Human Neuroblastoma IMR-32 Cells Research Articles

CdSe quantum dots induce apoptosis in human neuroblastoma cells via mitochondrial-dependent pathways and inhibition of survival signals

Quantum dots (QDs) may be useful as novel luminescent markers, but their cytotoxicity has not been fully investigated. In this report, we demonstrate that CdSe-core QDs can induce apoptotic biochemical changes, including JNK activation, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and activation of caspase-9 and caspase-3 in the IMR-32 human neuroblastoma cell line. Importantly, treatment of IMR-32 cells with CdSe-core QD triggered an increase in reactive oxygen species (ROS) and inhibited survival-related signaling events, such as decreased Ras and Raf-1 protein expression and decreased ERK activation. These apoptotic biochemical changes were not detected in cells treated with ZnS-coated CdSe QDs. Collectively, these results demonstrate that CdSe-core QD treatment of IMR-32 cells induced JNK activation and mitochondrial-dependent apoptotic processes while inhibiting Ras → ERK survival signaling and that a ZnS coating could effectively reduce QD cytotoxicity.

Cellular low zinc levels increase cell oxidants partially through a mechanism involving NADPH oxidase

Several studies have shown that a condition of zinc (Zn) deficiency increases the production of cell oxidants such as reactive oxygen species (ROS), reactive nitrogen species and carbon-centered free radicals. However, the mechanisms involved in the high levels of cellular ROS are still unclear. The objective of this study was to test the hypothesis that a decrease in cellular Zn levels can lead to an increased ROS production through a mechanism that involves NADPH oxidase. For this purpose, human IMR-32 neuroblastoma cells were cultured in control (C) or Zn-chelated media containing 1.5 (1.5 Zn) or 15 μM (15 Zn) Zn. Global cell oxidants, measured with DCDCDHF, H2O2 production, measured with Amplex Red, and the activation of the H2O2-responsive transcription factor AP-1 were determined. After 2 to 6 h of incubation, significantly higher levels of oxidants (DCDCDHF) were found in the 1.5 Zn (60–40 %, p < 0.005) compared to C and 15 Zn cells. This increase was reduced (p < 0.001) when cells were incubated simultaneously in the presence of NADPH oxidase inhibitor DPI. Similar results were observed when H2O2 was determined. We next evaluated the effect of Zn deficiency-induced increase in H2O2 on the activation of AP-1. A significant increase in AP-1-DNA binding activity (40%) and in the transactivation of an AP-1-driven gene (p-AP1-Luc) (60%) of the Zn deficient cells was observed after 2 and 6 h respectively. Results indicate that Zn deficiency could increase ROS production and trigger oxidant-responsive cell signals partially through a mechanism involving NADPH oxidase. Supported by grants from the University of California and NIEHS-CEHS, USA

Zinc Deficiency Increases the Susceptibility of Human Neuroblastoma Cells to Lead-Induced Activator Protein-1 Activation

Lead (Pb2+) is a major environmental pollutant that has severe adverse effects on the nervous system. Similar human populations are at risk of suffering both Pb2+ toxicity and zinc (Zn) deficiency. Thus, in the present study we investigated whether Zn deficiency can increase the susceptibility of human neuroblastoma IMR-32 cells to Pb2+-induced oxidative stress which could trigger the activation of the mitogen-activated protein kinases (MAPKs) c-Jun-N-terminal kinase (JNK) and p38 and subsequently activate transcription factor activator protein-1 (AP-1). After 24 h of incubation, 5-50 microM Pb2+ caused a decrease in cell viability that was markedly higher in the Zn-deficient cells compared to controls. Pb caused a time (2-24 h) and dose (5-50 microM)-dependent increase of cell oxidants, with a significantly higher effect in the Zn-deficient cells. Pb2+ treatment triggered the activation of JNK and p38, measured as the phosphorylation of JNK and p38, only in cells incubated in the Zn-deficient media. The exposure to Pb2+ (2-24 h) led to a higher AP-1 DNA-binding activity and AP-1-dependent gene transactivation, only in the Zn-deficient cells. Results show that Zn deficiency can increase the cytotoxicity of Pb2+ and the susceptibility of neurons to Pb2+-induced oxidative stress, leading to JNK and p38 phosphorylation and, subsequently, AP-1 activation.

Clusterin up-regulation following sub-lethal oxidative stress and lipid peroxidation in human neuroblastoma cells

Clusterin/apolipoprotein J is a multifunctional protein up-regulated during various pathophysiological states. Since oxidative stress plays an important role in brain aging, and in many neurodegenerative disorders, to further understand the mechanistic underpinnings of clusterin expression, in this study, we examined clusterin expression in human neuroblastoma cells under conditions of increased production of reactive oxygen species and lipid peroxidation. Specifically, we analyzed clusterin mRNA and protein levels in human neuroblastoma IMR-32 and SH-SY5Y cells following exposure to sub-lethal amounts of iron–ascorbate to induce an increase in reactive oxygen species generation and lipid peroxidation. Under such conditions, we observed a time-dependent up-regulation of clusterin protein and mRNA levels, detected by immunoblot analysis and RT-PCR, respectively. Given the known roles of clusterin, the results of the present study support the notion that an increase in clusterin expression may be a physiological defence mounted to reduce cell damage and maintain cell viability during periods of increased oxidative stress.

α-Lipoic acid and N-acetyl cysteine prevent zinc deficiency-induced activation of NF-κB and AP-1 transcription factors in human neuroblastoma IMR-32 cells

This work investigated the capacity of α-lipoic acid (LA) and N-acetyl-l-cysteine (NAC) to reduce zinc deficiency-induced oxidative stress, and prevent the activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and the cross-talk between both activated cascades through β-Transducin Repeat-containing Protein (β-TrCP). IMR-32 cells were incubated in control media or media containing variable concentrations of zinc, without or with 0.5 mM LA or 1 mM NAC. Relative to control and zinc supplemented (15 μM Zn) groups, Hydrogen peroxide (H2O2) and total oxidant cell concentrations were higher, and total glutathione concentrations were lower in the zinc deficient groups (1.5 and 5 μM Zn). Both, LA and NAC, markedly reduced the increase in cell oxidants and the reduction in glutathione concentrations in the zinc deficient cells. Consistent with this, LA and NAC prevented zinc deficiency-induced activation of the early steps of NF- κB (IκBα phosphorylation) and AP-1 [c-Jun-N-terminal kinase (JNK) and p38 phophorylation] cascades, and the high NF-κB- and AP-1-DNA binding activities in total cell extracts. Thus, LA and NAC can reduce the oxidative stress associated with zinc deficiency and the subsequent triggering of NF-κB- and AP-1-activation in neuronal cells.

A Norbergenin Derivative Inhibits Neuronal Cell Damage Induced by Tunicamycin

Several chemically synthesized compounds were examined for protective effects against the cell damage in tunicamycin-treated human neuroblastoma IMR-32 cells. Among the compounds tested, an antioxidant, Norbergenin-11-caproate (10 microM), exhibited complete protection against the cell growth inhibitory effect of tunicamycin but did not inhibit the induction of Bip/GRP78 mRNA by tunicamycin. Both norbergenin-11-caproate and alpha-tocopherol completely inhibited the production of reactive oxygen species induced by tunicamycin, however, alpha-tocopherol inhibited tunicamycin-induced cell damage only partially, even at 100 microM. These findings suggest the potential of Norbergenin-11-caproate for therapeutic application in endoplasmic reticulum (ER) stress-dependent diseases implicating a specific mechanism other than anti-oxidative one.

PHOX2B Regulates Its Own Expression by a Transcriptional Auto-regulatory Mechanism

The specification of neuronal identity is a result of interactions between the following two distinct classes of determinants: extrinsic factors that include secreted or cell membrane-associated signals in the local environment, and intrinsic factors that generally consist of ordered cascades of transcription factors. Little is known about the molecular mechanisms underlying the interplay between these extrinsic and intrinsic factors and the transcriptional processes that establish and maintain a given neuronal phenotype. Phox2b is a vertebrate homeodomain transcription factor and a well established intrinsic factor in developing autonomic ganglia, where its expression is triggered by the bone morphogenic proteins secreted by the dorsal aorta. In this study we characterized its proximal 5'-regulatory region and found that it contained five putative DNA sites that potentially bind homeodomain proteins, including PHOX2B itself. Chromatin immunoprecipitation assays showed that PHOX2B could bind its own promoter in vivo, and electromobility gel shift assays confirmed that four of the five sites could be involved in PHOX2B binding. Functional experiments demonstrated that 65% of the transcriptional activity of the PHOX2B promoter in neuroblastoma cells depends on this auto-regulatory mechanism and that all four sites were required for full self-transactivation. Our data provide a possible molecular explanation for the maintenance of PHOX2B expression in developing ganglia, in which initially its expression is triggered by bone morphogenic proteins, but may become independent of external stimuli when it reaches a certain nuclear concentration and sustains its own transcription.

A novel function of transcription factor α-Pal/NRF-1: Increasing neurite outgrowth

α-Pal/NRF-1 is a critical regulator of the promoter of human IAP/CD47 gene, a gene related to memory formation in rodents. However, its function in neurons was unknown. We found that stable or transient expression of full-length α-Pal/NRF-1 in human neuroblastoma IMR-32 cells significantly induced neurite outgrowth and increased the length of neurites both in medium containing 10% fetal bovine serum and in serum-free medium. In contrast, the dominant-negative mutant of α-Pal/NRF-1 inhibited the induction and extension of neurites. Ectopic expression of full-length α-Pal/NRF-1 also increased the induction of neurite outgrowth in primary mouse cortical neurons. The IAP antisense cDNA significantly inhibited the increase of neurite outgrowth by α-Pal/NRF-1. These findings indicate that a novel function of α-Pal/NRF-1 is to regulate neuronal differentiation, and that this function is mediated partly via its downstream IAP gene.

HIV-1 Tat Interactions with p300 and PCAF Transcriptional Coactivators Inhibit Histone Acetylation and Neurotrophin Signaling through CREB

The human immunodeficiency virus type-1 (HIV-1) infects microglia, macrophages, and astrocytes in the central nervous system (CNS) and may cause severe neurological diseases, such as AIDS-related dementias or progressive encephalopathies, as a result of CNS inflammation and neurotrophin signaling defects associated with expression of viral antigens and HIV-1 replication in the brain. The HIV Tat protein can be endocytosed by surrounding uninfected cells; interacts with transcriptional coactivators/acetyltransferases, p300/CREB-binding protein, and p300/CREB-binding protein-associated factor (PCAF); and induces neuronal apoptosis. Since nerve growth factor (NGF) receptor and brain-derived neurotrophic factor receptor signaling through CREB requires p300 and PCAF histone acetyltransferases, we sought to determine whether HIV-1 Tat coactivator interactions interfere with neurotrophin receptor signaling in neuronal cells. Here, we demonstrate that Tat-coactivator interactions inhibit NGF- and brain-derived neurotrophic factor-responsive CRE trans-activation and neurotrophin protection against apoptosis in PC12 and IMR-32 neuroblastoma cells. Purified recombinant Tat or Tat-derived synthetic peptides, spanning p300- and PCAF-binding sequences, inhibit histone H3/H4 acetylation in vitro. A Tat mutant, TatK28A/K50A, defective for binding p300 and PCAF, neither repressed NGF-responsive CRE transactivation nor inhibited histone acetylation. HIV-1 Tat interacts in PCAF complexes in post-mortem CNS tissues from donor neuro-AIDS patients, as determined by fluorescence resonance energy transfer immunoconfocal microscopy. Importantly, these findings suggest that HIV-1 Tat-coactivator interactions may contribute to neurotrophin signaling impairments and neuronal apoptosis associated with HIV-1 infections of the CNS.

Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection

Polyomavirus JC (JCV) infection causes the fatal human demyelinating disease, progressive multifocal leukoencephalopathy. Although the initial interaction of JCV with host cells occurs through direct binding of the major viral capsid protein (VP1) with cell-surface molecules possessing sialic acid, these molecules have not yet been identified. In order to isolate monoclonal antibodies which inhibit attachment of JCV, we established an immunoscreening system using virus-like particles consisting of the VP1. Using this system, among monoclonal antibodies against the cell membrane fraction from JCV-permissive human neuroblastoma IMR-32 cells, we isolated a monoclonal antibody designated as 24D2 that specifically inhibited attachment and infection of JCV to IMR-32 cells. The antibody 24D2 recognized a single molecule of around 60 kDa in molecular weight in the IMR-32 membrane fraction. Immunohistochemical staining with 24D2 demonstrated immunoreactivity in the cell membrane of JCV-permissive cell lines and glial cells of the human brain. These results suggested that the molecule recognized by 24D2 plays a role in JCV infection, and that it might participate as a receptor or a co-receptor in JCV attachment and entry into the cells.

177Lu-DOTA-lanreotide: a novel tracer as a targeted agent for tumor therapy

177Lu of specific activity ∼100–110 TBq/g and radionuclidic purity of ∼100% was obtained by irradiation of enriched Lu 2O 3 (60.6% 176Lu) target for 7 days at a thermal neutron flux of 3×10 13n/cm 2/sec. The 177Lu labeling of a macrocyclic bifunctional chelating agent viz. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) has been extensively studied. Lanreotide, [β-naphthyl-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH 2] a disulfide-linked cyclic octapeptide and a somatostatin analog, reported to bind with a wide variety of tumors expressing somatostatin receptors, was conjugated with DOTA. The peptide-BFCA conjugate was characterized with the help of high-resolution two-dimensional proton NMR spectroscopy. The 177Lu labeling of the DOTA-lanreotide conjugate has been standardized to give a radiolabeling yield of 85%. The tracer showed specific binding with A-431 human epidermoid carcinoma and IMR-32 human brain neuroblastoma cells.

Malignant blue naevus of the scalp

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Side‐Chain Length is Important for Shogaols in Protecting Neuronal Cells from β‐Amyloid Insult.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Effect of maprotiline on Ca 2+ movement in human neuroblastoma cells

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca 2+ concentrations ([Ca 2+] i) was explored by using the Ca 2+-sensitive probe fura-2. Maprotiline at concentrations greater than 100 μM caused a rapid rise in [Ca 2+] i in a concentration-dependent manner (EC 50 = 200 μM). Maprotiline-induced [Ca 2+] i rise was reduced by 50% by removal of extracellular Ca 2+. Maprotiline-induced [Ca 2+] i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. In Ca 2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2+-ATPase, caused a monophasic [Ca 2+] i rise, after which the increasing effect of maprotiline on [Ca 2+] i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca 2+] i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca 2+] i by stimulating extracellular Ca 2+ influx and also by causing intracellular Ca 2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.

Regulation of glycogen synthase kinase-3beta by products of lipid peroxidation in human neuroblastoma cells.

The potential role of 4-hydroxynonenal (HNE), a major product of membrane lipid peroxidation, in regulating glycogen synthase kinase-3beta (GSK3beta) activity was examined in human neuroblastoma IMR-32 cells. The inhibition of GSK3beta activity by HNE was observed by in vitro kinase assays with two substrates, the synthetic glycogen synthase peptide-2 and the human recombinant tau. GSK3beta activity is regulated by Ser9 (inhibitory) and Tyr216 (stimulatory) phosphorylation. By using specific activity-dependent phospho-antibodies, immunoblot analysis revealed that HNE induces an increase in phosphorylation of GSK3beta in Ser9, enhancing basal phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 2 (ERK2) signalling pathways. Ser9-GSK3beta phosphorylation induced by HNE was abolished by treatment with LY294002 or U0126, two inhibitors of PI3K/AKT and ERK pathways, respectively. These experiments provide evidence for a crucial role of the PI3K/AKT and ERK2 pathways as intracellular targets of HNE that mediate the inhibition of GSK3beta activity in regulating cellular response to HNE in viable cells under conditions in which membrane lipid peroxidation occurs. These data support a key role for GSK3beta as a mediator of the signalling pathways activated by oxidative stress, and therefore it may be included among the redox-sensitive enzymes.

α-Pal/NRF-1 Regulates the Promoter of the Human Integrin-associated Protein/CD47 Gene

Integrin-associated protein (IAP or CD47) is expressed in a variety of tissues, including the nervous system and immune system. To understand how cells control the expression of the IAP gene, we cloned the 5'-proximal region of the human IAP gene and investigated IAP promoter activity by transient transfection. RT-PCR confirmed the expression of IAP transcripts in human neuroblastoma IMR-32 and hepatoma HepG2 cells. Deletion analysis identified a core promoter of the human IAP gene located between nucleotide positions -232 and -12 relative to the translation initiation codon in these two cell lines. Site-directed mutagenesis and gel electrophoretic mobility shift assay identified a alpha-Pal/NRF-1 binding element within the IAP core promoter. Supershift assays using the alpha-Pal/NRF-1 antiserum confirmed the binding of this transcription factor on the alpha-Pal/NRF-1 site. Overexpression of the DNA binding domain of alpha-Pal/NRF-1 in cells enhanced DNA-alpha-Pal/NRF-1 binding in vitro. Furthermore, overexpression of full-length alpha-Pal/NRF-1 significantly enhanced IAP promoter activity while overexpression of dominant-negative mutant reduced promoter activity both in the cultured human cell lines and primary mouse cortical cells. These results revealed that alpha-Pal/NRF-1 is an essential transcription factor in the regulation of human IAP gene expression.

Side-chain length is important for shogaols in protecting neuronal cells from β-amyloid insult

Ten shogaols were synthesized to evaluate the importance of the side-chain length in protecting cells from βA(1-42) insult using PC12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. The compounds cell protectivity against βA insult was demonstrated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The efficacy of cell protection from βA insult by these shogaols was shown to improve as the length of the side chain increase.

Effects of 50 Hz electromagnetic fields on voltage-gated Ca 2+ channels and their role in modulation of neuroendocrine cell proliferation and death

Possible correlation between the effects of electromagnetic fields (EFs) on voltage-gated Ca 2+ channels, cell proliferation and apoptosis was investigated in neural and neuroendocrine cells. Exposure to 50 Hz EFs significantly enhanced proliferation in human neuroblastoma IMR32 (+40%) and rat pituitary GH3 cells (+38%). In IMR32 cells EF stimulation also inhibited puromycin- and H 2O 2-induced apoptosis (−22 and −33%, respectively). EF effects on proliferation and apoptosis were counteracted by Ca 2+ channel blockade. In whole-cell patch-clamp experiments 24–72 h exposure to EFs increased macroscopic Ba 2+-current density in both GH3 (+67%) and IMR32 cells (+40%). Single-channel recordings showed that gating of L and N channels was instead unaffected, thus suggesting that the observed enhancement of current density was due to increased number of voltage-gated Ca 2+ channels. Western blot analysis of plasma membrane-enriched microsomal fractions of GH3 and IMR32 cells confirmed enhanced expression of Ca 2+ channel subunit α 1 following exposure to EFs. These data provide the first direct evidence that EFs enhance the expression of voltage-gated Ca 2+ channels on plasma membrane of the exposed cells. The consequent increase in Ca 2+ influx is likely responsible for the EF-induced modulation of neuronal cell proliferation and apoptosis.

Effects of Camptothecin Conjugated to a Somatostatin Analog Vector on Growth of Tumor Cell Lines in Culture and Related Tumors in Rodents

Two CPT-SSA conjugates, JF-10-71 and JF-10-81, containing a chemically adjustable release-rate carbamate linker, have been reported previously by us to potently inhibit growth of human neuroblastoma IMR32 cells overexpressing somatostatin receptor type II (SSTR2) but are stable under buffer incubation conditions or in rat plasma. Further experiments now reveal that the conjugates performed well against many additional cell lines, particularly somatostatin receptor containing rat pancreatic CA20948 cells that were actually more sensitive to the conjugates than free camptothecin itself. JF-10-71 and JF-10-81 also were examined for their inhibitory effects on the growth of this and several other tumors transplanted into rats (CA20948) or nude mice. CA20948 tumors, known to overexpress SSTR2 and grown in Lewis rats, were treated, respectively, with nontoxic 400 nmol/kg intraperitoneal (ip) doses of JF-10-71 or JF-10-81. Also, SSTR2-positive human SCLC NCI-H69 tumors transplanted in nude mice were treated in a similar fashion. Human prostate PC-3 tumors, which do not contain high concentrations of SSTR2, also were grown in nude mice and treated with a 400 nmol/kg ip dose of JF-10-71. Both cytotoxic conjugates significantly inhibited growth of SSTR2-specific pancreatic and SCLC tumors, but JF-10-81 did not significantly affect PC-3 tumor growth. These experimental results suggested that CPT-SSA conjugates can effectively target and kill tumor cells growing in vivo and that the effect is mediated by somatostatin receptors resulting in either release of camptothecin at the cell surface or, more likely, after receptor-mediated cellular internalization.

Effects of MYCN antisense oligonucleotide administration on tumorigenesis in a murine model of neuroblastoma.

Human MYCN (hMYCN) oncogene amplification is a powerful predictor of treatment failure in childhood neuroblastoma, and dysregulation of hMYCN protein expression appears to be critically involved in the pathogenesis of this disease. We used hMYCN antisense (AS) oligonucleotides to investigate, both in vitro and in vivo, the therapeutic potential of inhibiting hMYCN expression. We transiently transfected human neuroblastoma IMR-32 cells, which have an amplified hMYCN gene, with fluorescently labeled hMYCN AS or scrambled (SCR) control oligonucleotides and used fluorescence-activated cell sorting to enrich for cell populations containing different levels of the oligonucleotides. We used fluorescence immunocytochemistry or reverse transcription polymerase chain reaction to assay gene expression levels and trypan blue exclusion to assay growth inhibition in the cell populations. We examined the effects of continuous treatment for 6 weeks with AS or SCR oligonucleotides via subcutaneously implanted microosmotic pumps on tumor growth in a transgenic mouse model of hMYCN-induced neuroblastoma (n = 20 mice per group). All statistical tests were two-sided. IMR-32 cells treated with AS oligonucleotides had approximately half as much hMYCN protein and cell proliferation as either SCR oligonucleotide-transfected or mock-transfected controls; the differences were statistically significant. Transgenic mice treated with AS oligonucleotides had lower tumor incidence and statistically significantly lower tumor mass than SCR-treated or untreated control mice. Compared with control treatments, AS oligonucleotide treatment in vitro and in vivo was associated with decreased expression of hMYCN and putative hMYCN target genes but not with that of closely related genes. Several AS oligonucleotide-treated mice developed tumors contralateral to the site of oligonucleotide administration, whereas SCR oligonucleotide-treated or untreated mice displayed bilateral tumor growth. Decreased expression of hMYCN protein is achievable with the use of AS oligonucleotide treatment, even in the presence of hMYCN oncogene amplification. Antisense strategies targeting the hMYCN oncogene in vivo decrease mouse neuroblastoma tumorigenesis. Investigation of their clinical effect in children with neuroblastoma is warranted.