite caused transient increase in MAP kinase activity, and resulted in cell death. PD98059, a selective inhibitor of MAP kinase kinase, reduced peroxynitrite-induced cell death. These results suggest that the activation of MAP kinase may be involved in cell death induced by peroxynitrite.     

Hypoxia and reoxygenation increased BACE1 mRNA and protein levels in human neuroblastoma SH-SY5Y cells

Ischemic cerebrovascular diseases, usually involved in hypoxia and reoxygenation, have been reported to increase the risk of dementia such as Alzheimer's disease (AD). β-site amyloid protein precursor (APP)-cleaving enzymes (BACE1) have been identified to participate in the secretion of β-amyloid peptides (Aβ), and its expressive alteration would contribute to the AD neuropathology. We have investigated the effect of hypoxia (0% O₂, 24 h) and reoxygenation (0 h, 12 h and 24 h after 24 h hypoxia) on BACE1 mRNA and protein levels in human neuroblastoma SH-SY5Y cells. At the same time, we also examined the effect of hypoxia and reoxygenation on APP mRNA and protein levels. We demonstrated that hypoxia and reoxygenation did not alter APP mRNA and protein level, However compared to those of controls, BACE1 mRNA levels were up-regulated by 31.5% (P = 0.028) and 35.1% (P = 0.005) at 12 h and 24 h and the protein levels increased to 22%(P = 0.021), 42% (P = 0.000) and 51.5% (P = 0.000) at 0 h, 12 h and 24 h after reoxygenation, respectively. Thus by up-regulating of BACE1 mRNA and protein level in the neuronal cell, hypoxia may be a linkage in the pathophysiology between cerebravascular diseases and AD.     

Nicotinic acetylcholine receptors in human neuroblastoma (SH-SY5Y) cells.

Whole-cell patch-clamp recordings were used to investigate nicotinic acetylcholine receptors (nAChRs) in the human neuroblastoma cell line, SH-SY5Y. Acetylcholine, nicotine and the neuronal nAChR agonist dimethylphenylpiperazinium iodide (DMPP), but not muscarine, all evoked inward currents in the cells (voltage-clamped at -60 mV). DMPP's actions were concentration- and voltage-dependent, and were antagonised by the neuronal nAChR antagonist mecamylamine (1-3 microM). Atropine was ineffective at 0.1 microM, but at 1 microM caused significant reductions in current amplitudes. Pre-incubation of cells with 2 microM alpha-cobratoxin had no effect on the actions of DMPP, and inward currents could also be induced when extracellular NaCl was replaced with CaCl2. DMPP also reversibly depolarized SH-SY5Y cells. These findings clearly identify nAChRs in SH-SY5Y cells, and provide two possible mechanisms by which receptor activation may lead to noradrenaline release, namely by triggering Ca2+ influx through the nAChR itself or by opening voltage-gated Ca2+ channels.     

Calpastatin reduces calpain and caspase activation in methamphetamine-induced toxicity in human neuroblastoma SH-SY5Y cultured cells

Mutations in presenilins are the major cause of early onset familial Alzheimer disease. It has recently been argued that clinical presenilin mutations work as loss-of-function but not toxic gain-of-function. To investigate whether presenilins are involved in the regulation of the distribution of neuronal membrane lipids, we treated neuronally differentiated PC12 cells with DAPT, an inhibitor of presenilin-dependent γ-secretase, and performed lipid analyses of neuritic terminals, which is an initial site of Aβ deposition in brains, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). With DAPT treatment, levels of sphingomyelin, phosphatidylcholine, and cholesterol remained unchanged. However, DAPT treatment increased the ganglioside levels in PC12 neuritic terminals. Together with a previous finding that accumulation of gangliosides at neuritic terminals facilitates Aβ assembly and deposition, the present data suggest that the loss-of-function of presenilins, i.e., a decrease in γ-secretase activity, has an impact on neuronal membrane architecture in a way that eventually exacerbates Alzheimer pathology.     

Calpastatin reduces calpain and caspase activation in methamphetamine-induced toxicity in human neuroblastoma SH-SY5Y cultured cells

Methamphetamine (METH) is an abused psychostimulant drug that can cause neurotoxicity to dopaminergic cells. It has been demonstrated that METH can induce caspase- and calpain-dependent death cascades. The purpose of the present study was to investigate the functional role of calpastatin, a specific endogenous calpain inhibitor protein, on caspase and calpain activation in METH-induced degeneration in neuroblastoma SH-SY5Y cell cultures. In this study, we found that METH significantly decreased cell viability, tyrosine hydroxylase phosphorylation and calpastatin levels. Supplementation of cells with exogenous calpastatin was able to reverse the toxic effect of METH on reduction in cell viability and tyrosine hydroxylase phosphorylation. METH also significantly increased calpain levels, the formation of calpain-specific breakdown products and cleaved caspase-3 levels; once again, these effects were diminished by pretreating the cells with calpastatin. These data suggest the contribution of calpastatin as a potential regulatory factor for calpain- and caspase-dependent death processes.     

Protein kinase-A activity in PRKAR1A-mutant cells,and regulation of mitogen-activated protein kinases ERK1/2

Carney complex (CNC) is caused by PRKAR1A-inactivating mutations. PRKAR1A encodes the regulatory subunit type I-alpha (RIalpha) of the cAMP-dependent kinase (PKA) holoenzyme; how RIalpha insufficiency leads to tumorigenesis remains unclear. In many cells PKA inhibits the extracellular receptor kinase (ERK1/2) cascade of the mitogen-activated protein kinase (MAPK) pathway leading to inhibition of cell proliferation. We investigated whether the PKA-mediated inhibitory effect on ERK1/2 is affected in CNC cells that carry germline PRKAR1A mutations. PKA activity both at baseline and after stimulation with cAMP was augmented in cells carrying mutations. Quantitative message analysis showed that the main PKA subunits expressed were type I (RIalpha and RIbeta) but RIalpha was decreased in mutant cells. Immunoblot assays of ERK1/2 phosphorylation by the cell- and pathway-specific stimulant lysophosphatidic acid (LPA) showed activation of this pathway in a time- and concentration-dependent manner that was prevented by a specific inhibitor. There was a greater rate of growth in mutant cells; forskolin and isoproterenol inhibited LPA-induced ERK1/2 phosphorylation in normal but not in mutant cells. Forskolin inhibited LPA-induced cell proliferation and metabolism in normal cells, but stimulated these parameters in mutant cells. These data were also replicated in a pituitary tumor cell line carrying the most common PRKAR1A mutation (c.578del TG), and an in vitro construct of mutant PRKAR1A that was recently shown to lead to augmented PKA-mediated phosphorylation. We conclude that PKA activity in CNC cells is increased and that its stimulation by forskolin or isoproterenol increases LPA-induced ERK1/2 phosphorylation, cell metabolism and proliferation. Reversal of PKA-mediated inhibition of this MAPK pathway in CNC cells may contribute to tumorigenesis in this condition.     

PPARγ stimulation promotes neurite outgrowth in SH-SY5Y human neuroblastoma cells

Several evidences indicate that PPARγ stimulation promotes neuronal differentiation. However, to date, no data describe the effects of PPARγ agonists on neurite outgrowth. Here we have evaluated the effects of pioglitazone, a synthetic PPARγ agonist, on differentiation and neurite outgrowth in SH-SY5Y human neuroblastoma cells. Our results show that pioglitazone promotes cell differentiation and the outgrowth of cell processes in a concentration-dependent manner with the maximal effect at 100 nM–1 μM. It significantly increases both the mean process length and the percentage of neurite-bearing cells. In addition, these effects are accompanied by significant activation of p42 and p44 mitogen-activated protein kinases. In conclusion, albeit preliminary, these findings suggest the possibility that PPARγ stimulation may contribute to the development and maintenance of a proper neuronal connectivity within neuronal networks.     

Modulation of tyrosine hydroxylase expression by melatonin in human SH-SY5Y neuroblastoma cells

We have previously reported in vivo preservation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment with physiological doses of melatonin, in a 6-hydroxydopamine model of Parkinson's disease. Based on these findings, we postulated that melatonin would similarly modulate the expression of TH in vitro. Therefore, using human SH-SY5Y neuroblastoma cells which can differentiate into dopaminergic neurons following treatment with retinoic acid, we first examined whether these cells express melatonin receptors. Subsequently, the physiological dose-dependent effects of melatonin on TH expression were examined in both undifferentiated and differentiated cells. The novel detection of the G protein-coupled melatonin MT(1) receptor in SH-SY5Y cells by RT-PCR was confirmed by sequencing and Western blotting. In addition, following treatment of SH-SY5Y cells with melatonin (0.1-100 nM) for 24h, Western analysis revealed a significant increase in TH protein levels. A biphasic response, with significant increases in TH protein at 0.5 and 1 nM melatonin and a reversal at higher doses was seen in undifferentiated cells; whereas in differentiated cells, melatonin was effective at doses of 1 and 100 nM. These findings suggest a physiological role for melatonin in modulating TH expression, possibly via the MT(1) receptor.     

Possible involvement of Ca2+ signaling in rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells

Rotenone, an inhibitor of mitochondrial respiratory chain complex I, is a useful tool to elicit animal model of Parkinson's disease. Rotenone-induced neuronal apoptosis may contribute to the etiology of Parkinson's disease. However, the mechanism of rotenone-induced apoptosis is not fully understood. In the present study, we show that Ca2+ signaling is essential for rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells. By using Fluo-3/AM and Fura-2/AM, the fluorescent calcium indicator, rotenone was found to cause a rise in intracellular free Ca2+ ([Ca2+]i). The intracellular Ca2+ chelator BAPTA attenuated rotenone-induced apoptosis. Notably, Ca2+ suppression also prevented rotenone-induced apoptotic related events including reactive oxygen species production, G2/M cell cycle arrest and caspase activation, suggesting that Ca2+ signaling is upstream to these events. In the absence of extracellular Ca2+, the rotenone-induced [Ca2+]i elevation was inhibited. Further, the voltage-dependent Ca2+ channel blocker nifedipine suppressed most of the elevation of [Ca2+]i induced by rotenone. These results demonstrate that rotenone leads to an elevation in [Ca2+]i through Ca2+ influx by the opening of voltage-gated Ca2+ channel. This study of rotenone may help to elucidate the neurodegenerative mechanims in Parkinson's disease.     

下调Notch1信号抑制人神经母细胞瘤SH-SY5Y的增殖能力

 目的 探讨Notch1基因与人神经母细胞瘤SH-SY5Y细胞增殖能力的关系。方法 应用?-分泌酶抑制剂DAPT或表达Notch1-shRNA慢病毒载体阻断SH-SY5Y细胞Notch1信号的活化,RT-PCR和Western blot法行Notch1及Hes1表达水平的检测,MTT法和裸鼠体内种植检测瘤细胞体内外增殖能力的变化。结果 DAPT处理和表达Notch1-shRNA慢病毒载体转染均能减少细胞内NICD和Hes1表达,其细胞体外增殖能力受到明显抑制(P<0.01),Notch1RNAi细胞体内种植瘤重为0.20±0.13g,明显轻于对照组的0.89±0.58g（P<0.01)。结论 Notch1信号的活化与人神经母细胞瘤SH-SY5Y细胞的增殖能力密切相关,有望成为神经母细胞瘤基因治疗的潜在靶点。     

白藜芦醇对神经母细胞瘤SH-SY5Y增殖的影响

目的:探讨白藜芦醇(Resveratrol,Res)对人神经母细胞瘤SH-SY5Y增殖变化规律和生长状况的影响。方法:通过不同浓度(0.15～500)μmol/LRes作用于SH-SY5Y,检测药物作用5d后各组细胞增殖率和细胞生长状态,同时通过增殖率曲线获得其半数生长抑制浓度(IC50)和杀伤浓度,并对各组细胞进行Hochest染色,检测其细胞凋亡情况。结果:Res5μmol/L组细胞增殖率为(117.00±15.30)%显著高于空白对照组(P0.05),15μmol/L组细胞增殖率为(31.33±2.89)%,50、150、500μmol/L组,细胞出现负增长。Res对SH-SY5Y的IC50为12.78μmol/L,细胞杀伤浓度(即增殖率为0%)为19.88μmol/L。Res50～500μmol/L各组内,部分细胞皱缩,突起减少或消失,并出现凋亡细胞。结论:Res在0.15～500μmol/L浓度范围内对SH-SY5Y的增殖影响存在浓度依赖性,并依次表现为促进增殖、抑制增殖和杀伤细胞三个方面效应。其中杀伤细胞效应与促进SH-SY5Y的凋亡启动相关。     

Okadaic acid protects human neuroblastoma SH-SY5Y cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis

1-Methyl-4-phenylpyridinium ion (MPP⁺) has been shown to selectively inhibit mitochondrial function and induce a parkinsonism-like syndrome. MPP⁺ stimulates the production of reactive oxygen species (ROS) and induces cell death in vitro. In this study, we investigated the protective effects of okadaic acid on MPP⁺-induced cell death in SH-SY5Y neuroblastoma cells. We found that MPP⁺-induced apoptosis and -ROS generation were blocked by okadaic acid. MPP⁺-mediated activation of AKT was also inhibited by okadaic acid. Taken together, these results demonstrate that okadaic acid protects against MPP⁺-induced apoptosis by blocking ROS stimulation and ROS-mediated signaling pathways in SH-SY5Y cells. These data indicated that okadaic acid could provide a therapeutic strategy for the treatment of neurodegenerative diseases including Parkinson's disease.     

Tissue transglutaminase is essential for neurite outgrowth in human neuroblastoma SH-SY5Y cells

Tissue transglutaminase is a normal constituent of the central and peripheral nervous systems and in rats transglutaminase activity in brain and spinal cord is highest during fetal stages when axonal outgrowth is occurring. Further, treatment of human neuroblastoma SH-SY5Y cells with retinoic acid results in the cells withdrawing from the cell cycle and extending neurites, in the same time frame that tissue transglutaminase expression significantly increases. Considering these and other previous findings, this study was carried out to determine whether tissue transglutaminase is involved in neuronal differentiation of SH-SY5Y cells. For these studies SH-SY5Y cells stably overexpressing wild-type tissue transglutaminase, an inactive tissue transglutaminase mutant (C277S) or an antisense tissue transglutaminase construct (which decreased endogenous tissue transglutaminase below detectable levels) were used. SH-SY5Y cells overexpressing wild-type tissue transglutaminase spontaneously differentiated into a neuronal phenotype when grown in low-serum media. In contrast, cells overexpressing inactive tissue transglutaminase or the antisense tissue transglutaminase continued to proliferate and exhibit a flat polygenic morphology even when maintained in low-serum conditions. In addition, increased tissue transglutaminase expression in response to retinoic acid was abolished in the antisense tissue transglutaminase cells, and antisense and mutant tissue transglutaminase expressing cells did not extend neurites in response to retinoic acid. Moreover, wild-type and inactive tissue transglutaminase exhibited differential intracellular localization.These data indicate that tissue transglutaminase is necessary and sufficient for neuronal differentiation of human neuroblastoma SH-SY5Y cells.     

Different mechanisms of lysophosphatidylcholine-induced Ca mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells

In mice, lysophosphatidylcholine (LPC) was found to be a physiological substrate of neuropathy target esterase, which is also bound by organophosphates that cause a delayed neuropathy in human and some animals. However, the mechanism responsible for causing the different symptoms in mice and humans that are exposed to neuropathic organophosphates still remains unknown. In the present study, we examined and compared the effect of exogenous LPC on intracellular Ca²⁺ overload in mouse N2a and human SH-SY5Y neuroblastoma cells. LPC caused an intracellular Ca²⁺ level ([Ca²⁺]_i) increase in both N2a and SH-SY5Y cells; moreover, the amplitude was higher in N2a cells than that in SH-SY5Y cells. Preincubation of the cells with verapamil, an L-type Ca²⁺ channel blocker, did not affect the LPC-induced Ca²⁺ increase in N2a cells, verapamil inhibited the response by 23% in SH-SY5Y cells. In Ca²⁺-free medium, LPC produced a significant [Ca²⁺]_i decrease in N2a cells, while it caused 64% of total [Ca²⁺]_i increase in SH-SY5Y cells. The results of a cell viability test suggest that N2a cells were more sensitive to LPC than were SH-SY5Y cells. These data suggested that the LPC-induced [Ca²⁺]_i increase was produced in each cell line through different mechanisms. In particular, the [Ca²⁺]_i increase occurred via entry through a permeabilized membrane in N2a cells, but through L-type Ca²⁺ channels as well as by Ca²⁺ release from intracellular Ca²⁺ stores in SH-SY5Y cells. Thus, the symptomatic differences of organophosphate-induced neurotoxicity between mice and humans are probably not related to the diverse amplitudes of intracellular Ca²⁺ overload produced by LPC. Moreover, the demyelination effect induced by LPC in mice may be a consequence of its detergent effect on membranes.     

Enhancement of Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by phorbol esters with and without activation of protein kinase C

The effects of phorbol esters on Ca²⁺ channel currents in human neuroblastoma SH-SY5Y cells were studied using whole-cell patch-clamp recordings. Bath application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu; 100 nM to 1 M), known activators of protein kinase C (PKC), enhanced Ca²⁺ channel currents in a voltage-dependent manner similar to that of Bay K 8644. TPA also enhanced Ca²⁺ channel currents during cell dialysis with the PKC pseudosubstrate, PKC(19–36), and in cells which had been pre-incubated with 500 nM staurosporine, and which were exposed to staurosporine during recordings. Application of 4-phorbol12, 13-didecanoate (4-PDD; 100 nM), which does not activate PKC, caused current enhancement similar to the effects of TPA. However, intracellular dialysis of TPA was without effect on Ca²⁺ channel currents. Residual Ca²⁺ channel currents recorded after exposure to 1 M -conotoxin GVIA were still enhanced by TPA, but in the presence of either Bay K 8644 (5 M) or nifedipine (5 M), TPA was without effect. When cells were pre-incubated for 10 min at 37° C with 100 nM TPA, currents subsequently recorded in its absence were enhanced as compared to untreated cells; 5 M nifedipine still inhibited currents to the same degree. This enhancement was not mimicked by 4PDD, and was inhibited by staurosporine. Our results indicate that acute applications of phorbol esters (at concentrations commonly used to activate PKC) enhance L-type Ca²⁺ channel currents in SH-SY5Y cells via a PKC-independent mechanism which appears similar to that induced by Bay K 8644. By contrast, pre-incubation with TPA enhances both L- and N-type currents via activation of PKC.     

The biologic role of ganglioside in neuronal differentiation--effects of GM1 ganglioside on human neuroblastoma SH-SY5Y cells.

Human neuroblastoma SH-SY5Y cell is a cloned cell line which has many attractive features for the study of neuronal proliferation and neurite outgrowth, because it has receptors for insulin, IGF-I and PDGF. Gangliosides are sialic acid containing glycosphingolipids which form an integral part of the plasma membrane of many mammalian cells. They inhibit cell growth mediated by tyrosine kinase receptors and ligand-stimulated tyrosine kinase activity, and autophosphorylation of EGF(epidermal growth factor) and PDGF receptors. The experiment was designed to study the effects of GM1 ganglioside on growth of human neuroblastoma SH-SY5Y cells stimulated with trophic factor in vitro. The cells were plated in Eagle''s minimum essential medium without serum. The number and morphologic change of SH-SY5Y cells were evaluated in the serum free medium added GM1 ganglioside with insulin or PDGF. SH-SY5Y cells were maintained for six days in serum-free medium, and then cultured for over two weeks in serum-free medium containing either insulin or PDGF. The effect of insulin on cell proliferation developed earlier and was more potent than that of PDGF. These proliferative effects were inhibited by GM1 ganglioside, and the cells showed prominent neurites outgrowth. These findings suggest that GM1 ganglioside inhibits the cell proliferation mediated by tyrosine kinase receptors and directly induces neuritogenesis as one of the neurotrophic factors.     

Resistance to geldanamycin-induced apoptosis in differentiated neuroblastoma SH-SY5Y cells

Geldanamycin (GA) is a specific inhibitor of the 90 kDs heat shock protein (Hsp90) in the cytoplasm of mammalian cells, which binds directly to Hsp90 and promotes proteolytic degradation of its client proteins. As an antitumor drug, GA antagonizes the protecting effects of Hsp90 on cell survival, while its mechanisms remain unclear. Here, we show that GA induces apoptosis in a human neuroblastoma cell line, SH-SY5Y. Treatment of the cells with all trans retinoic acid (RA) generates a neuron-like, morphological change of differentiation, and results in the activation of ERK and Akt pathways, an inhibition of the nuclear translocation of p53 induced by GA, and induces higher resistance to the GA-induced apoptosis. These results provide the first evidence for the requirement of p53 nucleation in SH-SY5Y cells to counteract GA in neuron survival.     

Quantification of vesicles in differentiating human SH-SY5Y neuroblastoma cells by automated image analysis

A new automated image analysis method for quantification of fluorescent dots is presented. This method facilitates counting the number of fluorescent puncta in specific locations of individual cells and also enables estimation of the number of cells by detecting the labeled nuclei. The method is here used for counting the AM1-43 labeled fluorescent puncta in human SH-SY5Y neuroblastoma cells induced to differentiate with all-trans retinoic acid (RA), and further stimulated with high potassium (K+) containing solution. The automated quantification results correlate well with the results obtained manually through visual inspection. The manual method has the disadvantage of being slow, labor-intensive, and subjective, and the results may not be reproducible even in the intra-observer case. The automated method, however, has the advantage of allowing fast quantification with explicitly defined methods, with no user intervention. This ensures objectivity of the quantification. In addition to the number of fluorescent dots, further development of the method allows its use for quantification of several other parameters, such as intensity, size, and shape of the puncta, that are difficult to quantify manually.     

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.