sb-415286 and Neuroblastoma

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.

Topics: Adjuvants, Immunologic; Aminophenols; Amyloid beta-Protein Precursor; Animals; Axons; Cell Line, Tumor; Copper; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Humans; Indoles; Lithium Chloride; Maleimides; Mice; Mutation, Missense; Neuroblastoma; Phosphorylation; Protein Transport; Synapses

In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca²⁺) homeostasis has been linked to presenilins, the catalytic core in γ-secretase complexes cleaving the amyloid precursor protein (APP), thereby generating amyloid-β (Aβ) peptides. Here we investigate whether APP contributes to ER Ca²⁺ homeostasis and whether ER Ca²⁺ could in turn influence Aβ production. We show that overexpression of wild-type human APP (APP(695)), or APP harboring the Swedish double mutation (APP(swe)) triggers increased ryanodine receptor (RyR) expression and enhances RyR-mediated ER Ca²⁺ release in SH-SY5Y neuroblastoma cells and in APP(swe)-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca²⁺ release leads to the reduction of both intracellular and extracellular Aβ load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aβ reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and β- and γ-secretases activities. Importantly, dantrolene diminishes Aβ load, reduces Aβ-related histological lesions, and slows down learning and memory deficits in Tg2576 mice. Overall, our data document a key role of RyR in Aβ production and learning and memory performances, and delineate RyR-mediated control of Ca²⁺ homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches.

Topics: Alzheimer Disease; Aminophenols; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Analysis of Variance; Animals; Brain; Caffeine; Calcium; Calcium Channel Blockers; Cells, Cultured; Cytosol; Dantrolene; Disease Models, Animal; Embryo, Mammalian; Endoplasmic Reticulum; Enzyme Inhibitors; Exploratory Behavior; Gene Expression Regulation; Humans; Inositol 1,4,5-Trisphosphate Receptors; Maleimides; Maze Learning; Membrane Potentials; Membrane Proteins; Memory Disorders; Mice; Mice, Transgenic; Muscle Relaxants, Central; Mutation; Nerve Tissue Proteins; Neuroblastoma; Neurons; Patch-Clamp Techniques; Peptide Fragments; Phosphorylation; Plaque, Amyloid; Purines; Reaction Time; Recognition, Psychology; RNA, Messenger; Roscovitine; Ryanodine Receptor Calcium Release Channel; Transfection

Neuroblastoma is the most common extracranial solid tumor of childhood. While survival rates are high for localized disease, treatment response remains poor for a subset of patients with large tumors or disseminated disease. Thus, there remains much room for improvement in treatment strategies for this disease. Using in vitro and in vivo systems, we present glycogen synthase kinase-3β (GSK-3β) inhibition as a potential mechanism to treat neuroblastoma. Using the specific GSK-3β inhibitor SB415286, we demonstrate that GSK-3β inhibition decreases the viability of Neuro-2A cells, as determined by cell proliferation assay and clonogenic survival. Moreover, we show that GSK-3β inhibition induces apoptosis in neuroblastoma cells, as determined by Annexin V staining and confirmed with DAPI staining. Using flow cytometry, we are able to demonstrate that SB415286 induces the accumulation of cells in the G2/M phase of the cell cycle. Finally, we show that these in vitro results translate into delayed tumor growth in vivo using a heterotopic tumor model in nude mice treated with SB415286. These findings suggest that GSK-3β is a potential molecular target for the treatment of neuroblastoma.

Topics: Aminophenols; Animals; Apoptosis; Biomarkers, Tumor; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorimetry; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Maleimides; Mice; Neuroblastoma; RNA, Small Interfering; Time Factors; Tumor Stem Cell Assay; X-Linked Inhibitor of Apoptosis Protein

Pharmacological GSK-3 inhibitors are potential drugs for the treatment of neurodegenerative diseases, cancer and diabetes. We examined the antiproliferative effects of two GSK-3 inhibitors, lithium and SB-415286, on B65 neuroblastoma cell line. Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G(2)/M cell cycle arrest. Cell-cycle proteins such as cyclins D, E, A, cdk4 and cdk2 were up-regulated. Since lithium and SB-415286-induced G(2)/M arrest we studied changes in the expression of proteins involved in this phase, specifically cyclin B, cdc2 and the phosphorylated form of this protein (tyr15-cdc2). Both drugs increased the expression of tyr15-cdc2, thus inhibiting mitosis. On the other hand, SB-415286 increased the expression of SIRT2, involved in the regulation of proliferation. Moreover, cell-cycle arrest mediated by SB-415286 was accompanied by apoptosis that was not prevented by 100 microM of zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), a pan-caspase inhibitor. Likewise, GSK-3 inhibitors did not affect the mitochondrial release of apoptosis inducing factor (AIF). We conclude that inhibitors of GSK-3 induced cell-cycle arrest, mediated by the phosphorylation of cdc2 and, in the case of SB-415286, SIRT2 expression, which induced apoptosis in a caspase-independent manner.

Topics: Aminophenols; Animals; Apoptosis; Apoptosis Inducing Factor; CDC2 Protein Kinase; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Lithium; Maleimides; Neuroblastoma; Rats; Sirtuin 2