lithium-chloride and 6-bromoindirubin-3--oxime

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

Definitive endoderm (DE) differentiation from mouse embryonic stem cell (mESC) monolayer cultures has been limited by poor cell survival or low efficiency. Recently, a combination of TGFβ and Wnt activation with BMP inhibition improved DE induction in embryoid bodies cultured in suspension. Based on these observations we developed a protocol to efficiently induce DE cells in monolayer cultures of mESCs. We obtained a good cell yield with 54.92% DE induction as shown by Foxa2, Sox17, Cxcr4 and E-Cadherin expression. These DE-cells could be further differentiated into posterior foregut and pancreatic phenotypes using a culture protocol initially developed for human embryonic stem cell (hESC) differentiation. In addition, this mESC-derived DE gave rise to hepatocyte-like cells after exposure to BMP and FGF ligands. Our data therefore indicate a substantial improvement of monolayer DE induction from mESCs and support the concept that differentiation conditions for mESC-derived DE are similar to those for hESCs. As mESCs are easier to maintain and manipulate in culture compared to hESCs, and considering the shorter duration of embryonic development in the mouse, this method of efficient DE induction on monolayer will promote the development of new differentiation protocols to obtain DE-derivatives, like pancreatic beta-cells, for future use in cell replacement therapies.

Topics: Activins; Animals; Carrier Proteins; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Embryonic Stem Cells; Endoderm; Humans; Indoles; Lithium Chloride; Mice; Mice, Inbred NOD; Mice, SCID; Models, Biological; Oximes; Pancreas; Stem Cell Transplantation; Wnt Signaling Pathway

Glycogen synthase kinase-3beta (GSK3beta) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3beta. However, the inactive form of GSK3beta which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3beta substrates, such as beta-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3beta at serine-9 and other substrates including tau, beta-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3beta inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3beta may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3beta inhibitors could be a valuable drug candidate in AD.

Topics: Alzheimer Disease; Animals; Cells, Cultured; Disease Models, Animal; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Indoles; Lithium Chloride; Neurons; Okadaic Acid; Oximes; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 2; Rats; Serine

In tauopathies such as Alzheimer's disease (AD), the molecular mechanisms of tau protein aggregation into neurofibrillary tangles (NFTs) and their contribution to neurodegeneration remain not understood. It was recently demonstrated that tau, regardless of its aggregation, might represent a key mediator of neurodegeneration. Therefore, reduction of tau levels might represent a mechanism of neuroprotection. Glycogen synthase kinase-3beta (GSK3beta) and protein phosphatase-2A (PP2A) are key enzymes involved in the regulation of tau phosphorylation, and have been suggested to be involved in the abnormal tau phosphorylation and aggregation in AD. Connections between PP2A and GSK3beta signaling have been reported. We have previously demonstrated that exposure of cultured cortical neurons to lithium decreased tau protein expression and provided neuroprotection against Abeta. Since lithium is not a specific inhibitor of GSK3beta (ID50=2.0 mM), whether or not the lithium-induced tau decrease involves GSK3beta remained to be determined. For that purpose, cultured cortical neurons were exposed to 6-bromo-indirubin-3'-oxime (6-BIO), a more selective and potent GSK3beta inhibitor (ID50=1.5 microM) or to lithium. Analysis of tau levels and phosphorylation by western-blot assays showed that lithium and 6-BIO dose-dependently decreased both tau protein levels and tau phosphorylation. Conversely, inhibition of cyclin-dependent kinase-5 (CDK5) by roscovitine decreased phosphorylated tau but failed to alter tau protein levels. These data indicate that GSK3beta might be selectively involved in the regulation of tau protein levels. Moreover, inhibition of PP2A by okadaic acid, but not that of PP2B (protein phosphatase-2B)/calcineurin by FK506, dose-dependently reversed lithium-induced tau decrease. These data indicate that GSK3beta regulates both tau phosphorylation and total tau levels through PP2A.

Topics: Analysis of Variance; Animals; Blotting, Western; Calcineurin; Calcineurin Inhibitors; Cells, Cultured; Cyclin-Dependent Kinase 5; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Indoles; Lithium Chloride; Neurons; Okadaic Acid; Oximes; Phosphorylation; Protein Phosphatase 2; Purines; Rats; Rats, Wistar; Roscovitine; Tacrolimus; tau Proteins

Our recent study has demonstrated that glucocorticoids (GCs) induce cyclooxygenase-1 (COX-1) gene expression in rat cardiomyocytes. While investigating the mechanism underlying corticosterone (CT) induced COX-1, we found that three structurally and mechanistically distinct GSK-3 inhibitors, LiCl, SB216763, and (2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO), inhibited COX-1 transcription and protein induction. A genetic approach of expressing wild type GSK-3beta increased COX-1 promoter activity, which was abolished by LiCl. LiCl increased inhibitory GSK-3alpha/beta phosphorylation at Ser21/Ser9, while BIO or SB216763 prevented stimulatory phosphorylation at Tyr279/Tyr216 of GSK-3alpha/beta. GSK inhibitors failed to block nuclear translocation of glucocorticoid receptor (GR) or activation of glucocorticoid response element (GRE) by CT treatment. While Sp3 transcription factor mediates CT induced COX-1 expression, GSK inhibitors did not change the level of Sp3 protein or binding of Sp3 transcription factor to COX-1 promoter. The observed effect of GSK-3 inhibitors appears to be unique to COX-1 since LiCl or BIO does not prevent CT from inducing COX-2 gene. We conclude that GSK-3 inhibitors block CT from inducing COX-1 gene expression via a mechanism beyond GR and Sp3 transcription factor.

Topics: Animals; Animals, Newborn; Cells, Cultured; Corticosterone; Cyclooxygenase 1; Cyclooxygenase 2; Enzyme Induction; Glycogen Synthase Kinase 3; Indoles; Lithium Chloride; Maleimides; Membrane Proteins; Myocytes, Cardiac; Oximes; Phosphorylation; Promoter Regions, Genetic; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Glucocorticoid; Sp3 Transcription Factor; Transfection