Effect Of GSK-3β Inhibitor Research Articles

Abstract 2862: Deregulated GSK3β sustains gastrointestinal cancer cells by modulating hTERT and telomerase

Abstract Purpose: Glycogen synthase kinase-3β (GSK3β) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3β in gastrointestinal, pancreatic, and liver cancers. Experimental Design: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3β expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3β activity by in vitro kinase assay. The effects of small-molecule GSK3β inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3β inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. Results: Cancer cell lines and most cancer tissues showed increased GSK3β expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3β resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3β inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. Conclusion: The results indicate that deregulated GSK3β sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2862. doi:1538-7445.AM2012-2862

Abstract LB-290: Glycogen synthase kinase-3β inhibits tumor growth and angiogenesis of gastric cancer

Abstract Objective: The mechanisms underlying angiogenesis in cancer are diverse. Although in vitro studies have shown that glycogen synthase kinase-3β (GSK-3β) inhibits the expressions of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in several cancer cells, the implication of GSK-3β in cancer angiogenesis in vivo has not been shown. We examined the effects of GSK-3β on angiogenesis and human gastric cancer growth. Methods: Human gastric cancer cell lines SNU-638 and SNU-484 were stably transfected with either human wild-type (WT)-GSK-3β or kinase-dead (KD)-GSK-3β, and the effects of GSK-3β on tumor growth and angiogenesis were examined in in vivo experiments. Gastric cancer cells were injected into nude mice subcutaneously and the size of xenografted tumors was measured. The effects of GSK-3β inhibition on angiogenesis and HIF-1α activation were assessed by immunohistochemistry, Western blotting and semiquantitative reverse transcription-polymerase chain reaction. In addition, GSK-3β effects on the HIF-1α degradation and synthesis were examined. Results: Xenografts in nude mice derived from stable gastric cell lines over-expressing KD-GSK-3β showed larger tumor volumes and higher microvessel density than those over-expressing WT-GSK-3β. Immunohistochemistry and Western blot analysis using xenografted tumor tissues showed that KD-GSK-3β over-expression increased immunoreactivity for Ki-67, HIF-1α, VEGF or CD31 compared with WT-GSK-3β over-expression. In addition, KD-GSK-3β over-expression enhanced HIF-1α expression under hypoxic conditions at the translational level, but not under normoxic conditions. Conclusion: These results indicate that GSK-3β inhibits gastric tumor growth and angiogenesis by regulating the expression of HIF-1α and VEGF in gastric cancer cells under hypoxic conditions. Our results suggest the potential usefulness of GSK-3β/HIF-1α/VEGF pathway to find molecular targets in gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-290. doi:10.1158/1538-7445.AM2011-LB-290

GSK3β negatively regulates oligodendrocyte differentiation and myelination in vivo

Glycogen synthase kinase 3β (GSK3β) is an essential integrating molecule for multiple proliferation and differentiation signals that regulate cell fate. Here, we have examined the effects of inhibiting GSK3β on the development of oligodendrocytes (OLs) from their oligodendrocyte precursors (OP) in vivo by injection into the lateral ventricle of postnatal mice and ex vivo in organotypic cultures of isolated intact rodent optic nerve. Our results show that a range of GSK3β inhibitors (ARA-014418, lithium, indirubin, and L803-mt) increase OPs and OLs and promote myelination. Inhibition of GSK3β stimulates OP proliferation and is prosurvival and antiapoptotic. The effects of GSK3β inhibition in OPs is via the canonical Wnt signaling pathway by stimulating nuclear translocation of β-catenin. However, direct comparison of the effects of Wnt3a and GSK3β inhibition in optic nerves shows that they have opposing actions on OLs, whereby GSK3β inhibition strikingly increases OL differentiation, whereas Wnt3a inhibits OL differentiation. Notably, GSK3β inhibition overrides the negative effects of Wnt3a on OLs, indicating novel GSK3β signaling mechanisms that negatively regulate OL differentiation. We identify that two mechanisms of GSK3β inhibition are to stimulate cAMP response element binding (CREB) and decrease Notch1 signaling, which positively and negatively regulate OL differentiation and myelination, respectively. A key finding is that GSK3β inhibition has equivalent effects in the adult and stimulates the regeneration of OLs and remyelination following chemically induced demyelination. This study identifies GSK3β as a profound negative regulator of OL differentiation that contributes to inefficient regeneration of OLs and myelin repair in demyelination.

Mechanical Loading Regulates NFATc1 and β-Catenin Signaling through a GSK3β Control Node

Mechanical stimulation can prevent adipogenic and improve osteogenic lineage allocation of mesenchymal stem cells (MSC), an effect associated with the preservation of beta-catenin levels. We asked whether mechanical up-regulation of beta-catenin was critical to reduction in adipogenesis as well as other mechanical events inducing alternate MSC lineage selection. In MSC cultured under strong adipogenic conditions, mechanical load (3600 cycles/day, 2% strain) inactivated GSK3beta in a Wnt-independent fashion. Small interfering RNA targeting GSK3beta prevented both strain-induced induction of beta-catenin and an increase in COX2, a factor associated with increased osteoprogenitor phenotype. Small interfering RNA knockdown of beta-catenin blocked mechanical reduction of peroxisome proliferator-activated receptor gamma and adiponectin, implicating beta-catenin in strain inhibition of adipogenesis. In contrast, the effect of both mechanical and pharmacologic inhibition of GSK3beta on the putative beta-catenin target, COX2, was unaffected by beta-catenin knockdown. GSK3beta inhibition caused accumulation of nuclear NFATc1; mechanical strain increased nuclear NFATc1, independent of beta-catenin. NFATc1 knockdown prevented mechanical stimulation of COX2, implicating NFATc1 signaling. Finally, inhibition of GSK3beta caused association of RNA polymerase II with the COX2 gene, suggesting transcription initiation. These results demonstrate that mechanical inhibition of GSK3beta induces activation of both beta-catenin and NFATc1 signaling, limiting adipogenesis via the former and promoting osteoblastic differentiation via NFATc1/COX2. Our novel findings suggest that mechanical loading regulates mesenchymal stem cell differentiation through inhibition of GSK3beta, which in turn regulates multiple downstream effectors.

Abstract 240: Inhibition of Glycogen Synthase Kinase 3 Beta Improves EPC Yield, Function and Ameliorates Arterial Repair Post-injury and Stenting

Endothelial progenitor cells (EPCs) are circulating undifferentiated pluripotent cells capable of reconstituting endothelium in vivo and diminishing lesion formation following arterial injury. We tested the hypothesis that treatment with a specific soluble GSK 3β-inhibitor (GSK3βi) would improve the in vitro yield and function of EPCs from patients with coronary artery disease (CAD) and augment their in vivo potential to improve arterial repair after injury and stent implantation. In vitro treatment with GSK3βi at 1× (IC50) and 2× (IC50) increased the number of cells that dual labelled for acLDL and UEA-1 lectin on day 7 (13.4±3.2 vs. 39.9±5.3 vs. 43.8±10.1; p < 0.05). Furthermore, GSK3βi significantly augmented CAD-EPC survival to levels equal to those of healthy controls, increased VEGF secretion (7.1±1.6 vs 30.0±8.9, p<0.05) and resulted in a 4× increase in adhesion (p<0.05). Using Q-PCR, mRNA copy numbers for VLA-1,2,3,4 and 5 were assayed; however, only VLA-4 levels increased (4×, p<0.01). Use of a VLA-4 specific blocking antibody confirmed that the improved EPC adhesion was specifically mediated by VLA-4. Finally, the effects of GSK 3β inhibition were tested in two in vivo models of arterial injury. CD-1 athymic nude mice underwent femoral artery wire injury and injection of CAD-EPCs that were either GSK3βi treated, untreated, or injection with vehicle alone. GSK3βi treated EPCs improved the ability of xenotransplanted cells to inhibit lesion formation when compared to untreated and vehicle arteries respectively (intima/media ratios: 0.3±0.1 vs. 5.3±1.2 vs 11.2±2.7 μm, p<0.01). GSK3βi coated stents were inserted into rabbit carotid arteries. Fourteen days after stent deployment, GSK3βi stents resulted in a 44% increase in re-endothelialization (assessed by scanning electron microscopy and compared to vehicle coated stents; p<0.01) and a 16% trend towards a reduction in neointima formation (p<0.09). In summary, our data demonstrates that GSK3βi improves EPC function in vitro and ameliorates arterial repair following injury and stent insertion in vivo .

83 Dual effects of GSK-3β inhibition on death receptor-mediated apoptosis in hepatoma cells

83 Dual effects of GSK-3β inhibition on death receptor-mediated apoptosis in hepatoma cells

Glycogen synthase kinase-3β negatively regulates group IIA phospholipase A 2 expression in human aortic smooth muscle and HepG2 hepatoma cells

The present study shows that the IFN-γ-mediated upregulation of secretory phospholipase A 2 of group IIA (sPLA 2-IIA) in HASMC and HepG2 cells is synergistically increased after simultaneous inhibition of glycogen synthase kinase-3β (GSK-3β) by indirubin-3 ′-monoxime, 5-iodo or AR-A014418. The effect of GSK-3β inhibition was dose- and time-dependent and can be further augmented by its concomitant incubation with Clostridium difficile toxin B, an inhibitor of small Rho proteins, or H-1152, an inhibitor of Rho-associated kinase. Using AG-490 and caffeic acid phenethyl ester (CAPE), it is further demonstrated that the effect of GSK-3β inhibition on sPLA 2-IIA expression depends on Janus kinase-2 and NF-κB-signaling.

Glycogen Synthase Kinase 3-Beta (GSK3-β) inhibitors decrease protein degradation in L6 myotubes

Introduction. Previous reports suggest that inhibition of GSK3-β results in muscle hypertrophy. Muscle hypertrophy may result from increased protein synthesis, decreased protein degradation, or a combination of both processes. The effect of GSK3-β inhibition on protein degradation in skeletal muscle has not been reported. We tested the hypothesis that the GSK3-β inhibitors lithium chloride (LiCl), SB216763, and SB415286 decrease protein degradation in cultured L6 myotubes. Methods. Fully-differentiated, cultured L6 myotubes, a rat skeletal muscle cell line, were treated with LiCl, SB216763, or SB415286 for 24 h. Protein degradation was assessed by measuring the release of TCA-soluble 3H-tyrosine from proteins that had been pre-labeled with the amino acid. Results. Treatment of cultured myotubes with the GSK3-β inhibitors resulted in a dose-dependent decrease in protein degradation. Results in the Table show the maximal inhibition (24–30%) caused by the GSK3-β inhibitors. Conclusions. This study provides evidence that inhibition of GSK3-β reduces protein degradation in skeletal muscle cells. The hypertrophy reported previously following inhibition of GSK3-β may, at least in part, be due to a decrease in protein breakdown. TABLE—ABSTRACT P26 − Inhibitor + Inhibitor LiCl 25 mM 13.7 ± 0.2 10.0 ± 0.1 ∗ SB216763 25 μM 11.5 ± 0.3 8.8 ± 0.5 ∗ SB415286 100 μM 12.8 ± 0.3 8.9 ± 0.8 ∗ Note. Protein degradation (%/24 hours) in myotubes treated with GSK3-β inhibitors. Results are means ± SEM with n = 6 per group. ∗ P < 0.001 versus no inhibitor by ANOVA.

Inhibition of GSK-3β leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium

WNT-7a induces axonal spreading and branching in developing cerebellar granule neurons. This effect is mediated through the inhibition of GSK-3beta, a serine/threonine kinase and a component of the WNT pathway. Lithium, an inhibitor of GSK-3beta, mimics WNT-7a in granule cells. Here we examined further the effect of GSK-3beta inhibition on cytoskeletal re-organisation. Lithium induces axonal spreading and increases growth cone area and perimeter. This effect is associated with the absence or reduction of stable microtubules in spread areas. Lithium induces the loss of a phosphorylated form of MAP-1B, a microtubule associated protein involved in axonal outgrowth. Down-regulation of the phosphorylated MAP-1B, MAP-1B-P, from axonal processes occurs before axonal remodelling is evident. In vitro phosphorylation assays show that MAP-1B-P is generated by direct phosphorylation of MAP-1B by GSK-3beta. WNT-7a, like lithium, also leads to loss of MAP-1B-P from spread axons and growth cones. Our data suggest that WNT-7a and lithium induce changes in microtubule dynamics by inhibiting GSK-3beta which in turn lead to changes in the phosphorylation of MAP-1B. These findings suggest a novel role for GSK-3beta and WNTs in axonal remodelling and identify MAP-1B as a new target for GSK-3beta and WNT.