微波辐射诱导人神经母细胞瘤SH-SY5Y细胞凋亡的可能机制

目的:观察微波辐射对人神经母细胞瘤SH-SY5Y细胞凋亡的影响,并探讨其机制.方法:采用10、30和50 mW·cm-2辐射强度的微波辐射SH-SY5Y细胞5 min,以假辐射组(0 mW·cm-2)为对照,光镜下观察细胞形态变化;荧光显微镜下观察DAPI染色细胞核的形态;CTAB法提取细胞基因组DNA,电泳观察DNA ladder;台盼蓝拒染法检测细胞存活率;AnnexinV-FITC和PI双染色,流式细胞仪检测细胞凋亡率;MTT法检测细胞相对活性;蛋白质印迹检测细胞凋亡相关蛋白的表达.结果:微波辐射后SH-SY5Y细胞形态即刻改变,胞核、胞质结构欠清,细胞皱缩甚至脱壁;细胞核内的染色质出现不规则凝集,碎裂成2～5个微核;细胞DNA出现明显的梯状条带;细胞存活率随微波辐射强度增大逐渐降低,10 mW·cm-2辐射组与假辐射组比较,细胞存活率差异无统计学意义(P>0.05),但30、50 mW·cm-2辐射组细胞存活率明显低于假辐射组(P<0.05);微波辐射后6 h的细胞凋亡率随辐射强度的增加逐渐升高,各辐射组细胞凋亡率均明显高于假辐射组(P<0.05);微波辐射后24和48 h,细胞相对活力随辐射强度的增加明显降低,各辐射组的细胞相对活力均明显低于假辐射组(P<0.05).微波辐射后,细胞凋亡相关蛋白Bcl-2、survivin表达水平逐渐下降,Bax、caspase-3 和caspase-7表达水平逐渐升高,caspase-8和-9表达水平无明显变化.结论:微波辐射可诱导SH-SY5Y细胞凋亡,其作用机制可能与survivin、Bcl-2蛋白表达水平下调、Bax蛋白表达水平上调及caspase-3和caspase-7的作用有关,而与caspase-8和caspase-9介导的caspase-3活化这一作用无密切关系.     

Differential regulation of insulin receptor substrate-2 and mitogen-activated protein kinase tyrosine phosphorylation by phosphatidylinositol 3-kinase inhibitors in SH-SY5Y human neuroblastoma cells

Insulin-like growth factor I (IGF-I) is a potent neurotropic factor promoting the differentiation and survival of neuronal cells. SH-SY5Y human neuroblastoma cells are a well characterized in vitro model of nervous system growth. We report here that IGF-I stimulated the tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and insulin receptor substrate-2 (IRS-2) in a time- and concentration-dependent manner. These cells lacked IRS-1. After being tyrosine phosphorylated, IRS-2 associated transiently with downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-K) and Grb2. Treatment of the cells with PI 3-K inhibitors (wortmannin and LY294002) increased IGF-I-induced tyrosine phosphorylation of IRS-2. We also observed a concomitant increase in the mobility of IRS-2, suggesting that PI 3-K mediates or is required for IRS-2 serine/threonine phosphorylation, and that this phosphorylation inhibits IRS-2 tyrosine phosphorylation. Treatment with PI 3-K inhibitors induced an increased association of IRS-2 with Grb2, probably as a result of the increased IRS-2 tyrosine phosphorylation. However, even though the PI 3-K inhibitors enhanced the association of Grb2 with IRS-2, these compounds suppressed IGF-I-induced mitogen-activated protein kinase activation and neurite outgrowth. Together, these results indicate that although PI 3-K participates in a negative regulation of IRS-2 tyrosine phosphorylation, its activity is required for IGF-IR-mediated mitogen-activated protein kinase activation and neurite outgrowth.     

Cytotoxic effects of MPTP on SH-SY5Y human neuroblastoma cells

Morphological and metabolic endpoints were used to evaluate MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity to SH-SY5Y human neuroblastoma cells. After 8 hours of exposure, MPTP was found to affect cell viability only at a very high concentration (3 x 10(-3) M), but its metabolite MPP+ could decrease viability at 10(-4) M. MPTP, via its metabolite MPP+, inhibited NADH dehydrogenase activity when concentrations exceeded 10(-4) M (for MPP+ 10(-5)M). The Ki were 2.4 x 10(-3) M and 3 x 10(-4)M for MPTP and MPP+, respectively. MPTP at concentrations greater than 10(-4) M altered cell morphology as early as one hour after exposure. These changes included formation of cell surface blebs and attenuated neurites. After 8 hours at 10(-3) M and 24 hrs at 10(-4) M, MPTP caused ultrastructural changes of mitochondria with increased electron-density of the matrix and disorganization of cristae, as well as abnormal aggregation of filamentous material of the cytoskeleton. Because these changes of structure and function took place at concentrations lower than those needed to affect cell viability, they may play a role in MPTP neurotoxicity in SH-SY5Y cell culture.     

Characterization of phospholipase D activation by muscarinic receptors in human neuroblastoma SH-SY5Y cells

The cholinergic regulation of phospholipase D activity was studied in SH-SY5Y human neuroblastoma cells with phosphatidylethanol formation as a specific marker for the enzyme activity. The muscarinic antagonists, hexahydrosiladifenidol and pirenzepine, inhibited carbachol-induced phosphatidylethanol formation in a concentration-dependent manner and the inhibitory constants indicated that muscarinic M1 receptors are responsible for the major part of the phospholipase D activation. The mechanism of receptor-mediated phospholipase D activation varies between different cell types and receptors. In SH-SY5Y cells, the carbachol-induced phospholipase D activity was inhibited by protein kinase C inhibitors. Since both phospholipases D and C are activated by muscarinic stimulation in SH-SY5Y cells, most of the phospholipase D activation is probably secondary to the protein kinase C activation that follows phospholipase C-mediated increase in diacylglycerols. Other kinases may be involved in the regulation since also a tyrosine kinase inhibitor decreased the phosphatidylethanol formation. Stimulation of G-protein(s) and increase in the intracellular Ca2+ concentration activated phospholipase D and may be additional mechanisms for the muscarinic regulation of phospholipase D in SH-SY5Y cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, increased the carbachol-induced formation of phosphatidic acid at the expense of 1,2-diacylglycerol. This indicates that phospholipase D contributes to the formation of 1,2-diacylglycerol after carbachol stimulation in SH-SY5Y cells.     

(-)-Deprenyl protects human dopaminergic neuroblastoma SH-SY5Y cells from apoptosis induced by peroxynitrite and nitric oxide

In Parkinson's disease the cell death of dopamine neurons has been proposed to be mediated by an apoptotic death process, in which nitric oxide may be involved. This article reports the induction of apoptosis by nitric oxide and peroxynitrite in human dopaminergic neuroblastoma SH-SY5Y cells and the antiapoptotic activity of (-)-deprenyl. After the cells were treated with a nitric oxide donor, NOR-4, or a peroxynitrite donor, SIN-1, DNA damage was quantitatively studied using a single-cell gel electrophoresis (comet) assay. NOR-4 and SIN-1 induced DNA damage dose-dependently. Cycloheximide and alkaline treatment of the cells prevented the DNA damage, indicating that the damage is apoptotic and that it depends on the intracellular signal transduction. Superoxide dismutase and the antioxidants reduced glutathione and alpha-tocopherol protected the cells from the DNA damage. (-)-Deprenyl protected the cells from the DNA damage induced by nitric oxide or peroxynitrite almost completely. The protection by (-)-deprenyl was significant even after it was washed from the cells, indicating that (-)-deprenyl may activate the intracellular system against apoptosis. These results suggest that (-)-deprenyl or related compounds may be neuroprotective to dopamine neurons through its antiapoptotic activity.     

The neurotoxic effect of 24-hydroxycholesterol on SH-SY5Y human neuroblastoma cells

PURPOSE: The purpose of this study was to compare two commercially available accelerometers with indirect calorimetry in a group of older adults (x +/- SD; 70.6+/-3.7 yr; N = 86, 44 males and 42 females). METHODS: The accelerometers (Caltrac and Tritrac, Hemokinetics, Madison, WI) were worn while performing three submaximal, discontinuous (5 min exercise, 2 min recovery), progressive levels of treadmill walking and bench stepping. The treadmill exercise averaged 3.4 mph, at 0.4% grade, 3.0% grade, and 5.1% grade, while the stepping work rates (24 steps x min(-1)) were performed on 15.2-, 20.3-, and 25.4-cm steps. Estimated energy expenditure (EE) from the two accelerometers was compared with EE as measured by indirect calorimetry. RESULTS: The Caltrac significantly (P < 0.05) overestimated EE at the three treadmill work rates (10-52% difference) and underestimated EE at the three stepping work rates (-19% to -28% difference). When comparing the changes in EE between work rates one, two and three, the Caltrac was not sensitive to the changes (increase in EE) that occurred during graded treadmill walking but did detect some changes in the stepping exercise. The Tritrac significantly (P < 0.05) underestimated EE for the three work rates of both the treadmill and stepping exercise when compared with indirect calorimetry but did detect differences in EE among work rates during stepping exercise (P < 0.05). CONCLUSIONS: These data indicate that the magnitude of the differences between measured and estimated EE is affected by exercise mode and intensity and that caution is warranted when using the accelerometers in an attempt to quantify EE in older adults.     

Induction of apoptotic cell death in differentiating neuroblastoma SH-SY5Y cells by colchicine

In retinoic acid (RA)-differentiated SH-SY5Y cell cultures, colchicine could induce cell death, accompanied by the typical ladder pattern of DNA fragmentation found in apoptotic cells. This effect could be attenuated by the protein synthesis inhibitor cycloheximide. The protein kinase inhibitor H-89 efficiently reduced colchicine-induced cell death. These results suggest that the mechanism for colchicine-induced cell death may act, at least in part, through the activation of apoptosis in differentiating SH-SY5Y cells.     

Activation of protein kinase C-alpha and translocation of the myristoylated alanine-rich C-kinase substrate correlate with phorbol ester-enhanced noradrenaline release from SH-SY5Y human neuroblastoma cells

The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the alpha-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-epsilon from cytosolic and membrane-associated to cytoskeleton- and membrane-associated. TPA had no effect on the cytosolic location of PKC-zeta. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 microM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-alpha (but not -epsilon or -zeta). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-alpha.     

Calcium/calmodulin-dependent protein kinase II and arrestin phosphorylation in Limulus eyes

In rhabdomeral photoreceptors, light stimulates the phosphorylation of arrestin, a protein critical for quenching the photoresponse, by activating a calcium/calmodulin-dependent protein kinase (CaM PK). Here we present biochemical evidence that a CaM PK that phosphorylates arrestin in Limulus eyes is structurally similar to mammalian CaM PK II. In addition, cDNAs encoding proteins homologous to mammalian and Drosophila CaM PK II in the catalytic and regulatory domains were cloned and sequenced from a Limulus lateral eye cDNA library. The Limulus sequences are unique, however, in that they lack most of the association domain. The proteins encoded by these sequences may phosphorylate arrestin.     

Ethanol exposure potentiates fosB and junB expression induced by muscarinic receptor stimulation in neuroblastoma SH-SY5Y cells

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been shown to play a role in host defense and pathogenesis of chronic HCV infection. Our aim was to test the hypothesis that intrahepatic HCV-specific CTL activity may impact subsequent response to interferon alfa (IFN-alpha) therapy. Of the 37 patients that we have prospectively evaluated for HCV-specific CTL activity in liver, 21 received IFN therapy, and 19 completed a 6-month course and attended 6 to 18 months of follow-up. Intrahepatic CD8+ cells were isolated from liver biopsy tissue and tested against target cells expressing HCV antigens to determine intrahepatic CTL activity. The relationship between treatment response and HCV-specific CTL activity and other factors known to associate with response (genotype, viremia, histology) was analyzed. HCV-specific CTL activity was detected in 9 of 21 patients (and 9 of 19 who completed therapy). After 6 months of IFN therapy, 8 of 19 (42%) patients had normal serum alanine transaminase (ALT) (complete responders). After 18 months of follow-up, only 3 patients (16%) had a sustained biochemical response. Of the 9 patients with detectable HCV-specific CTL activity in their liver before treatment, 7 (78%) developed a complete response. In contrast, only 1 of the 10 patients with no detectable HCV-specific CTL activity developed a complete response to IFN (P < .01). In 6 of 8 patients with a complete response, including the 3 sustained responders, the CTL response appeared to be directed predominately to the HCV core region. These data suggest that the host immune response, particularly that mediated by CD8+ CTL, may be important in determining the outcome of IFN therapy for chronic HCV infection. Further understanding of the mechanism of action of IFN should impact the design of better therapeutic strategies against chronic HCV infection.     

Determination of a cleavage site of presenilin 2 protein in stably transfected SH-SY5Y human neuroblastoma cell lines

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes are associated with early-onset autosomal dominant familial Alzheimer's disease, and the gene products are endoproteolytically processed to yield N-terminal fragments (NTF) and C-terminal fragments (CTF). We have studied the cleavage site of the PS2 protein in stably transfected human neuroblastoma cells. The 23 kD PS2-CTF was isolated by a combination of anion exchange chromatography and affinity chromatography and directly sequenced. The N-terminus of the PS2-CTF started at residue 307, which indicated that the cleavage occurs between Lys306 and Leu307 in the proximal portion of the large hydrophilic loop. This site is close to the cleavage positions observed in the PS1 protein.     

Role of protein kinase C (PKC) in agonist-induced mu-opioid receptor down-regulation: I. PKC translocation to the membrane of SH-SY5Y neuroblastoma cells is induced by mu-opioid agonists

Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (mu, delta, and kappa), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of mu- and delta-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which mu-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed "reverse translocation." The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L-type calcium channel antagonist nimodipine attenuated opioid-mediated effects on PKC and mu-receptor down-regulation, suggesting that this is a process partially regulated by Ca2+-dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-alpha) and novel (PKC-epsilon) isoforms, SH-SY5Y cells also contain a DAG- and Ca2+-independent, atypical PKC isozyme (PKC-zeta), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-zeta is similarly sensitive to activation by mu-opioids. PKC-zeta translocates from the cytosol to the membrane with kinetics similar to those of PKC-alpha and epsilon in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by mu-opioid agonists is involved in the processes that result in mu-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.     

Hypoxia enhances [3H]noradrenaline release evoked by nicotinic receptor activation from the human neuroblastoma SH-SY5Y

We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K+]o (100 mM) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [3H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K+-evoked release was unaffected by hypoxia (PO2 approximately 30-38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca2+o with 1 mM EGTA abolished NA release. K+-evoked release was also dramatically reduced in the presence of 200 microM Cd2+ to block voltage-gated Ca2+ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd2+-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca2+ influx through the nAChR pore, or by activating an unidentified Cd2+-resistant Ca2+-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.     

MPP+ induced SH-SY5Y apoptosis is potentiated by cyclosporin A and inhibited by aristolochic acid

The purpose of the study was evaluate hepatic (portal and arterial) and splenic perfusion at parenchymal level using dynamic computed tomography (CT) in patients with diffuse liver disease and controls. Nineteen patients and 30 normal controls underwent dynamic CT of the liver. Single-location dynamic sequences were carried out after intravenous administration of a 40 ml bolus of contrast medium (Ioversol: 5 cc/s). Hepatic arterial perfusion, hepatic portal perfusion and splenic perfusion were calculated from time-attenuation curves. Hepatic portal perfusion was significantly lower in patients (0.63 +/- 0.33 ml/min/ml (mean +/- SD); P = 0.003) compared with that in controls (1.03 +/- 0.43), although hepatic arterial perfusion did not differ (0.090 +/- 0.044 versus 0.102 +/- 0.114). Splenic arterial perfusion was significantly lower in patients (0.92 +/- 0.31; P = 0.0009) than in controls (1.35 +/- 0.44). The decreased hepatic portal perfusion in patients with diffuse liver disease was not supplemented by an increase in hepatic arterial perfusion. The decreased splenic perfusion in patients may be due to increased portal pressure. Dynamic CT enables quantification of hepatic and splenic perfusion.     

ATP-activated chloride permeability in biliary epithelial cells is regulated by calmodulin-dependent protein kinase II

Previous studies in freshly isolated rat biliary epithelial cells and in the human cholangiocarcinoma cell line Mz-ChA-1 have demonstrated that ATP activates a calcium-dependent chloride conductance. The coupling between the rise in intracellular calcium and activation of chloride channels has not previously been investigated. In the present study, we evaluated the potential role of calmodulin-dependent protein kinase II (CaMKII) in ATP-activated chloride permeability in Mz-ChA-1 cells. ATP stimulated [125I] efflux, a marker for Cl- movement. Peak efflux rates were inhibited by approximately 60% in cells pretreated with the calmodulin antagonist, W-7. In whole-cell patch clamp recordings, ATP and ionomycin activated calcium-dependent Cl- currents. Pretreatment of cells with the CaMKII inhibitor KN-62 blocked activation by either agent. It is concluded that calcium-dependent activation of chloride currents in Mz-ChA-1 cells is coupled to a CaMKII-dependent process.     

The effect of benomyl on neurite outgrowth in mouse NB2A and human SH-SY5Y neuroblastoma cells in vitro

The commercial fungicide methyl 1-[(butylamino) carbonyl]-1H-benzimidazol-2-ylcarbamate (benomyl) is teratogenic in rats. Its mode of action is believed to be related to its ability to inhibit the polymerization of brain tubulin. In this study its effects were studied in cultured neuronal cells during differentiation and neurite outgrowth. Mouse NB2a and human SH-SY5Y neuroblastoma cells were induced to differentiate by addition of dibutyryl cyclic AMP and at the same time were exposed to various concentrations of benomyl. Benomyl significantly inhibited neurite outgrowth in both cell lines at concentrations of 10(-8) M and above with IC50 values of 5.9 x 10(-7) M and 1.0 x 10(-6) M in the NB2a and SH-SY5Y cells respectively. The results show that benomyl inhibits neuronal cell differentiation at concentrations likely to be achieved during the development of fetal abnormalities in rats in vivo.     

A neurotoxin N-methyl(R)salsolinol induces apoptotic cell death in differentiated human dopaminergic neuroblastoma SH-SY5Y cells

The coagglutination test was standardised for detection of fowl pox antigen in infected scabs and chorioallontoic membrane of chicken embryos. The Staphylococcus aureus Cowan I strain, containing large amounts of Protein A in their cell wall, coated with fowl pox antibodies was found specific and sensitive for detection of fowl pox antigen. The test is easy to perform and rapid as the positive results can be read within 15 seconds.