sincalide and Neuroblastoma

The aim of the study was to investigate the effects of the GLP-1 analog liraglutide on beta-amyloid (Aβ)-induced neurotoxicity in the human neuroblastoma cell line SH-SY5Y and study the underlying mechanisms.. Cultured SH-SY5Y cells in vitro were randomly divided into normal control group, beta-amyloid (Aβ) group (20, 40, and 80 uM), and liraglutide pre-treatment group (10, 100, and 200 nM). Cell viability was determined by CCK-8 and lactate dehydrogenase (LDH). Based on its higher protection potentials, the effect of the liraglutide (100 nM) and wortmannin (200 nM) on beta-amyloid (Aβ) group (40 uM) damage in human SH-SY5Ycells was examined by DAPI fluorescence staining and flow cytometry. Caspase-3, Bcl-2, Bax, Cyt-C, Akt, and P-Akt expression were detected by western blotting.. We found that exposure of SH-SY5Y to Aβ (25-35)-induced cytotoxicity, increased lactate dehydrogenase (LDH) leakage, and cellular apoptosis. Interestingly, pre-treatment with liraglutide reversed these reactions. Liraglutide afforded protection against Aβ (25-35)-induced toxicity by inhibiting apoptosis, which was also confirmed by the activated caspase-3 assay. P-Akt and Bcl-2/Bax expression increased after pre-treatment with liraglutide in SH-SY5Y cells exposed to Aβ (25-35), whereas cytochrome-c release decreased. This effect could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase).. These findings suggest that liraglutide prevented Aβ (25-35)-induced neurotoxicity by inhibiting neuronal apoptosis and liraglutide may have a neuroprotective effect through activation of the PI3K/Akt signaling pathway. Thus, liraglutide may be a preventive or therapeutic agent for Alzheimer's disease.

Topics: Amyloid beta-Peptides; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Drug Administration Schedule; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation; Humans; L-Lactate Dehydrogenase; Liraglutide; Neuroblastoma; Neuroprotective Agents; Peptide Fragments; Phosphatidylinositol 3-Kinases; Signal Transduction; Sincalide

Drug-loaded nanoparticles from 'Ershiwuwei Shanhu' Pill (ESP) inducing cellular swelling of the SH-SY5Y neuroblastoma cells were investigated. Electron microscope was used to observe nanoparticles existing in the freeze-dried supernatant of 'Ershiwuwei Shanhu' Pill. Drug-free nanoparticles were obtained from the solution of drug-loaded nanoparticles via dialysis. The size and zeta potential of two kinds of nanoparticles were tested by granularmetric analysis and surface charge analysis. Results showed that nanoparticles could penetrate into cellular nucleus and caused cell swelling. CCK8 analysis implied that low concentration of drug-free nanoparticles from 'Ershiwuwei Shanhu' Pill can induce cell proliferation of the SH-SY5Y neuroblastoma cells, while drug-loaded nanoparticles can reduce cell viability through NF-κB pathway. Drug-loaded nanoparticles existed in 'Ershiwuwei Shanhu' pill might play a vital role during pharmacotherapy, which served as nanocarriers in delivering drugs into cells.

Topics: Cell Line, Tumor; Cholecystokinin; Drug Carriers; Humans; Microscopy, Electron, Transmission; Nanoparticles; Neuroblastoma; Peptide Fragments; Plant Extracts

Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and the AD transgenic mouse models. Here, we investigated whether down-regulation of PTPA affects cell viability and the underlying mechanisms. We found that PTPA was located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines. PTPA knockdown decreased mitochondrial membrane potential and induced Bax translocation into the mitochondria with a simultaneous release of Cyt C, activation of caspase-3, cleavage of poly (DNA ribose) polymerase (PARP), and decrease in Bcl-xl and Bcl-2 protein levels. Over-expression of Protein phosphatase 2A (PP2A) catalytic subunit (PP2AC ) did not rescue the apoptosis induced by PTPA knockdown, and PTPA knockdown did not affect the level of and their phosphorylation of mitogen-activated protein kinases (MAPKs), indicating that PP2A and MAPKs were not involved in the apoptosis induced by PTPA knockdown. In the cells with over-expression of tau, PTPA knockdown induced PP2A inhibition and tau hyperphosphorylation but did not cause significant cell death. These data suggest that PTPA deficit causes apoptotic cell death through mitochondrial pathway and simultaneous tau hyperphosphorylation attenuates the PTPA-induced cell death. Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and AD transgenic mouse models. Here, we investigated whether down-regulation of PTPA affects cell viability. We found that PTPA located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines by decreasing mitochondrial membrane potential, which leads to translocation of Bax and a simultaneous release of Cyt C. In the cells with tau over-expression, PTPA knockdown inactivated PP2A to phosphorylate tau to avoid cell apoptosis which induced by PTPA knockdown.

Topics: Adenosine Triphosphate; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cell Nucleus; Cytochromes c; Cytoplasm; Gene Knockdown Techniques; HEK293 Cells; Humans; Mice; Mitochondria; Neuroblastoma; Phosphoprotein Phosphatases; Phosphorylation; Sincalide; tau Proteins

Mutations in the parkin gene underlie a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinsonism (AR-JP). Dysfunction of parkin, a ubiquitin E3 ligase, has been implicated in the accumulation of ubiquitin proteasome system-destined substrates and eventually leads to cell death. However, regulation of parkin enzymatic activity is incompletely understood. Here we investigated whether the ubiquitin E3 ligase activity of parkin could be regulated by neddylation. We found that parkin could be a target of covalent modification with NEDD8, a ubiquitin-like posttranslational modifier. In addition, NEDD8 attachment caused an increase of parkin activity through the increased binding affinity for ubiquitin-conjugating E2 enzyme as well as the enhanced formation of the complex containing parkin and substrates. These findings point to the functional importance of NEDD8 and suggest that neddylation is one to the diverse modes of parkin regulation, potentially linking it to the pathogenesis of AR-JP.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Cells, Cultured; Cycloheximide; Embryo, Mammalian; Gene Expression Regulation; Hippocampus; Humans; Immunoprecipitation; Leupeptins; Mutation; NEDD8 Protein; Neuroblastoma; Neurotoxins; Protein Binding; Protein Synthesis Inhibitors; Rats; RNA, Small Interfering; Sincalide; Stem Cells; Subcellular Fractions; Time Factors; Transfection; Ubiquitin-Protein Ligases; Ubiquitination; Ubiquitins

Cholecystokinin octapeptide (CCK-8), the most potent endogenous anti-opioid peptide, has been shown to regulate the processes of morphine dependence. In our previous study, we found that exogenous CCK-8 attenuated naloxone induced withdrawal symptoms. To investigate the precise effect of exogenous CCK-8 and the role of cholecystokinin (CCK) 1 and/or 2 receptors in morphine dependence, a SH-SY5Y cell model was employed, in which the μ-opioid receptor, CCK1/2 receptors, and endogenous CCK are co-expressed.. Forty-eight hours after treating SH-SY5Y cells with morphine (10 μM), naloxone (10 μM) induced a cAMP overshoot, indicating that cellular morphine dependence had been induced. The CCK receptor and endogenous CCK were up-regulated after chronic morphine exposure. The CCK2 receptor antagonist (LY-288,513) at 1-10 μM inhibited the naloxone-precipitated cAMP overshoot, but the CCK1 receptor antagonist (L-364,718) did not. Interestingly, CCK-8 (0.1-1 μM), a strong CCK receptor agonist, dose-dependently inhibited the naloxone-precipitated cAMP overshoot in SH-SY5Y cells when co-pretreated with morphine. The L-364,718 significantly blocked the inhibitory effect of exogenous CCK-8 on the cAMP overshoot at 1-10 μM, while the LY-288,513 did not. Therefore, the CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence in SH-SY5Y cells. An additional inhibitory effect of CCK-8 at higher concentrations appears to involve the CCK1 receptor.. This study reveals the difference between exogenous CCK-8 and endogenous CCK effects on the development of morphine dependence, and provides the first evidence for the participation of the CCK1 receptor in the inhibitory effects of exogenous CCK-8 on morphine dependence.

Topics: Analysis of Variance; Cell Line, Tumor; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Hormone Antagonists; Humans; Morphine; Naloxone; Narcotic Antagonists; Narcotics; Neuroblastoma; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Receptors, Opioid, mu; RNA, Messenger; Sincalide; Up-Regulation

To observe the effect of cholecystokinin (CCK) on lipofusin value, neuronal dendrite and spine ultrastructure, and total cellular protein during the process of experimental neuronal aging.. Experimental neuronal aging study model was established by NBA2 cellular serum-free culture method. By using single intracellular lipofusin value from microspectrophotometry, morphology of neuronal dendrites and spines from the scanner electron microscopy, and total cellular protein as the indexes of experimental neuronal aging, we observed the effect of CCK8 on the process of experimental neuronal aging.. Under the condition of serum-free culture, intracellular fluorescence value (%) increased with the extension of culture time (1 d 8.51+/-3.43; 5 d 10.12+/-3.03; 10 d 20.54+/-10.3; 15 d 36.88+/-10.49; (b)P<0.01). When CCK was added to serum-free culture medium, intracellular lipofusin value (%) decreased remarkably after consecutive CCK reaction for 10 and 15 d (control 36.88+/-10.49; 5 d 32.03+/-10.01; 10 d 14.37+/-5.55; 15 d 17.31+/-4.80; (b)P<0.01). As the time of serum-free culturing was prolonged, the number of neuronal dendrite and spine cells decreased. The later increased in number when CCK8 was added. CCK8 could improve the total cellular protein in the process of experimental neuronal aging.. CCK8 may prolong the process of experimental neuronal aging by maintaining the structure and the number of neuronal dendrite and spine cells and changing the total cellular protein.

Topics: Animals; Cell Line, Tumor; Cellular Senescence; Culture Media, Serum-Free; Flow Cytometry; Lipofuscin; Mice; Microscopy, Electron; Microvilli; Neuroblastoma; Neurons; Proteins; Sincalide

The effect of ¿2-[4-(4-chloro-2, 5-dimethoxy-phenyl)-5-[2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoy l]-5, 7-dimethyl-indol-1-yl¿-acetic acid (SR146131), a novel non-peptide agonist of cholecystokinin (CCK) CCK(1) receptors, was compared to the effect of sulphated cholecystokinin octapeptide (CCK-8-S) on CCK(1) receptors of the human neuroblastoma cell line IMR-32. SR146131 inhibited [125I]CCK-8-S binding to IMR-32 cells at nanomolar concentrations. SR146131 and CCK-8-S increased intracellular free Ca(2+) levels ([Ca(2+)](i)) in the same concentration range (EC(50)=6+/-2.3 and 1.3+/-0.14 nM, respectively). Although the shape of the [Ca(2+)](i) increase induced by CCK-8-S and SR146131 was slightly different, extracellular Ca(2+) removal affected the response of both compounds to a similar degree, and the response of both compounds was essentially due to Ca(2+) release from intracellular stores. This was also confirmed by measuring the [Ca(2+)](i) response of single cells: both compounds induced [Ca(2+)](i) oscillations at subnanomolar concentrations and elicited a large peak increase in [Ca(2+)](i) at higher concentrations (EC(50)=0.5+/-0.04 and 5.7+/-1.9 nM for CCK-8-S and SR146131, respectively). Both CCK-8-S and SR146131 induced a sustained increase of phosphoinositide turnover in these cells, and acted at similar concentrations (EC(50)=2.7+/-0.7 and 6+/-3.1 nM, respectively), although the maximal effect of SR146131 was somewhat lower than the effect of CCK-8-S. These data show that SR146131 activates human CCK(1) receptors on IMR-32 cells in a manner and with a potency similar to that of CCK-8-S.

Topics: Benzodiazepinones; Binding, Competitive; Calcium; Devazepide; Dose-Response Relationship, Drug; Humans; Indoles; Iodine Radioisotopes; Neuroblastoma; Phenylurea Compounds; Phosphatidylinositols; Radioligand Assay; Receptors, Cholecystokinin; Sincalide; Thiazoles; Tumor Cells, Cultured

SR146131 inhibited the binding of [125I]-Bolton Hunter (BH)-sulfated cholecystokinin octapeptide (CCK-8S) for the human recombinant cholecystokinin subtype 1 (CCK1) receptor (IC50 = 0.56 nM) with high (300-fold) selectivity to the CCK2 receptor. The biological activity of SR146131 was characterized in vitro in a NIH-3T3 cell line expressing the human recombinant CCK1 receptor (3T3-hCCK1). Measuring intracellular calcium release, SR146131 behaved as a full agonist with an efficacy comparable with that of CCK-8S (EC50 = 1.38 +/- 0.06 nM). On individual cells, SR146131 induced, like CCK-8S, Ca2+ oscillations at subnanomolar concentrations and sustained responses at higher concentrations. Like CCK-8S, SR146131 also fully stimulated inositol monophosphate formation (EC50 = 18 +/- 4 nM). SR146131 partially activated mitogen-activated protein kinase and enhanced the expression of the immediate early gene krox 24. In the human CHP212 and IMR32 neuroblastoma cell lines, which constitutively express the CCK1 receptor, SR146131 behaved as a partial agonist on intracellular calcium release and inositol monophosphate formation. All of these effects of SR146131 were inhibited by the CCK1 receptor antagonists SR27897B and devazepide, suggesting that the effects of SR146131 were entirely mediated by the CCK1 receptor. In contrast, high concentrations (>1 microM) of SR146131 had only minimal effects on CCK-8S-stimulated and unstimulated Chinese hamster ovary (CHO) cells expressing the human CCK2 receptor, indicating that SR146131 is functionally inactive on the CCK2 receptor. In conclusion, these in vitro experiments show that SR146131 is a highly potent and selective agonist of the CCK1 receptor.

Topics: 3T3 Cells; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; CHO Cells; Cricetinae; Devazepide; DNA-Binding Proteins; Early Growth Response Protein 1; Genes, Immediate-Early; Hormone Antagonists; Humans; Immediate-Early Proteins; Indoleacetic Acids; Indoles; Inosine Monophosphate; Mice; Neuroblastoma; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Recombinant Proteins; Sincalide; Thiazoles; Transcription Factors; Tumor Cells, Cultured

Agonist stimulation of cells often results in desensitization of the response, to protect the cell from overstimulation. We have previously shown that the type A cholecystokinin (CCK) receptor on the pancreatic acinar cell and on the model CHO-CCKR cell line undergoes desensitization in response to CCK, with receptor phosphorylation and internalization playing key roles. Although these mechanisms contribute in a cell-specific manner, no analogous information exists for the CCK receptor expressed on neuronal cells, where in vivo data demonstrate a particularly sensitive response to CCK. The present study was designed to explore CCK receptor desensitization in the CHP212 neuroblastoma cell line, focusing on inositol 1,4,5-trisphosphate (IP3) responses to CCK and on recognized molecular and cellular mechanisms of desensitization. CCK promptly stimulated IP3 responses in these cells, with hormonal responsiveness rapidly and completely desensitized. Both receptor phosphorylation and internalization were observed to occur, with the former occurring most rapidly and likely being responsible for the earliest desensitization observed. Although the time course of receptor phosphorylation and dephosphorylation, and the groups of kinases involved in the neuroblastoma cell line, were most similar to those in the pancreatic cell, the movement of the agonist-bound receptor in these cells was quite different from that in the pancreatic cell and most similar to that in the CHO-CCKR cell line. This hybrid response supports the cell-specific nature of CCK receptor regulation and provides an important system to explore the molecular determinants of these processes.

Topics: Dose-Response Relationship, Drug; Endocytosis; Enzyme Inhibitors; Fluorescent Dyes; GTP-Binding Proteins; Humans; Hypertonic Solutions; Inositol 1,4,5-Trisphosphate; Neuroblastoma; Phosphorylation; Receptors, Cholecystokinin; Rhodamines; Sensitivity and Specificity; Sincalide; Staurosporine; Tumor Cells, Cultured

[125I]Cholecystokinin-8-S (CCK-8-S) bound to a single class of saturable binding sites on the human neuroblastoma cell line IMR-32 (KD = 4 +/- 1.5 nM, Bmax = 10,500 +/- 3,500 sites/cell (n = 6)). These binding sites were of the CCKA type, as demonstrated by the differential inhibition of the binding of [125I]CCK-8-S and CCK-8-S-induced 45Ca2+ efflux by the specific CCKA antagonist SR 27897 and the specific CCKB antagonist PD 134,308. CCK-JMV-180, an analogue of CCK-8-S which has been shown to activate 45Ca2+ efflux in rat cells in a manner similar to CCK-8-S, acted as a potent antagonist of CCK-8-S-induced 45Ca2+ efflux (IC50 = 50 nM) and inhibited [125I]CCK-8-S binding to IMR-32 cells (IC50 = 1.7 nM). These results show that, unlike its CCK-like effect in various animal systems, CCK-JMC-180 acts as an antagonist of CCKA receptors in the human neuroblastoma cell line IMR-32.

Topics: Calcium Radioisotopes; Humans; Kinetics; Neuroblastoma; Receptors, Cholecystokinin; Sincalide; Tumor Cells, Cultured

The modulation of neuropeptide Y (NPY) and peptide YY (PYY) receptors by dynorphin, luteinizing hormone-releasing hormone (LHRH), corticotropin-releasing factor (CRF), and cholecystokinin octapeptide has been studied in human neuroblastoma cell lines SK-N-MC and SMS-MSN, which express Y1 and Y2 receptors for NPY/PYY. Dynorphin A and LHRH inhibited the binding of NPY/PYY to SK-N-MC cell membranes at concentrations ranging from 10(-7) to 10(-5) M, whereas dynorphin A and CRF were effective in SMS-MSN cells. The inhibitory effect of dynorphin A on NPY/PYY binding was observed in the presence of guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable GTP analogue, as well as H-7 and H-8, novel inhibitors of protein kinases C and A. However, U-50488, the most potent kappa-selective compound did not mimic the dynorphin action. Dynorphin A showed neither effect on the dissociation of NPY/PYY from their receptors nor inhibition on the basal as well as forskolin-stimulated adenosine 3',5'-cyclic monophosphate response. These results indicate that the interaction of dynorphin A with Y1 and Y2 receptors is not mediated by changes in receptor-G protein interaction, receptor phosphorylation, and allosteric binding to NPY/PYY receptors but that dynorphin A binds to NPY/PYY receptors at high concentrations, probably in an antagonistic manner.

Topics: Corticotropin-Releasing Hormone; Dynorphins; Gonadotropin-Releasing Hormone; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Neuroblastoma; Protein Kinase Inhibitors; Receptors, Gastrointestinal Hormone; Receptors, Neuropeptide Y; Sincalide; Tumor Cells, Cultured

Topics: Astrocytoma; Cell Line; Cell Membrane; Cholecystokinin; Glycopeptides; Humans; Hydrolysis; Kinetics; Neprilysin; Neuroblastoma; Sincalide; Tumor Cells, Cultured