Laboratory Study
Regulation of glycogen synthase kinase-3 beta (GSK-3β) by the Akt pathway in gliomas

https://doi.org/10.1016/j.jocn.2012.07.002Get rights and content

Abstract

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.

Introduction

Glioblastoma multiforme (GBM) represents the most lethal form of brain tumour which, despite intensive research, remains among the most deadly of all human cancers,1 with almost 100% mortality, irrespective of the interventions used.2, 3, 4 Patients treated with surgery, radiotherapy and chemotherapy survive an average of 14 months, with only 5% to 10% of patients surviving 2-year post-diagnosis.3, 5, 6 Gliomas are characterized by their diffuse and infiltrative nature7 which renders these cancers incurable by surgical intervention alone.8, 9, 10 Novel therapeutic treatments are required to improve patient outcomes. Glycogen synthase kinase-3 beta (GSK-3β) is a kinase at the crossroads of a plethora of signaling pathways that have been implicated in gliomagenesis and cancer in general. GSK-3β has roles in cell growth, proliferation, survival, cytoskeletal stability, microtubule dynamics, gene expression, neuronal polarization, nuclear export, differentiation, mood stabilization, neurodegeneration, apoptosis, but primarily for the process after which it was named, glycogen synthesis.[11, 12 GSK-3β’s input in both the Wnt signaling pathway and the phosphoinositide 3-kinase (PI3K)/Akt pathway (which are both commonly dysregulated in gliomagenesis) point to the possibility of this ubiquitously expressed kinase having a role in many biological factors regulated in gliomagenesis, including cell proliferation, apoptosis and tumour cell migration. The evidence that GSK-3β has a role in the myriad downstream effects of Akt suggests that selective inhibition of this kinase may permit the development of targeted molecular therapies for glioma. The present study investigated the effects of a non-specific, small-molecule inhibitor of GSK-3β, lithium chloride (LiCl), and a specific Akt inhibitor, AktX, on GSK-3β phosphorylation, proliferation, survival and morphology in adherent U87MG and U87MG.Δ2-7 cell lines, and one tumour stem cell-like cell line (TSC#004) grown as “neurospheres”.

Section snippets

Culture of glioma cells and neurospheres

Human glioblastoma cell lines U87MG and U87MG.Δ2-7 (containing the Δ2-7 epidermal growth factor receptor [EGFR] mutation, EGFRvIII)13 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) + 5% fetal calf serum + penicillin/streptomycin (P/S) at 37 °C and 10% carbon dioxide (CO2) as described by Rubin et al.14 The human glioblastoma stem cell line TSC#004 was established in our laboratory directly from a freshly resected brain tumour. Cells were cultured in “neurosphere conditions” (DMEM/F12 + P/S

Drug treatment of adherent cells and neurospheres

Cells were treated with increasing concentrations of LiCl and AktX to determine if these compounds affected cell morphology. Treatment with LiCl (50 mM) and AktX (25 μM) resulted in an altered cell morphology for the U87MG and TSC#004 cells (Fig. 1). The effects of AktX were minimal at the lower concentration (10 μM) (Fig. 1E) with no clear morphological changes observed. High AktX concentrations of 25 μM (Fig. 1F) and 50 μM caused U87MG cells to lose their ability to adhere to the plate. Reduced

Discussion

Our study demonstrated that LiCl reduced the spreading of astrocytic processes in a dose-dependent manner, which is in contrast to the literature that implicates GSK-3β inhibition in axon and dendrite growth in neurons.19, 20, 21 As seen in Figs. 1A–C, increasing concentrations of LiCl resulted in U87MG cells withdrawing their processes and adopting a rounder conformation. Active GSK-3β has been shown to phosphorylate and inactivate both CRMP-2, a protein involved in determining neuronal

References (29)

Cited by (52)

  • RRAGB-mediated suppression of PI3K/AKT exerts anti-cancer role in glioblastoma

    2023, Biochemical and Biophysical Research Communications
View all citing articles on Scopus
View full text