GSK3α/ß: A Novel Therapeutic Target for Neuroendocrine Tumors.

INTRODUCTION: Glycogen synthase kinase 3α/ß (GSK3α/ß) is a serine/threonine kinase that plays a critical role in cancer. AIMS: In this study, we evaluated the effects of the specific GSK3α/ß inhibitor AR-A014418 in vitro to gain novel insights into GSK3α/ß signaling in neuroendocrine tumors (NETs). MATERIALS AND METHODS: Human NET cell lines (BON1, QGP1, H727, and GOT1) were treated with different concentrations of AR-A014418 alone and in combination with lovastatin, everolimus, 5-fluorouracil (5-FU), and γ-irradiation. RESULTS: AR-A014418 significantly dose- and time-dependently decreased cell viability in all 4 NET cell lines through inhibition of epithelial growth factor receptor and mTORC1/p70S6K signaling, as well as cyclin D3 downregulation and induction of pChk1. In all cell lines tested, FACS analysis showed an AR-A014418-induced increase in the sub-G1 phase, reflecting cell death. Apoptosis induction was observed in H727, GOT1 and QGP1 cells, but not in BON1 cells. Furthermore, significant antimigratory effects upon GSK3α/ß inhibition were found and were associated with ß-catenin downregulation in all cell lines tested. Compensatory upregulation of pAkt and pERK in response to GSK3α/ß inhibition was prevented by combining AR-A014418 with the ERK and Akt inhibitor lovastatin. Accordingly, the lovastatin/AR-A014418 combination was synergistic in BON1 and QGP1 cells. Moreover, AR-A014418 displayed promising chemosensitizing effects on 5-FU in QGP1 and slight radiosensitizing properties in BON1 and QGP1 cells. CONCLUSION: Our data provide new insights into the role of GSK3α/ß in NETs and suggest that GSK3α/ß inhibition could be a novel therapeutic option in NETs, especially in combination with lovastatin or 5-FU, depending on tumor entity.

Impact of glycoprotein VI and platelet adhesion on atherosclerosis--a possible role of fibronectin.

Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly via fibronectin-could relevantly contribute to platelet-triggered atheroprogression.

Tumor necrosis factor-alpha triggers cell death of sensitized potassium chloride-stimulated cholinergic neurons.

Cell death of cholinergic neurons of the basal forebrain plays an important role in neurodegenerative disorders, such as Alzheimer's disease. Inflammatory cytokines, such as, for example, tumor necrosis factor-alpha (TNF-alpha), may be involved in these neurodegenerative processes. The aim of this project was to study the role of TNF-alpha in the survival and nerve fiber growth of cholinergic neurons of the basal nucleus of Meynert in organotypic brain slices and in adult rats. Cholinergic neurons were visualized by immunohistochemistry for the enzyme choline acetyltransferase and nerve fibers by histochemistry for the enzyme acetylcholinesterase. When co-slices of basal nucleus of Meynert and neocortex were sensitized for 15 min with 30 mM potassium chloride and subsequently incubated for 1 week with 20 ng/ml TNF-alpha, cholinergic neurons and nerve fibers markedly degenerated. Incubation with different growth factors rescued the loss of cholinergic cell bodies and cholinergic nerve fibers. Injection of 30 mM potassium chloride and 50 ng TNF-alpha into four defined cortical regions of anesthetized adult rats resulted in predominant cell death of cholinergic neurons on the ipsilateral side. In conclusion, our data show that TNF-alpha potentiated cell death of cholinergic neurons possibly via retrograde axonal damage in vitro and in vivo. Cortical overactivation combined with an increased expression of pro-inflammatory cytokines may contribute to the cell death observed in Alzheimer's disease and ageing.

Glial cell line-derived neurotrophic factor enhances survival of GM-CSF dependent rat GMIR1-microglial cells.

Microglial activation and proliferation occur in nearly all forms of brain injury. The aim of this study was to investigate the influence of glial cell-line derived neurotrophic factor (GDNF) on proliferation and/or survival in a GMIR1 rat microglial cell line, which proliferates in response to granulocyte-macrophage-colony stimulating factor (GM-CSF). Endogenous GDNF and its receptor, GFRalpha-1, were detected in GMIR1 cells by ELISA and immunohistochemistry/Western blot, respectively. Recombinant GDNF strongly enhanced GMIR1 cell numbers and BrdU-incorporation, an effect inhibited by GDNF blocking antibodies. Inhibition of cAMP/cGMP dependent protein kinase enhanced the GDNF-induced GMIR1 cell number. The results suggest that GDNF has synergistic survival promoting effects on microglia potentially via autocrine mechanisms.

Evidence that toxicity of lipopolysaccharide upon cholinergic basal forebrain neurons requires the presence of glial cells in vitro.

The cholinergic system of the basal forebrain is affected in brains of dementia patients and during neuroinflammation. The aim of this study was to establish a method to cultivate basal forebrain cholinergic neurons in dissociated, pure neuronal cultures and to apply this method to study the effect of acute and chronic experimentally-induced inflammation using lipopolysaccharide. Purity of the cultures, degrees of neuronal dissociation, connectivity and neuronal survival were investigated by immunocytochemistry for microtubule-associated protein-2 (neurons), glial fibrillary acidic protein (astroglia), complement receptor 3 (microglia), choline acetyltransferase and the neurotrophin receptor p75 (cholinergic neurons). Neuronal cultures only contained <7% astrocytes and <1% microglia when using a "sandwich-technique". Acute (1, 10 microg/ml) as well as chronic (0.1, 1 microg/ml) treatment with lipopolysaccharide did neither affect total number of neurons, nor number of p75-positive neurons or enhance expression of major histocompatibility complex I or II. Our results suggest that lipopolysaccharide-induced degeneration of both microtubule-associated protein-2-like immunoreactive as well as specific killing of cholinergic forebrain neurons in vitro are mediated by glial cells.

Nerve growth factor plays a divergent role in mediating growth of rat C6 glioma cells via binding to the p75 neurotrophin receptor.

Dysregulation of proliferation, differentiation and cell death play a major role in glial tumors, and there is evidence for regulatory mechanisms involving nerve growth factor (NGF) and its receptors in various CNS-derived tumor cell lines. The aim of our study was to observe the effect of exogenous recombinant NGF on C6 rat glioma growth, to characterize the role of endogenous NGF and the p75 neurotrophin receptor (p75) and to rule out whether p75 is necessary to mediate the effect of exogenous NGF. Recombinant exogenous NGF (1-100 ng/ml) was applied under different serum conditions (0%, 1%, 5%) and knockdown of endogenous NGF and p75 was achieved by lipid-mediated antisense oligonucleotide treatment. In presence of serum, NGF had a positive whereas in absence of serum NGF produced a negative effect on C6 cell number. A knockdown of NGF or p75 increased cell numbers and enhanced BrdU incorporation. In p75-knocked down cells NGF did not enhance C6 glioma growth in presence of serum. We conclude that (1) exogenous recombinant NGF enhances C6 glioma growth under serum conditions but decreases cell number in absence of serum, that (2) the effect of exogenous NGF is mediated by p75 alone or by heterodimers containing p75 and that (3) either basal levels of endogenous NGF or basal levels of p75 receptor moderate C6 glioma growth and represent an autoregulatory potential of C6 glioma cells.