lithium-chloride and Glioma

Tripartite motif 47 (TRIM47), a member of the TRIM protein family, plays a crucial role in tumor development and progression. However, the role of TRIM47 in glioma has not been investigated. In the present study, we investigated the expression of TRIM47 in glioma and explored the role of TRIM47 in glioma proliferation and migration both in vitro and in vivo. Our results showed that TRIM47 expression was significantly increased in glioma tissues compared to the normal brain tissues. Knockdown of TRIM47 in U87 and U251 cells inhibited cell proliferation, as well as cell migration and invasion. TRIM47 knockdown caused significant increase in E-cadherin expression and remarkable decrease in N-cadherin and vimentin expressions in both U87 and U251 cells. In vivo assay proved that knockdown of TRIM47 prevented tumor growth of glioma. Furthermore, TRIM47 silencing significantly inhibited the activation of Wnt/β-catenin pathway. Additionally, treatment with LiCl reversed the inhibitory effects of TRIM47 knockdown on cell proliferation and migration in U87 cells. In conclusion, these findings indicated that knockdown of TRIM47 suppressed cell proliferation and metastasis of glioma both in vitro and in vivo. TRIM47 exerted an oncogenic role in glioma and might be a therapeutic target for the treatment of glioma.

Topics: Animals; Brain Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; Humans; Lithium Chloride; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Transplantation; Nuclear Proteins; Up-Regulation; Wnt Signaling Pathway

Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed.. Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays.. All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging.. Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Movement; Child; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Glioma; Glycogen Synthase Kinase 3; Humans; Indoles; Lithium Chloride; Molecular Targeted Therapy; Neoplasm Invasiveness; Oximes; Protein Kinase Inhibitors; Spheroids, Cellular

The gene encoding isocitrate dehydrogenase (IDH) is somatically mutated predominantly in secondary glioblastoma multiforme. Mutations of IDH1 and IDH2 lead to simultaneous loss and gain of activities in the production of α-ketoglutarate and 2-hydroxyglutarate, respectively. Lithium chloride was recently proved efficient in inhibiting glioma cell migration. The mechanism of lithium chloride on C6 glioma cells harboring IDH2 mutation has not been studied. Here, we found lithium chloride induced inhibitive effects on cell proliferation of both C6 glioma cells with and without IDH2 mutation, although IDH2 mutation increased the stability of HIF-1α. GSK-3β could be phosphorylated at Ser9 and its activity was inhibited when C6 glioma cells were treated by lithium chloride. The degree of phosphorylation in IDH2(R172G) treatment group was lower than that as compared to the control and IDH2 treatment groups. At the same time, the accumulation of β-catenin in C6 cell nucleus was decreased. Moreover, although the β-catenin and HIF-1α increased the secretion of metalloproteinase-2,-9 in C6 glioma cells harboring IDH2 mutation, the migration potential of lithium chloride-treated C6 glioma cells harboring the IDH2 and its mutant was uniform. These results indicated lithium chloride could decrease the proliferation and migration potential of C6 glioma cells harboring IDH2 mutation.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Glioma; Glutarates; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Isocitrate Dehydrogenase; Ketoglutaric Acids; Lithium Chloride; Mutation

Therapies for high-grade gliomas (HHG) that have strong tendency of infiltration and resistance to chemotherapies are currently unavailable. Here, we report that lower-dose combination therapy of Buthus martensii Karsch (BmK) CT, a type of scorpion toxin peptide, and LiCl, clinically used as mood stabilizer, could synergistically inhibit the migration, invasion and proliferation of C6 glioma cells. The decreased invasiveness of C6 glioma cells was accompanied by inhibited activation, catalytic activity and/or expression of matrix metalloproteinase-2. Moreover, TOPfalsh luciferase reporter and immunofluorescence staining showed altered localization pattern of β-catenin at the leading edge of 2D scratch. Our results suggested that the combination treatment of lithium and BmK CT may constitute a novel and potential strategy for HHG therapy.

Topics: Antineoplastic Agents; beta Catenin; Cell Line, Tumor; Drug Synergism; Glioma; Humans; Lithium Chloride; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Organ Culture Techniques; Scorpion Venoms

Gliomas are aggressive brain tumours that, despite advances in multimodal therapies, continue to portend a dismal prognosis. Glioblastoma multiforme (GBM) represents the most aggressive glioma and patients have a median survival of 14 months, even with the best available treatments. The phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3 beta (GSK-3β) and Wnt/β-catenin pathways are dysregulated in a number of cancers, and these two pathways share a common node protein, GSK-3β. This protein is responsible for the regulation/degradation of β-catenin, which reduces β-catenin's translocation to the nucleus and influences the subsequent transcription of oncogenes. The non-specific small-molecule GSK-3β inhibitor, lithium chloride (LiCl), and the specific Akt inhibitor, AktX, were used to treat U87MG and U87MG.Δ2-7 human glioma cell lines. LiCl treatment significantly affected cell morphology of U87MG and U87MG.Δ2-7 cells, while also increasing levels of phospho-GSK-3β in a dose-dependent manner. Increased cell proliferation was observed at low-to-mid LiCl concentrations as determined by MTT cell growth assays. Treatment of U87MG and U87MG.Δ2-7 cells with AktX resulted in reduced levels of phospho-GSK-3β through its inhibition of Akt, in addition to decreased levels of phosphorylated (active) Akt in a dose-dependent fashion. We have shown in this study that GSK-3β regulation by phosphorylation is important for cell morphology and growth, and that LiCl enhances growth of U87MG and U87MG.Δ2-7 cells by inhibiting GSK-3β through its phosphorylation, whereas AktX reduces growth via activation of GSK-3β by inhibiting Akt's kinase activity.

Topics: Blotting, Western; Brain Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Coloring Agents; ErbB Receptors; Gene Expression Regulation, Enzymologic; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Oncogene Protein v-akt; Phosphorylation; Tetrazolium Salts; Thiazoles; Wnt Proteins

Therapies targeting glioma cells that diffusely infiltrate normal brain are highly sought after. Our aim was to identify novel approaches to this problem using glioma spheroid migration assays. Lithium, a currently approved drug for the treatment of bipolar illnesses, has not been previously examined in the context of glioma migration. We found that lithium treatment potently blocked glioma cell migration in spheroid, wound-healing, and brain slice assays. The effects observed were dose dependent and reversible, and worked using every glioma cell line tested. In addition, there was little effect on cell viability at lithium concentrations that inhibit migration, showing that this is a specific effect. Lithium treatment was associated with a marked change in cell morphology, with cells retracting the long extensions at their leading edge. Examination of known targets of lithium showed that inositol monophosphatase inhibition had no effect on glioma migration, whereas inhibition of glycogen synthase kinase-3 (GSK-3) did. This suggested that the effects of lithium on glioma cell migration could possibly be mediated through GSK-3. Specific pharmacologic GSK-3 inhibitors and siRNA knockdown of GSK-3alpha or GSK-3beta isoforms both reduced cell motility. These data outline previously unidentified pathways and inhibitors that may be useful for the development of novel anti-invasive therapeutics for the treatment of brain tumors.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Glioma; Glycogen Synthase Kinase 3; Humans; Lithium Chloride; Mice; Neoplasm Invasiveness; Neurons; Organ Culture Techniques

In this study, the authors have demonstrated the effect of lithium, a typical mood stabilizer, on thrombin-evoked Ca2+ mobilization in C6 cells to elucidate the action mechanisms of the drug. Thrombin-induced Ca2 mobilization was reduced 24 hr after 1 or 10 mM lithium chloride (LiCl) pretreatment. The Ca2+ rise was reduced in a time-dependent manner, and the significant inhibition was observed 9 hr pretreatment with 10 mM LiCl. On the other hand, pretreatment of the cells with 10 mM LiCl for 24 hr did not alter the amount of Galphaq/11 significantly. Pretreatment with 10 mM LiCl for 24 hr failed to reduce the 5-HT-induced Ca2+ mobilization or to affect the desensitization of the 5-HT signal. Finally, thrombin-elicited Ca2+ rise was markedly inhibited in the presence of 0.05 U/ml plasmin, however, the Ca2+ rise was not further attenuated in the presence of plasmin in C6 cells pretreated with LiCl for 24 hr. These results indicate that pretreatment with LiCl attenuated thrombin-evoked intracellular Ca2+ mobilization in plasmin sensitive manner in C6 rat glioma cells. Thus, it is important to investigate the effect of lithium on thrombin-induced cellular responses to clarify the action mechanism of lithium in relation to some abnormality in thrombin-evoked Ca2+ rise observed in bipolar disorders.

Topics: Animals; Blotting, Western; Brain Neoplasms; Calcium; Cell Line; Dose-Response Relationship, Drug; Fibrinolysin; Fibrinolytic Agents; Glioma; Lithium Chloride; Rats; Receptor, Serotonin, 5-HT2A; Receptors, Serotonin; Serotonin; Thrombin; Time Factors; Tumor Cells, Cultured

Differential display PCR was used to identify genes regulated by mood stabilizer lithium in rat cerebral cortex. A differentially displayed lithium regulated gene fragment was isolated in rat cerebral cortex after chronic treatment with lithium (1.69 g/kg, p.o. ) for three weeks. A 1216-nucleotide cDNA for a novel lithium regulated gene (NLRG) was isolated from a rat brain cDNA library with RACE (rapid amplification of 5' cDNA end) PCR using a prime from the differentially displayed NLRG gene fragment. The deduced protein sequence was 321 amino acids long, and shows a significant homology with yeast nitrogen permease regulator 2 (NPR2). NLRG expression induced by lithium was confirmed by Northern and slot blot analysis in rat cerebral cortex and neuroblastomaxglioma NG108-15 cells, respectively. In situ hybridization revealed that chronic treatment with lithium increased NLRG gene expression in frontal cortex and hippocampus, but not in striatum, hypothalamus and thalamus regions of rat brain. These results suggest a novel target for lithium which may be relevant to its mechanism of action.

Topics: Amino Acid Sequence; Animals; Antimanic Agents; Base Sequence; Brain; Cerebral Cortex; DNA, Complementary; Gene Expression Regulation; Glioma; Hybrid Cells; In Situ Hybridization; Lithium Chloride; Male; Mice; Molecular Sequence Data; Nerve Tissue Proteins; Neuroblastoma; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Sequence Alignment; Sequence Homology, Amino Acid; Subtraction Technique; Trans-Activators; Tumor Cells, Cultured

The mechanisms underlying the antimanic effects of lithium are largely unknown but may involve long-term changes in brain gene expression. To determine if lithium could modify gene expression in astrocytes, the predominant cell type in brain, we tested the effects of LiCl on expression of nitric oxide synthase type 2 (NOS-2) in cultured glial cells. Incubation of primary rat astrocytes with endotoxin [lipopolysaccharide (LPS)] and proinflammatory cytokines induced NOS-2 gene and protein expression, as assessed by nitrite production and measurement of L-citrulline synthesis in whole cell lysates. Incubation with LiCl, but not KCl, increased NOS-2 activity up to 1.6-fold. LiCl also potentiated (up to 2.7-fold) the induction of NOS-2 expression by LPS plus interferon-gamma in C6 glioma cells but had little effect on LPS-induced nitrite accumulation from mouse RAW 264.7 macrophages. LiCl increased NOS-2 mRNA steady-state levels, suggesting an effect on mRNA stability and/or NOS-2 gene transcription. These results demonstrate that LiCl can modify astroglial gene expression and suggest that chronic treatment with lithium could exacerbate inflammatory responses in brain glial cells.

Topics: Animals; Antimanic Agents; Astrocytes; Cerebral Cortex; Citrulline; Enzyme Activation; Gene Expression Regulation, Enzymologic; Glioma; Interferon-gamma; Lipopolysaccharides; Lithium Chloride; Macrophages; Mice; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; RNA, Messenger; Tumor Cells, Cultured

Lithium, a monovalent cation, is the mainstay in the treatment of manic-depressive (MDI) illness, but despite extensive research, its mechanism of action remains to be elucidated. Since lithium requires chronic administration for therapeutic efficacy, and because its beneficial effects last well beyond its discontinuation, it has been postulated that lithium may exert major effects at the genomic level. In the present study we found that lithium, at therapeutically relevant concentrations, increases AP-1 DNA binding activity in human SH-SY5Y cells and rat C6 glioma cells. Additionally, in both SY5Y and C6 cells transiently transfected with a reporter gene vector driven by an SV40 promoter, lithium increased the activity of the reporter gene in a time- and concentration-dependent manner. Furthermore, mutations in the AP-1 sites of the reporter gene promoter significantly attenuated lithium's effects. These data indicate that lithium stimulates gene expression through the AP-1 transcription factor pathway, effects which may play a role in its long-term mood-stabilizing effects.

Topics: Animals; Base Sequence; Binding Sites; Chlorides; Consensus Sequence; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioma; Humans; Lithium Chloride; Luciferases; Molecular Sequence Data; Mutagenesis, Site-Directed; Neuroblastoma; Potassium Chloride; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; Rubidium; Sequence Alignment; Sequence Homology, Nucleic Acid; Simian virus 40; Transcription Factor AP-1; Transfection; Tumor Cells, Cultured

The effects of chronic treatment with antidepressants, verapamil, or lithium on serotonin (5-HT)-induced Ca2+ increase were investigated in single C6BU-1 glioma cells with digital imaging microscopy. Clomipramine and citalopram, at a concentration of 100 nM, decreased the peak values of 5-HT-induced [Ca2+]i changes. Verapamil (100 nM), a calcium antagonist, and lithium (1 mM) also inhibited the peak amplitudes in the same way. The present findings suggest that chronic treatment with antidepressants, verapamil, or lithium, at therapeutic concentrations, have the common action of inhibiting 5-HT-mediated [Ca2+]i increase.

Topics: Animals; Antidepressive Agents; Calcium; Calcium Channel Blockers; Citalopram; Clomipramine; Drug Interactions; Glioma; Intracellular Fluid; Lithium Chloride; Rats; Selective Serotonin Reuptake Inhibitors; Serotonin; Serotonin Antagonists; Tumor Cells, Cultured; Verapamil

Bipolar disorder is associated with increased levels and function of the G-protein, Gs alpha which may be normalized by treatment with mood stabilizing medications (i.e. lithium salts and the anticonvulsants, valproic acid and carbamazepine). In C6 glioma cells, endogenous ADP ribosylation was markedly increased by lithium chloride (+83%, P < 0.005), decreased by valproic acid (-48%, P = 0.07) whereas carbamazepine had no effect. Since ADP ribosylation of Gs alpha has been shown to increase turnover of this protein these results suggest a possible mechanism of action for lithium chloride. These results also suggest that lithium salts and the anticonvulsant mood stabilizers may have distinct mechanisms of action.

Topics: Adenosine Diphosphate Ribose; Animals; Carbamazepine; Glioma; Lithium Chloride; Rats; Tumor Cells, Cultured; Valproic Acid

Previous reports have provided conflicting evidence as to whether the response to TRH desensitizes. Here we show that TRH stimulation of phosphoinositide (PPI) hydrolysis, measured as inositol phosphate accumulation in the presence of LiCl, desensitizes in rat pituitary GH3 cells and in rat glioma C6 cells stably transfected with mouse pituitary TRH receptor complementary DNA. In GH3 cells, the rate of stimulation by 1000 nM TRH of PPI hydrolysis was maximal initially and then decreased by 44 +/- 13% after 20 min. In an experimental paradigm in which PPI hydrolysis was measured by adding 20 mM LiCl at different times after TRH, desensitizations caused by 3, 10, and 1000 nM TRH were 33 +/- 5%, 41 +/- 6%, and 69 +/- 2%, respectively. In transfected C6 cells, TRH-induced desensitization of 76 +/- 9% was found. In GH3 cells, 1 microM phorbol myristate acetate (PMA), an activator of protein kinase C, inhibited the initial response to TRH by 75 +/- 6% and preexposure to PMA and TRH decreased the rate of PPI hydrolysis by 98 +/- 1% after 60 min. One hundred micromolar H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine), an inhibitor of protein kinases, abolished the effect of PMA but did not inhibit TRH-induced desensitization. Elevation of cytoplasmic free Ca2+ by K+ depolarization increased TRH stimulation of PPI hydrolysis. We conclude that TRH stimulation of PPI hydrolysis acutely desensitizes and that this effect is not specific to pituitary cells. TRH-induced desensitization, moreover, does not appear to be mediated by protein kinase C or by elevation of cytoplasmic free Ca2+.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Cells, Cultured; Chlorides; Dose-Response Relationship, Drug; Drug Tolerance; Glioma; Hydrolysis; Isoquinolines; Lithium; Lithium Chloride; Phosphatidylinositols; Piperazines; Pituitary Gland; Protein Kinase C; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone