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Shouliang Wang,1 Bin Xia,1 Zonglei Qiao,2 Lian Duan,3 Gongming Wang,1 Wenjun Meng,1 Zhifei Liu,1 Yu Wang,1 Mengyuan Zhang11Department of Anesthesiology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, People’s Republic of China; 2Department of Anesthesiology, Qingyun County People’s Hospital, Dezhou 253700, Shandong Province, People’s Republic of China; 3Department of Ophthalmology, Qianfoshan Hospital Affiliated to Medical School of Shandong University, Jinan 250014, Shandong Province, People’s Republic of ChinaBackground: Bupivacaine (BUP) acts as a local anesthetic, which is extensively used for clinical patients but could generate neurotoxicity in neurons. Tetramethylpyrazine (TET) exhibits strong neuron protective effects against neurotoxicity. Hence, we investigate the effect of TET on BUP-induced neurotoxicity in SH-SY5Y cells.Methods: CCK-8 assay was used to detect cell proliferation in SH-SY5Y cells. In addition, Western blotting was used to examine Bax, Bcl-2, active caspase 3, LC3II, Beclin 1 and p-62 protein levels in cells. Moreover, ELISA assay was used to detect the levels of total glutathione (GS), superoxide dismutase (SOD) and malondialdehyde (MDA) in cells.Results: In this study, we found that TET attenuated the neurotoxicity of BUP on SH-SY5Y cells. Meanwhile, TET alleviated BUP-induced apoptosis in SH-SY5Y cell via decreasing the expressions of active caspase-3 and Bax and increasing the expression of Bcl-2. In addition, monodansylcadaverine staining assay and Western blotting results confirmed that TET induced autophagy in SH-SY5Y cells via increasing the LC3II/I and Beclin 1 levels. Furthermore, TET attenuated BUP-induced oxidative damage in SH-SY5Y cells via upregulation of the levels of total GS and SOD and downregulation of the level of MDA. Interesting, the protective effects of TET against BUP-induced neurotoxicity in SH-SY5Y cells were reversed by autophagy inhibitor 3-methyladenine (3MA).Conclusion: These data indicated that TET may play a neuroprotective role via inhibiting apoptosis and inducing autophagy in SH-SY5Y cells. Therefore, TET may be a potential agent for the treatment of human neurotoxicity induced by BUP.Keywords: tetramethylpyrazine, bupivacaine, neurotoxicity, human neuroblastoma cell

Enterovirus A71 (EV-A71) is one of the causative agents causing the hand-foot-mouth disease which associated with fatal neurological complications. Several sporadic outbreaks of EV-A71 infections have been recently reported from Asia-Pacific regions and potentially established endemicity in the area. Currently, there is no effective vaccine or antiviral drug for EV-A71 available. This may be attributable to the limited information about its pathogenesis. In this study, the recombinant nonstructural 2B protein of EV-A71 was successfully produced in human neuroblastoma SH-SY5Y cells and evaluated for its effects on induction of the cell apoptosis and the pathway involved. The EV-A71 2B-transfected SH-SY5Y cells showed significantly higher difference in the cell growth inhibition than the mock and the irrelevant protein controls. The transfected SH-SY5Y cells underwent apoptosis and showed the significant upregulation of caspase-9 (CASP9) and caspase-12 (CASP12) genes at 3- and 24-h post-transfection, respectively. Interestingly, the level of cytosolic Ca2+ was significantly elevated in the transfected SH-SY5Y cells at 6- and 12-h post-transfection. The caspase-9 is activated by mitochondrial signaling pathway while the caspase-12 is activated by ER signaling pathway. The results suggested that EV-A71 2B protein triggered transient increase of the cytosolic Ca2+ level and associated with ER-mitochondrial interactions that drive the caspase-dependent apoptosis pathways. The detailed mechanisms warrant further studies for understanding the implication of EV-A71 infection in neuropathogenesis. The gained knowledge is essential for the development of the effective therapeutics and antiviral drugs. [ABSTRACT FROM AUTHOR]

Objective: To explore the mechanisms of whey protein hydrolysate reducing the damage of hydrogen peroxide on neu- roblastoma cells through inhibiting hydrogen peroxide. Methods: The human neuroblastoma cells were randomly divided into four groups. The cells in control group were cultured in normal medium without the medium containing H2O2 and whey protein hydrolysate, the cells in hydrogen peroxide group were treated with 50 μL H202. and the cells in whey protein observation group were treated with 50 μL H202+200 μg/mL whey protein hydrolysate. and the cells in whey protein control group were treated with 200 μg/mL whey protein hydrolysate. The survival and apoptosis of the four groups and the changes of intracellular calcium and mitochondrial membrane potential were observed. Results: 1 Compared with the other three groups, the differences for the survival rate and apoptosis rate of cells in the hydrogen peroxide group were statistically significant (F=24.62 and 32.82. P<0.01): The survival rate and apoptosis rate of cells in the whey protein observation group had significant differences compared with hydrogen peroxide group (t=7.65 and 9.82. P<0.05). 2 The fluorescence intensity of calcium ion in hydrogen peroxide group was the highest (Z=5.12. P<0.05). while the fluorescence intensity of the control group and the whey protein control group was the weakest (Z=5.57 and 4.S2. P<0.05). The fluorescence intensity of the whey protein observation group was stronger than those of the two control groups (Z=6.68 and 4.15. P<0.05). which was weaker than that of the hydrogen peroxide group (Z=6.12. P<0.05). 3 The fluorescence intensity of the normal control group and the whey protein control group were the highest, which indicated that the change of membrane potential was the least. The fluorescence intensity of the hydrogen peroxide group was the weakest, which indicated that the change of membrane potential was the sharpest. The fluorescence intensity of whey protein group was lower than that of the control group, which was higher than tliat of the hydrogen peroxide group, suggesting that the whey protein hydrolysate could prevent the change of mitochondrial membrane potential. Conclusions: Whey protein hydrolysate can reduce the increase of calcium ion induced by hydrogen peroxide in neuroblastoma cells, and reduce the potential difference between the inner and outer membrane of mitochondria, so it can inhibit the apoptosis of cells. [ABSTRACT FROM AUTHOR]

Isatin has been shown to initiate apoptotic processes in SH-SY5Y neuroblastoma cells. The aim of the present study was to investigate whether isatin is also able to alter the proliferation and migratory ability of SH-SY5Y cells. The results demonstrated that the proportion of SH-SY5Y cells in G1 phase was significantly increased following treatment with isatin for 48 h with simultaneous downregulation of cyclin D1 expression. In addition, isatin significantly inhibited cell migration and invasion, along with decreases in matrix metalloproteinase (MMP)2 and MMP9 expression. In addition, isatin reduced the levels of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in a concentration-dependent manner. These results demonstrated that isatin induces G1-phase arrest in SH-SY5Y cells, possibly by decreasing cyclin D1 expression as well as inhibiting their migration and invasiveness, probably by reducing MMP2 and MMP9. These effects may be exerted by isatin via a downregulating the levels of pSTAT3. [ABSTRACT FROM AUTHOR]

G q/11 -coupled muscarinic acetylcholine receptors (mAChRs) belonging to M 1 , M 3 and M 5 subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca 2 + /calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca 2 + required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca 2 + entry (SOCE) in AMPK activation by pharmacologically defined M 3 mAChRs in human SH-SY5Y neuroblastoma cells. In Ca 2 + -free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2 min. Conversely, in the presence of extracellular Ca 2 + CCh-induced AMPK phosphorylation lasted for at least 180 min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50 μM) that suppressed CCh-induced intracellular Ca 2 + ([Ca 2 + ] i ) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca 2 + sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca 2 + ] i plateau and AMPK activation. M 3 mAChRs increased glucose uptake and this response required extracellular Ca 2 + and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M 3 mAChRs and suggests that SOCE is a critical process connecting M 3 mAChRs to the control of neuronal energy metabolism. [ABSTRACT FROM AUTHOR]

Highlights: [•] Blonanserin affects DNA methylation profiles of human neuroblastoma cells. [•] About 3,000 CpG sites show changes in methylation levels, regardless of dose. [•] CpG sites related to axonogenesis and neuron differentiation are hypermethylated. [•] With high-dose blonanserin, most CpG sites far from promoters are hypermethylated. [•] Hypermethylation of DRD2 and HTR2A suggests suppressing effects on receptor function. [Copyright &y& Elsevier]

Abstract: An age-dependent increase in mRNA levels of the amyloid precursor protein (APP), the microtubule-associated protein Tau, and voltage-dependent anion channel 1 (VDAC1) genes are reported to be toxic to neurons affected by Alzheimer's disease (AD). However, the underlying toxic nature of these genes is not completely understood. The purpose of our study was to determine the effects of RNA silencing of APP, Tau, and VDAC1 genes in AD pathogenesis. Using human neuroblastoma (SHSY5Y) cells, we first silenced RNA for APP, Tau, and VDAC1 genes, and then performed real-time RT-PCR analysis to measure mRNA levels of 34 genes that are involved in AD pathogenesis. Using biochemical assays, we also assessed mitochondrial function by measuring levels of H2O2 production, lipid peroxidation, cytochrome c oxidase activity, ATP production, and GTPase enzymatic activity. We found that increased mRNA expression of synaptic function and mitochondrial fission genes, and reduced levels of mitochondrial fusion genes in RNA silenced the SHSY5Y cells for APP, Tau and VDAC1 genes relative to the control SHSY5Y cells. In addition, RNA-silenced APP, Tau, and VDAC1 genes in SHSY5Y cells showed reduced levels of H2O2 production, lipid peroxidation, fission-linked GTPase activity, and increased cytochrome oxidase activity and ATP production. These findings suggest that a reduction of human APP, Tau, and VDAC1 may enhance synaptic activity, may improve mitochondrial maintenance and function, and may protect against toxicities of AD-related genes. Thus, these findings also suggest that the reduction of APP, Tau, and VDAC1 mRNA expressions may have therapeutic value for patients with AD. [Copyright &y& Elsevier]

Abstract: Recently, it has been suggested that anesthetic agents may have neuroprotective potency. The notion that anesthetic agents can offer neuroprotection remains controversial. Propofol, which is a short-acting intravenous anesthetic agent, may have potential as a neuroprotective agent. In this study, we tried to determine whether propofol affected the viability of human neuroblastoma SH-SY5Y cells by using the MTT assay. Surprisingly, our results showed that propofol at a dose of 1–10μM could improve cell proliferation. However, at higher doses (200μM), propofol appears to be cytotoxic. On the other hand, propofol could up-regulate the expression of key proteins involved in neuroprotection including B-cell lymphoma 2 at a dose range of 1–10μM, activation of phospho-serine/threonine protein kinase at a dose range of 0.5–10μM, and activation of phospho-extracellular signal-regulated kinases at a dose range of 5–10μM. Similarly, we demonstrate that propofol (10μM) could elevate protein levels of heat shock protein 90 and heat shock protein 70. Therefore, we choose to utilize a 10μM concentration of propofol to assess neuroprotective activities in our studies. In the following experiments, we used dynorphin A to generate cytotoxic effects on SH-SY5Y cells. Our data indicate that propofol (10μM) could inhibit the cytotoxicity in SH-SY5Y cells induced by dynorphin A. Furthermore, propofol (10μM) could decrease the expression of the p-P38 protein as well. These data together suggest that propofol may have the potential to act as a neuroprotective agent against various neurologic diseases. However, further delineation of the precise neuroprotective effects of propofol will need to be examined. [Copyright &y& Elsevier]

Abstract: The purpose of the study was to investigate the antitumor effects of Isatin and the related mechanism. Human neuroblastoma cells (SH-SY5Y) were exposed to Isatin at different concentrations for 48 h. Apoptotic features were demonstrated by means of nuclei staining with Hoechst 33258 and flow cytometry with Propidium Iodide (PI). Expressions of Bcl-2, Bax and Vascular endothelial growth factor (VEGF) mRNA were analyzed via RT-PCR. Expressions of Bcl-2, Bax proteins and phosphorylated extracellular signal regulated protein kinases (ERKs, p42/p44) were analyzed via Western blot. Activation of caspase-3 was assayed by flow cytometry with anti-active caspase-3-McAb-PE. VEGF protein was determined by ELISA kits. And the results showed that apoptosis of SH-SY5Y cells were induced by Isatin in a dose-dependent manner. Expressions of Bcl-2, VEGF mRNA and Bcl-2, VEGF proteins were down-regulated, while expressions of Bax mRNA and Bax protein were not changed obviously. Expression of phosphorylated ERKs decreased, but the level of activated caspase-3 increased after treatment of Isatin. These results suggest that Isatin promotes the apoptosis of neuroblastoma cells, therefore, it might be a potential candidate for the treatment of neuroblastoma. [Copyright &y& Elsevier]

Abstract: Neuropathy target esterase (NTE) is inhibited and aged by organophosphorus compounds that induce delayed neuropathy in human and some sensitive animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation. However, to date, there is no direct evidence of the relevance of NTE in neurodifferentiation under physiological conditions. In this study, we have investigated a possible role for NTE in the all-trans retinoic acid-induced differentiation of neuroblastoma cells. The functional inactivation of NTE by RNA interference indicated that reduction of NTE does not affect process outgrowth or differentiation of the cells, although moderate expression of NTE by expression of the NTE esterase domain accelerates the elongation of neurite processes. Mipafox, a neurotoxic organophosphate, was shown to block process outgrowth and differentiation in cells that have lowered NTE activity due to RNA interference, suggesting that mipafox may interact with other molecules to exert its effect in this context. [Copyright &y& Elsevier]

Abstract: Methylmercury (MeHg) is well known as a neurotoxic chemical. However, little is mentioned about its neurotoxic mechanism or molecular target in human neuronal cells in particular. We show in this study that exposure of human neuronal cell line, SH-SY5Y, to MeHg dose- and time-dependently impairs viability and mRNA expression of selenoprotein W (SeW) with a significant difference, unlike other selenoenzymes such as, SeP, GPX4, 5DI, and 5′DI. Using real-time RT PCR, the influence of selenium (Se) and glutathione (GSH) on SeW expression was also investigated. While Se depletion caused a weakly reduced SeW mRNA levels, additional Se caused an increase of SeW mRNA levels. Although 2mM GSH had induced a weak shift on SeW level, the expression of SeW mRNA was down-regulated in SH-SY5Y cells treated with 25μM BSO, an inhibitor of GSH synthesis. To understand the relationship between a decrease of SeW expression and intracellular GSH and ROS, we measured the concentration of intracellular GSH and ROS in cells treated to 1.4μM MeHg using fluorescence based assays. A positive correlation was found between SeW mRNA level and intracellular GSH but no significant correlation was observed between intracellular ROS and SeW mRNA level or intracellular GSH contents. Therefore, we suggest that SeW is the novel molecular target of MeHg in human neuronal cells and down-regulation of this selenoenzyme by MeHg is dependent not on generation of ROS but on depletion of GSH. [Copyright &y& Elsevier]

Secretin is a neuropeptide that is expressed in distinct central neurones. As there is no information on how the secretin gene is regulated in neuronal cells, a well established neuronal differentiation cell model, SH-SY5Y, was used to study transcriptional regulation of the human secretin gene. High secretin transcript and peptide levels were found in this cell, and secretin gene expression and promoter activity were up-regulated uponall-transretinoic acid (RA) treatment. Within the promoter, a functional GC-box 1 (−131 from ATG, relative to the ATG initiation codon) was found to be regulated by a brain-specific Sp protein, Sp4, and ubiquitous factors Sp1 and Sp3. The human secretin gene in SH-SY5Y cells is controlled by the (Sp1 + Sp4)/Sp3 ratio and the RA-induced activation is a partial result of a decrease in Sp3 levels. In addition to the GC-box 1, an N1 motif in close proximity was also responsible for RA-induced secretin gene activation. Competitive gel mobility shift and southwestern blot studies revealed binding of Nuclear Factor I (NFI) with the N1 motif. Overexpression of NFI-C increased promoter activity upon RA treatment. Consistent with this observation, NFI-C transcript levels were augmented after RA treatment. We conclude that RA induction of the secretin gene in neuronal cells is regulated by the combined actions of reducing Sp3 and increasing NFI-C expression. [ABSTRACT FROM AUTHOR]

The recently discovered endogenous peptide orphanin FQ/nociceptin (OFQ/N) activates the opioid receptor-like 1 (ORL1) receptor and produces diverse effects on pain perception. In addition to producing spinal analgesia, OFQ/N also exhibits an ‘anti-opioid activity’ against functional (supraspinal analgesia) and behavioral (conditioned place preference and withdrawal) properties of morphine. One manifestation of the behavioral changes resulting from chronic use of morphine is the upregulation of tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine biosynthesis), which contributes to the dramatic increases in catecholamine release in the target regions of the locus coeruleus (LC) and the ventral tegmental area (VTA). The present study sought to determine the molecular mechanism(s) by which OFQ/N modulates the chronic actions of morphine by utilizing human neuroblastoma cell lines [BE(2)-C and SH-SY5Y] that endogenously express TH, and μ and ORL1 receptors. Activation of μ or ORL1 receptors in these cells in turn activates extracellular signal-regulated protein kinases (ERKs), ERK1 and ERK2. Chronic activation of μ, but not ORL1, receptors upregulated TH levels in these cells as previously reported in rat brain. Morphine-induced TH upregulation was blocked upon inclusion of a MEK-1 (mitogen-activated protein kinase kinase-1) inhibitor (PD98059), confirming the role for ERKs in this adaptive response to morphine. Inclusion of OFQ/N during chronic morphine exposure also blocked morphine-induced TH upregulation. Furthermore, chronic OFQ/N exposure increased levels of the TH gene repressor, Oct-2, irrespective of the presence or absence of morphine. This report suggests a potential role for Oct-2 in mediating the anti-opioid actions of OFQ/N against the behavioral manifestations resulting from chronic use of morphine. [Copyright &y& Elsevier]

Peptide nucleic acid (PNA) sequences are synthetic versions of naturally ocurring oligonucleotides which display improved binding properties to DNA and RNA, but are still poorly internalized across cell membranes. In an effort to employ the rapid binding/internalization properties of somatostatin agonist analogs and the over-expression of somatostatin receptors on many types of tumor cells, PNAs complementary to target sites throughout 5′-UTR, translation start site and coding region of the n-myc oncogene were conjugated to a somatostatin analog (SSA) with retention of high somatostatin biological potency. IMR32 cells, which over-express somatostatin receptor type 2 (SSTR2) and contain the n-myc oncogene, were treated with these PNA–SSA conjugates. The results show that PNA conjugates targeted to the 5′-UTR terminus and to regions at or close to the translation start site could effectively inhibit n-myc gene expression and cell growth, whereas the non-conjugate PNAs were without effect at similar doses. The most potent inhibition of cell growth was achieved with PNAs binding to the translation start site, but those complementary to the middle coding region or middle upstream site between 5′-UTR and translation start site displayed no inhibition of gene expression. These observations were extended to four other cell lines: GH3 cells which express SSTRs with the n-myc gene, SKNSH cells containing a silent n-myc gene without SSTR2, HT-29 cells carrying the c-myc but no n-myc gene, and CHO-K1 cells lacking SSTR2 with n-myc gene. The results show that there was almost no effect on these four cell lines. Our study indicates that PNAs conjugated to SSA exhibited improved inhibition of gene expression possibly due to facilitated cellular uptake of the PNAs. These conjugates were mRNA sequence- and SSTR2-specific suggesting that many other genes associated with tumor growth could be targeted using this approach and that SSA could be a novel and effective transportation vector for the PNA antisense strategy. [Copyright &y& Elsevier]

A significant number of adult male patients with acquired immunodeficiency syndrome develop cerebral atrophy and progressive brain disorders such as dementia complex and neuropsychiatric problems. Upon entering the brain via activated macrophages or microglias, the human immunodeficiency type 1 virus (HIV-1) may produce cytotoxic factors such as HIV-1 envelope protein (gp120) and protease. Owing to significant proteolysis of nonviral proteins, the protease derived from HIV-1 may be detrimental to brain cells and neurons. Our results revealed that HIV-1 protease, at nanomolar concentrations, was as potent as gp120 in causing neurotoxicity in human neuroblastoma neurotypic SH-SY5Y cells. As shown by the Oncor ApopTag staining procedure, HIV-1 protease significantly increased the number of apoptotic cells over the serum-free controls. Moreover, HIV-1 protease-induced neurotoxicity was blocked by a selective protease inhibitor, kynostatin (KNI-272). Antioxidants such as 17β-estradiol, melatonin, and S-nitrosoglutathione also prevented protease-induced neurotoxicity. These findings indicate that oxidative proteolysis may mediate HIV-1 protease-induced apoptosis and the degeneration of neurons and other brain cells. Centrally active protease inhibitors and antioxidants may play an important role in preventing cerebral atrophy and associated dementia complex caused by HIV-1. [ABSTRACT FROM AUTHOR]

DNA transfection conditions were investigated by calcium phosphate-DNA co-precipitation in SK-N-BE(2)C human neuroblastoma cells. The DNA plasmid of TH2400CAT was used in which rat tyrosine hydroxylase gene was inserted into chloramphenicol acetyltransferase reporter gene. The transfection efficiency was 25≈30% and the method was simple and reproducible. So, the method will be a good tool for transient transfection analysis. [ABSTRACT FROM AUTHOR]