metallothionein and Neuroblastoma

Mangiferin (MGN), a C-glucosylxanthone was investigated for its ability to protect against methylmercury (MeHg) induced neurotoxicity by employing IMR-32 (human neuroblastoma) cell line. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and clonogenic cell survival assays confirmed the efficacy of MGN supplementation in attenuating MeHg-induced cytotoxicity. Pre-treatment with MGN significantly (p<0.01) inhibited MeHg-induced DNA damage (micronuclei, olive tail moment and % tail DNA) thereby demonstrating MGN's antigenotoxic potential. Also, pre-treatment with MGN significantly reduced MeHg-induced oxidative stress, intra-cellular Ca(2+) influx and inhibited depolarization of mitochondrial membrane. MGN pre-treated cells demonstrated a significant (p<0.05) increase in the GSH and GST levels followed by a significant (p<0.05) decrease in malondialdehyde (MDA) formation. In addition, inhibition of MeHg induced apoptotic cell death by MGN was demonstrated by microscopic, Annexin-V FITC and DNA fragmentation assays and further confirmed by western blot analysis. The present findings indicated the protective effect of MGN against MeHg induced toxicity, which may be attributed to its anti-genotoxic, anti-apoptotic and anti-lipid peroxidative potential plausibly because of its free radical scavenging ability, which reduced the oxidative stress and in turn facilitated the down-regulation of mitochondrial apoptotic signalling pathways.

Topics: Annexin A5; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Cell Line, Tumor; Cell Nucleus; Cell Survival; Comet Assay; Cytochromes c; Cytokinesis; Cytoprotection; DNA Damage; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Interactions; Free Radical Scavengers; Humans; Inhibitory Concentration 50; Intracellular Space; Membrane Potential, Mitochondrial; Metallothionein; Methylmercury Compounds; Micronucleus Tests; Necrosis; Neuroblastoma; NF-E2-Related Factor 2; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tubulin; Tubulin Modulators; Tumor Stem Cell Assay; Xanthones

Tumour markers are substances produced by malignant cells or by the organism as a response to cancer development. Determination of their levels can, therefore, be used to monitor the risk, presence and prognosis of a cancer disease or to monitor the therapeutic response or early detection of residual disease. Time-consuming imaging methods, examination of cerebrospinal fluid or tumour tissue and assays for hormones and tumour markers have been used for cancer diagnosis. However, no specific marker for diagnosis of childhood solid tumours has been discovered yet. In this study, metallothionein (MT) was evaluated as a prospective marker for such diseases. Serum metallothionein levels of patients with childhood solid tumours were determined using differential pulse voltammetry - Brdicka reaction. A more than 5-fold increase in the amount of metallothionein was found in sera of patients suffering from cancer disease, compared with those in sera of healthy donors. The average metallothionein level in the sera of healthy volunteers was 0.5 ± 0.2 μmol · dm⁻³ and was significantly different (p<0.05, determined using the Schefe test) from the average MT level found in serum samples of patients suffering from childhood solid tumours (3.4 ± 0.8 μmol · dm⁻³). Results found in this work indicate that the MT level in blood serum can be considered as a promising marker for diagnostics, prognosis and estimation of therapy efficiency of childhood tumours.

Topics: Biomarkers, Tumor; Blotting, Western; Child; Electrochemistry; Humans; Medulloblastoma; Metallothionein; Neoplasms; Neuroblastoma; Osteosarcoma; Risk Factors; Sarcoma, Ewing

The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug.

Topics: Animals; Antineoplastic Agents; Carboplatin; Cell Line, Tumor; Cisplatin; Humans; Metallothionein; Models, Biological; Neuroblastoma; Rabbits; Retinoblastoma

To identify markers of response to therapy in neuroblastic tumors.. A total of 58 patients with neuroblastic tumor (38 neuroblastomas, 13 ganglioneuroblastomas and 7 ganglioneuromas) were included in the study. TP53, BCL-2, p21Waf1/Cip1 and metallothionein were included as a biologic approach to tumor differentiation, response to therapy and prognosis.. Patients who died of disease had the following immunophenotype: BCL-2 (9 of 10), nuclear TP53 (7 of 10) and metallothionein (7 of 10). TP-53 expression was related to clinical stage (p = 0.062) and disease outcome (p = 0.0218). All patients in whom treatment failed expressed metallothionein (3 of 3).. TP53, BCL-2, p21Waf1/Cip1 and metallothionein had limited value reflecting tumor maturation (differentiation) or predicting response to therapy. Only nuclear TP53 accumulation may be relevant in patient's prognosis.

Topics: Adolescent; Adult; Biomarkers, Tumor; Cell Differentiation; Child; Cyclin-Dependent Kinase Inhibitor p21; Female; Ganglioneuroblastoma; Humans; Male; Metallothionein; Neuroblastoma; Prognosis; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

In this study, we examined the effect of Cilostazol to induce metallothionein (MT) in vivo and in vitro. Intraperitoneal injection of Cilostazol increased the expression of both MT-1 and MT-2 mRNA and total MT protein in the mouse liver. Cilostazol also augmented MT-1 mRNA levels in the murine brain. In vitro exposure to Cilostazol significantly augmented intracellular MT protein levels in cultured human brain microvascular endothelial cells (HBMEC) and in the neuroblastoma cell line IMR32. Taken together, these findings suggest that Cilostazol is an inducer of MT in the murine liver and brain, and that it has the potential to directly induce MT in cells. The contribution of the anti-oxidative effect of MT to the anti-stroke effect of Cilostazol was discussed.

Topics: Animals; Brain Chemistry; Cell Line, Tumor; Cilostazol; Dose-Response Relationship, Drug; Humans; Injections, Intraperitoneal; Male; Metallothionein; Mice; Mice, Inbred C57BL; Neuroblastoma; Neurons; Neuroprotective Agents; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazoles; Time Factors

Regional distribution of coenzyme Q10 and mitochondrial complex-1 activity were estimated in the brains of control-(C57BL/6), metallothionein knock out-, metallothionein transgenic-, and homozygous weaver mutant mice; and human dopaminergic (SK-N-SH) cells with a primary objective to determine the neuroprotective potential of coenzyme Q10 in Parkinson's disease. Complex-1 activity as well as coenzyme Q10 were significantly higher in the cerebral cortex as compared to the striatum in all the genotypes examined. Complex-1 activity and coenzyme Q10 were significantly reduced in weaver mutant mice and metallothionein knock out mice, but were significantly increased in metallothionein transgenic mice. The reduced complex-1 activity and 18F-DOPA uptake occurred concomitantly with negligible differences in the coenzyme Q10 between in the cerebral cortex and striatum of weaver mutant mice. Administration of coenzyme Q10 increased complex-1 activity and partially improved motoric performance in weaver mutant mice. Direct exposure of rotenone also reduced coenzyme Q10, complex-1 activity, and mitochondrial membrane potential in SK-N-SH cells. Rotenone-induced down-regulation of complex-1 activity was attenuated by coenzyme Q10 treatment, suggesting that complex-1 may be down regulated due to depletion of coenzyme Q10 in the brain. Therefore, metallothionein-induced coenzyme Q10 synthesis may provide neuroprotection by augmenting mitochondrial complex-1 activity in Parkinson's disease.

Topics: Analysis of Variance; Animals; Brain; Cell Line, Tumor; Chromatography, High Pressure Liquid; Coenzymes; Dihydroxyphenylalanine; Disease Models, Animal; Electron Transport Complex I; Fluorine Radioisotopes; Humans; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Mice, Transgenic; Neuroblastoma; Neuroprotective Agents; Parkinson Disease; Positron-Emission Tomography; Statistics as Topic; Tissue Distribution; Ubiquinone

Metallothionein-3(MT-3), also known as growth inhibitory factor (GIF), is predominantly expressed in central nervous system (CNS). It belongs to the family of metallothionein(MT) but has several unique properties that are not shared by other family members such as MT-1/MT-2. In the past few years, MT-3 had been postulated to be a multipurpose protein which could play important neuromodulatory and neuroprotective roles in CNS besides the common roles of MTs. However, the primary function of MT-3 and the mechanism underlying its multiple functions were not elucidated so far. In present study, human neuroblastoma cell line SH-SY5Y was employed to study the overall cellular protein changes induced by transient transfection of MT-3 gene, based on comparative proteome analysis. Averagely about 750 spots were visualized by Coomassie staining in one 2D gel, in which 17 proteins were shown to display significant and reproducible changes by semiquantitative analysis with ImageMaster 2D Elite software. Among them, 12 proteins were up-regulated while other 5 proteins were down-regulated. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 10 proteins were further identified to be zinc finger protein, glutamate transporter, and enhancer protein, etc., which were involved in several important pathways regulating the functions of central nervous system. The results showed that MT-3 might exert its unique functions by regulating the expression of these proteins.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Transport System X-AG; Apoptosis Regulatory Proteins; Cell Line, Tumor; DNA-Binding Proteins; Electrophoresis, Gel, Two-Dimensional; Gene Expression Profiling; Humans; Intracellular Signaling Peptides and Proteins; Metallothionein; Metallothionein 3; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Proteome; Proteomics; Recombinant Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transfection; Zinc Fingers

1. A class of compounds, 9-aminoacridines, have long been known to be reversible inhibitors of acetylcholinesterase (AChE-EC 3.1.1.7), the most familiar of which is 9-amino-1,2,3,4-tetrahydroacridine (Tacrine). 2. A novel aminoacridine was synthesised: -2-tertiary-butyl-9-amino-1,2,3,4- tetrahydroacridine (2tBuTHA). 3. In vitro comparisons of the acetylcholinesterase inhibitory potential and neurotoxicity compared to Tacrine were performed using a chemically differentiated neuroblastoma cell line (Neuro 2A). 2tBuTHA, but not Tacrine, was cytotoxic to the neural cell following 20 h exposure, despite being the least potent AChE inhibitor (IC80 AChE 12.53 microM +/- 1.14 s.e.m., Neutral Red Uptake IC50 9.53 microM +/- 0.98 s.e.m., MTT Reduction IC80 14.6 microM +/- 1.43 s.e.m.). 4. In vivo studies used a novel application of a five arm radial maze to assess neuropharmacological effects on working memory in control and Scopolamine (1 mg kg-1 i.p.) treated mice. There was an impairment of short term cognitive function with 2tBuTHA (15 mg kg-1 i.p.), but not Tacrine (10 mg kg-1 i.p.) which improved the Scopolamine deficit as expected. 5. This combined in vitro and in vivo data infers a neurotoxic property for the novel compound 2tBuTHA, a close structural analogue of Tacrine.

Topics: Aminoacridines; Analysis of Variance; Animals; Behavior, Animal; Cholinesterase Inhibitors; Exploratory Behavior; Memory; Metallothionein; Mice; Neuroblastoma; Neurotoxins; Neutral Red; Oxidation-Reduction; Scopolamine; Structure-Activity Relationship; Tacrine; Tumor Cells, Cultured

Since immunohistochemical studies indicated the presence of interleukin-6 in the cortices of patients with Alzheimer's disease, we were interested in the eventual biological effects of this cytokine on neuronal cells. We found that interleukin-6 and interleukin-1 induced metallothionein expression in a human neuronal (SH-SY5Y neuroblastoma) cell line. In contrast to metallothionein, amyloid precursor protein expression was unaffected by both cytokines. When searching in the same cell line for the expression of the classical 80-kDa interleukin-6 binding protein, which is part of the dimeric interleukin-6 receptor, we were unable to detect the respective mRNA. Our findings either indicate that the interleukin-6 receptor in these cells is expressed in extremely low levels or that interleukin-6 may act upon neuronal cells via a different, yet unknown neuronal receptor.

Topics: Amyloid beta-Protein Precursor; Base Sequence; DNA; Humans; Interleukin-1; Interleukin-6; Metallothionein; Molecular Probes; Molecular Sequence Data; Neuroblastoma; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Immunologic; Receptors, Interleukin-6; Tumor Cells, Cultured

We have recently succeeded in producing transgenic mice carrying a hybrid gene consisting of mouse metallothionein promoter-enhancer and the ret oncogene (MT/ret). (Iwamoto et al., 1991b). A retroperitoneal tumour developed in one of 17 MT/ret transgenic founder mice. Histological analysis revealed that the tumour consisted of undifferentiated neuroblasts and differentiated ganglion cells, the latter of which were strongly positive for neuron specific enolase. Expression of the ret transgene was observed at high levels in RNA from the tumour, but not in those of other normal tissues. In addition, a 100kDa ret protein was detected in the cell lysate of the tumour. Taken together with our previous data, these results suggest a possible role for the ret oncogene in the proliferation of neural crest cells.

Topics: Animals; Cloning, Molecular; Drosophila Proteins; Male; Metallothionein; Mice; Mice, Transgenic; Neuroblastoma; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases; Retroperitoneal Neoplasms

Growth factor-dependent neurons die when they are deprived of their specific growth factor. This "programmed" cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD.

Topics: Animals; Apoptosis; Cell Survival; DNA; Flow Cytometry; Ganglia, Spinal; Hybrid Cells; Metallothionein; Methionine; Mice; Neuroblastoma; Oxidation-Reduction; Rats; Sulfur Radioisotopes

Administration of ethanol induces the synthesis of hepatic metallothionein and metallothionein mRNA in the liver but not in the brain. Furthermore, ethyl alcohol, methyl alcohol and isopropyl alcohol enhance the synthesis of metallothionein in Chang cells but not in neuroblastoma IMR-32 cells in culture. The results of this study are interpreted to suggest that the mechanisms of synthesis of metallothionein and the utilization of essential metal nutrients in the brain and peripheral tissues are not identical.

Topics: 1-Propanol; Animals; Brain; Cadmium; Cycloheximide; Ethanol; Humans; Liver; Male; Metallothionein; Methanol; Neuroblastoma; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tumor Cells, Cultured; Zinc

Since cadmium exposure results in neuropathological alterations in central nervous system, we investigated the effects of cadmium on the gene expression of neuroblastoma (GOTO) cells. We observed an increase in mRNA levels of heat-shock protein (hsp) 70, hsp 90, hsp 32 and metallothionein after treatment of GOTO cells with cadmium, although the time courses of the changes of individual mRNA of the heat-shock proteins and metallothionein were somewhat different from each other. An accumulation of N-myc and multidrug-resistance gene (MDR1) mRNA was detected in the presence of cadmium. This is contrary to the previous report, in which an inverse correlation between the expression of MDR1 gene and N-myc oncogene in human neuroblastoma had been described. However, the increase of N-myc and MDR1 mRNA in the present study is not likely due to the loss of regulatory mechanism of these genes by cytotoxic effects of cadmium, because active protective mechanisms such as heat-shock proteins and metallothionein could be induced under these conditions.

Topics: Cadmium; Drug Resistance; Gene Expression; Genes; Genes, myc; Heat-Shock Proteins; Humans; Metallothionein; Neuroblastoma; RNA, Messenger; Tumor Cells, Cultured

Topics: Amino Acids; Animals; Brain; Brain Chemistry; Carrier Proteins; Cattle; Cell Line; Cerebral Ventricles; Chromatography, Gel; Chromatography, High Pressure Liquid; Injections, Intraventricular; Metalloproteins; Metallothionein; Models, Biological; Neuroblastoma; Pineal Gland; Rats; Receptors, N-Methyl-D-Aspartate; Sulfates; Zinc; Zinc Sulfate

Metallothioneins are a class of cysteine-rich and low molecular weight, metal-binding proteins that are inducible by a wide variety of agents, including metal ions, such as cadmium and zinc, glucocorticoid hormones, interferon, and tumor promoters. In an effort to delineate the regulation of the synthesis of the recently identified brain metallothionein-like protein, a study was undertaken to compare the induction of metallothionein in human neuroblastoma IMR-32 cells by zinc, cadmium, and dexamethasone using the human Chang liver cells as a control. Both cadmium (1 microM) and zinc (100 microM) significantly enhanced the incorporation of [35S]cysteine into metallothioneins isolated from both neuroblastoma and Chang liver cells. Dexamethasone in concentrations of 10 microM stimulated the synthesis of metallothionein in the Chang cells, whereas it had no effects on the synthesis of metallothionein in the neuroblastoma cells at concentrations ranging from 2.5--100 microM. The degree of stimulation of metallothionein synthesis in the Chang cells by cadmium and zinc was significantly higher than seen in neuroblastoma cells. The neuroblastoma IMR-32 exhibited less tolerance to the toxicity of both cadmium and zinc than the Chang cells, which may correlate with the inherent ability of these ions to induce metallothioneins in these dissimilar cells. The results of these studies are interpreted to indicate that the factors regulating the synthesis of metallothioneins in the Chang and neuroblastoma cells are not identical, suggesting also of the presence of dissimilar regulatory mechanisms in the liver and brain.

Topics: Animals; Cadmium; Dexamethasone; Liver; Male; Metallothionein; Neuroblastoma; Rats; Rats, Inbred Strains; Sulfur Radioisotopes; Tumor Cells, Cultured; Zinc

Eighteen cDNAs, cloned from interferon-treated T98G neuroblastoma cells, correspond to seven different mRNAs induced up to 40-fold by interferon. One codes for metallothionein II and another for a class I HLA. The others do not code for proteins of known sequence. In the continued presence of interferon, accumulation of the mRNAs continues for about 1 day but ceases whenever interferon is removed. Once induced, the mRNAs are stable. Synthesis of new proteins is not required for induction. The rate of transcription of one of the genes doubles 5 min after treatment with interferon and reaches a maximum by 60 min. This rate begins to fall after 4-6 hr, reaching the uninduced level by 8-12 hr. Since the mRNA continues to accumulate after 8-12 hr, posttranscriptional events must also play a role in increasing its level.

Topics: Animals; Cell Line; Cell Nucleus; Cloning, Molecular; Cycloheximide; DNA; Genes; HLA Antigens; Interferon Type I; Kinetics; Metallothionein; Mice; Neuroblastoma; Nucleic Acid Hybridization; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcription, Genetic