metallothionein and Glioma

Although metallothionein-3 (MT3), a brain-enriched form of metallothioneins, has been linked to Alzheimer's disease, little is known regarding the role of MT3 in glioma. As MT3 plays a role in autophagy in astrocytes, here, we investigated its role in irradiated glioma cells. Irradiation increased autophagy flux in GL261 glioma cells as evidenced by increased levels of LC3-II but decreased levels of p62 (SQSTM1). Indicating that autophagy plays a cytoprotective role in glioma cell survival following irradiation, measures inhibiting autophagy flux at various steps decreased their clonogenic survival of irradiated GL261 as well as SF295 and U251 glioma cells. Knockdown of MT3 with siRNA in irradiated glioma cells induced arrested autophagy, and decreased cell survival. At the same time, the accumulation of labile zinc in lysosomes was markedly attenuated by MT3 knockdown. Indicating that such zinc accumulation was important in autophagy flux, chelation of zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), induced arrested autophagy in and reduced survival of GL261 cells following irradiation. Suggesting a possible mechanism for arrested autophagy, MT3 knockdown and zinc chelation were found to impair lysosomal acidification. Since autophagy flux plays a cytoprotective role in irradiated glioma cells, present results suggest that MT3 and zinc may be regarded as possible therapeutic targets to sensitize glioma cells to ionizing radiation therapy.

Topics: Animals; Autophagy; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Chelating Agents; Ethylenediamines; Gene Knockdown Techniques; Glioma; Humans; Lysosomes; Metallothionein; Metallothionein 3; Mice; Nerve Tissue Proteins; Photons; Radiation Tolerance; RNA, Small Interfering; Zinc

Gliomas are the most common primary brain tumors in adults and a significant cause of cancer-related mortality. A 9-gene signature was identified as a novel prognostic model reflecting survival situation obviously in gliomas.. To identify an mRNA expression signature to improve outcome prediction for patients with different glioma grades.. We used whole-genome mRNA expression microarray data of 220 glioma samples of all grades from the Chinese Glioma Genome Atlas (CGGA) database (http://www.cgga.org.cn) as a discovery set and data from Rembrandt and GSE16011 for validation sets. Data from every single grade were analyzed by the Kaplan-Meier method with a two-sided log-rank test. Univariate Cox regression and linear risk score formula were applied to derive a gene signature with better prognostic performance. We found that patients who had high risk score according to the signature had poor overall survival compared with patients who had low risk score. Highly expressed genes in the high-risk group were analyzed by gene ontology (GO) and gene set variation analysis (GSVA). As a result, the reason for the divisibility of gliomas was likely due to cell life processes and adhesion.. This 9-gene-signature prediction model provided a more accurate predictor of prognosis that denoted patients with high risk score have poor outcome. Moreover, these risk models based on defined molecular profiles showed the considerable prospect in personalized cancer management.

Topics: Brain Neoplasms; Cell Cycle Proteins; DNA-Binding Proteins; Endosomal Sorting Complexes Required for Transport; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Variation; Genomic Library; Glioma; Histone Deacetylases; Humans; Inhibitor of Apoptosis Proteins; Kaplan-Meier Estimate; Membrane Proteins; Metallothionein; Microarray Analysis; Microtubule-Associated Proteins; Nuclear Proteins; rab1 GTP-Binding Proteins; Repressor Proteins; RNA, Messenger; Survivin; TEA Domain Transcription Factors; Transcription Factors; Tubulin

Metallothionein 1E (MT1E) has been found to be highly expressed in motile cell lines. We investigated whether MT1E actually modulates the migration and invasion of human glioma cell lines and the types of factors that have an effect on MT1E. RNA differential display was performed using Genefishing™ technology in the human glioma cell lines U343MG-A, U87MG and U87MG-10'; the results were validated by RT-PCR and northern blot analysis, in order to detect possible genetic changes as the determining factors for migration ability in malignant glioma. MT1E was identified in U87MG, a highly motile cell line. The migration and invasion abilities of human glioma cell lines, and MT1E transfectants were investigated using simple scratch testing and Matrigel invasion assays. Morphological and cytoskeletal (actin, vimentin) changes were documented by light and confocal microscopy. The expression of MT1E in four glioma cell lines was assessed by RT-PCR and western blotting. In addition, the effects of MT1E on the activity of the NF-κB p50/p65 transcription factor, MMP-2 and -9 were examined by western blotting and zymography. The endogenous MT1E expression in the human glioma cell lines was statistically correlated with their migratory abilities and invasion. The U87-MT-AS cells became more round and had decreased stress fibers, compared with the U87MG cells. Endogenous MT1E expression in the four human glioma cell lines was directly correlated with migration. Two antisense MT1E-transfected cell lines showed decreased NF-κB p50 translocation into the nucleus, which led to decreased activity of MMP-9 in conditioned media. It may be postulated that MT1E can enhance the migration and invasion of human glioma cells by inducing MMP-9 inactivation via the upregulation of NF-κB p50.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Glioma; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metallothionein; Neoplasm Invasiveness; NF-kappa B p50 Subunit; Transcriptional Activation; Transfection

Metallothioneins (MT) are low-molecular weight, cysteine-rich metal-binding proteins. MT play a role in the homeostasis of essential metals such as zinc (Zn) and copper (Cu), detoxification of toxic metals such as cadmium (Cd) and protection against oxidative stress. In this study, we examined the expression of MT in HaCaT and C6 cells as a strategy to enhance protection against Cd-mediated toxicity. At basal level, HaCaT cells showed higher MT level than C6 cells which could explain the resistance of HaCaT cells. Western blot showed that C6 cells treated with 20micromol/L Cd for 24h did not express any MT. MT were initially expressed in the cytoplasmic or periplasmic compartment and were then translocated in the nucleus after 24h treatment by Cd both in HaCaT and C6 cells. In addition, the cell treatment with Cd was followed by an increase in the cellular zinc level but the electrophoretic mobility shift assay (EMSA) experiment did not show any translocation of metal-responsive transcription factor-1 (MTF-1) to the nucleus of HaCaT cells. These absence of translocation could be due to the presence of MT in these cells at the basal state. The translocation study in HaCaT cells suggested that the MT translocation in the nucleus was greater than observed in C6 cells. The latter observation could explain HaCaT cells resistance to Cd concentrations up to 50micromol/L. Our results suggested that the C6 cell sensitivity was correlated with the decrease in MT level at 20micromol/L Cd occurring after the transcription of MT gene.

Topics: Analysis of Variance; Animals; Cadmium; Cell Death; Cell Line, Transformed; Cell Line, Tumor; DNA; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Glioma; Humans; Keratinocytes; Metallothionein; Promoter Regions, Genetic; Protein Transport; Rats; Transcription Factor MTF-1; Transcription Factors; Zinc

Metallothioneins (MT) are common proteins in animal tissues. These proteins take part in the homeostasis of the ions of the metals which are necessary for the proper metabolism of the organism (zinc, copper), biosynthesis regulation and zincprotein activity (for example the activity of the zinc-dependant transcription factors) and they also take part in the detoxication of the tissue from toxic metals. Apart from these, they also protect the tissue from reactive oxygen species, radiation, electrophilic pharmacological agents used in the cancer therapy and the mutagens. The aim of this work was to obtain cellular sub-fractions of brain tumors and to separate these proteins by SDS-polyacrylamide gel electrophoresis and Western Blotting technique and to determine the level of metallothioneins and to determine the level of metallthioneins in the cellular sub-fraction. The experimental materials were the brain neoplastic tissues resected during neurosurgical procedures. The brain tumors were divided into two groups; astrocytoma G-2 and malignant gliomas (astrocytoma GM-4, glioblastoma multiforme). The cellular fractions of tumour tissues were obtained according to Clark and Nicklas and Chauveau et al. methods. The level of the metallothioneins was determined by the cadmium-hemoglobin affinity assay using the cadmium isotope (109Cd). By performing protein dissection on polyacrylamide gel (PAGE) and applying immunoidentifying technique we proved the presence of metallothioneins in all the cell sub-fractions. In GM-4 patients the total MT level increased by 12.06% whereas in the cytosol sub-fraction it increased by 17.02% in comparison with astrocytoma G-2 patients. In the mitochondrial sub-fraction the level increased by 49.09% while in the nuclear and microsomal sub-fractions the increase was by 22.38 and 8.24%, respectively.

Topics: Astrocytoma; Blotting, Western; Brain Neoplasms; Electrophoresis, Polyacrylamide Gel; Glioma; Humans; Metallothionein; Subcellular Fractions

Metallothioneins (MTs) are metal binding proteins overexpressed in various human neoplasms which are associated with resistance to cytotoxic drugs. A series of 156 archival human brain tumours were investigated immunohistochemically for expression of MTs; these included 10 low-grade gliomas, 44 high-grade gliomas, 98 meningeal tumours (19 classical, 30 atypical, 38 anaplastic meningiomas, and 11 haemangiopericytomas or papillary meningiomas), and 4 other tumours. Low-grade gliomas showed heterogeneous MT expression; 32 high-grade gliomas (72.7%) showed MT expression of more than 25% of tumour cells without statistically significant differences between first operations and recurrent tumours. In 2 glioblastomas, the presence of MT was confirmed by Western blotting. The extent of MT immunoexpression showed a statistically significant inverse relationship to the degree of p53 immunoreactivity. In meningiomas, a tendency to a higher percentage of MT-expressing cells was observed from classical over atypical to anaplastic meningiomas, but these differences were not statistically significant. In conclusion, MT expression is present in a significant portion of, especially malignant, brain tumours and might be involved in their poor response to antineoplastic drugs.

Topics: Adult; Aged; Blotting, Western; Brain Neoplasms; Child; Child, Preschool; Female; Glioma; Humans; Immunohistochemistry; Male; Meningioma; Metallothionein; Middle Aged; Neoplasm Staging; Tumor Suppressor Protein p53

The C6 rat glioma cell line is response to glucocorticoid hormones. C6 variants that are hyper-responsive (ST1) and resistant (P7) to hormone treatment have been derived previously. Glucocorticoid treatment of ST1 cells leads to complete reversion of the transformed phenotype and loss of tumorigenic potential. Production of C type retrovirus particles is also induced by glucocorticoids in ST1 cells. Cloning of the genes regulated by glucocorticoids in this cell system was used here as a strategy to uncover the gene products involved in the transformed-to-normal phenotypic change. Construction of a cDNA library from glucocorticoid-treated ST1 cells and screening by differential hybridization resulted in the isolation of three cellular sequences that code for rat metallothioneins (C27 and C41) and alpha 1-acid glycoprotein (C36). Northern blot analysis revealed that expression of these genes was dramatically induced by hydrocortisone in ST1 but not in P7 cells. Viral genomic RNA was used to isolate and characterize retrovirus-related sequences that could also be responsible for the phenotypic reversion phenomenon.

Topics: Animals; Blotting, Northern; Cell Line, Transformed; Cloning, Molecular; Glioma; Hydrocortisone; In Situ Hybridization; Metallothionein; Orosomucoid; Phenotype; Rats; Retroviridae; RNA; Tumor Cells, Cultured; Viral Envelope Proteins; Virus Activation

The mRNA content of the brain-specific metallothionein-III (MT-III) protein was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in two transformed glial cell lines: rat C6-glial and human U-373 MG cells. Low levels of MT-III mRNA were detected compared to a high expression of this mRNA in primary cultures of rat astrocytes. C6-glial cell lines stably transfected with a human 5-HT1D alpha or 5-HT1D beta receptor gene showed a decrease (87 to 93%) in basal [3H]thymidine incorporation, whereas their MT-III mRNA content was more than 30-fold increased compared to plasmid transfected C6-glial cells. The inverse proportion between mitogenic activity and MT-III mRNA content suggests that MT-III may act as a growth inhibitory factor in rat C6-glial cells.

Topics: Actins; Animals; Animals, Newborn; Astrocytes; Base Sequence; Brain; Cell Line; Cells, Cultured; DNA Primers; DNA Replication; Gene Expression; Glioma; Humans; Kinetics; Metallothionein; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Receptor, Serotonin, 5-HT1B; Receptors, Serotonin; Recombinant Proteins; RNA, Messenger; Thymidine; Transfection; Tumor Cells, Cultured