Browsing by Subject "SH-SY5Y"
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(2010)Neuronal nicotinic acetylcholine receptors are ligand-gated ion channel receptors that consist of five transmembral receptor subunits. They can form a functional receptor subtype solely out of alfa-subunits or out of a combination of alfa- and beta-subunits. The number of potential assembly of nicotinic receptor subunits is high because at least nine alfa-subunits (α2-α10) and three beta-subunits (β2-β4) have been recognized. The composition, location and pharmacological properties of different subtypes have not yet been fully characterized. However, it has been shown that the neuronal nicotinic receptors are involved in a wide range of physiological and pathofysiological processes especially in the central nervous system. Toxins from snakes and Conus -sea snails have proved to be important tools in neuropharmacological research, from which considerable information on structure and subunit combinations of the nicotinic receptors have been received. Receptors binding assays or autoradiographic experiments exploiting toxins have also been useful methods to locate the neuronal nicotinic receptors in mammalian brain. The aim of the experimental part of the Pro gradu was to characterize in vitro binding affinities of α-contoxin MII, α-conotoxin Vc1.1, neurotoxin II, α-cobratoxin and weak-toxin synthetized in Moscow and of well-known receptor ligands cytisine and methyllycaconitine to neuronal nicotinic receptors in SH-SY5Y- and SH-EP1-hα7-cell membrane preparations. SH-SY5Y-cells are known to express various neuronal receptor subtypes (α3* or α7) endogenously. For the SH-EP1-hα7-cells part, the cell line has been transfected with α7 nAChR-genes and it expresses only α7 receptor subtypes. Receptor competition studies were performed with [3H]-epibatidine (400 pM SH-SY5Y, 2000 pM SH-EP1-hα7) and the radioactivity was measured with a Microbeta- scintillation counter. [3H]-epibatidine was perceived to be displaced almost completely by cytisine and methyllycaconitine in both cell lines. In addition to this, the toxins were shown to bind to two distinct receptor-binding sites in SH-SY5Y-cells. Also α-contoxin MII and α-conotoxin Vc1.1 inhibited [3H]-epibatidine binding by biphasic manner, but the maximal displacement failed to be complete. From α-conotoxins only MII had affinity to α7 receptors in SH-EP1-hα7-cells. Neurotoxin II, α-cobratoxin and weak-toxin were not found to compete with [3H]-epibatidine for the same binding sites in SH-SY5Y-cells. The results confirm the assumption that cytisine and methyllycaconitine label various nAChR-subtypes. Instead, based on SH-SY5Y-cell assays α-conotoxins used in this study would seem to label spesifically only particular nAChR-subtypes. The receptor competition studies also confirm the prevalent conception that neurotoxin II, α-cobratoxin and weak-toxin do not bind to neuronal nicotinic receptor subtypes containing α3-subunits.
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(2013)Alzheimer's disease is a neurodegenerative brain disease and it is the leading cause of dementia worldwide. However, there are not any medical treatments available to slow down or cure the disease. The typical microscopic changes in Alzheimer patients' brain are extracellular amyloid deposits and intracellular neurofibrillary tangles. Serine/threonine kinases are protein kinases that take part in the regulation of cellular functions. At least protein kinase C (PKC), glycogen synthase kinase 3 (GSK-3), cyclin-dependent kinase 5 (CDK5) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) are involved in the pathogenesis of Alzheimer's disease. There are currently molecules in development that either activate or inhibit these protein kinases in order to stop the progression of the disease. PKC is an interesting kinase considering this project. It has been shown that PKC activation prevents the formation of amyloid deposits and protects neurons from premature death. This could slow down or prevent the progression of the disease. The purpose of this study was to investigate the effects of dialkyl 5-(hydroxy-methyl)isophthalates (HMI-1a3 and HMI-1b11) on SH-SY5Y-neuroblastoma cell proliferation and morphology with live cell imaging and to Alzheimer's disease-related Wnt, ERK1/2 and PKC signaling pathways with Western blotting. The main purpose was to evaluate the potential of the compounds for further in vitro and in vivo experiments. According to the results of this study both isophthalates, HMI-1a3 and HMI-1b11, had good binding affinities to PKCα and PKCδ. Both of them caused a dramatic increase in ERK1/2 phosphorylation which may be due to PKC activation and may thus suggest a PKC-dependent mechanism of action. However, the possible PKC activation did not cause downregulation of the PKC-isoforms α, β and δ. In addition, both HMI-1a3 and HMI-1b11 inhibited SH-SY5Y cell proliferation. HMI-1a3 was cytotoxic at 20 µM, while HMI-1b11 did not cause any cell death. Both compounds also induced neurite outgrowth. In addition, HMI-1a3 increased the amount of β-catenin. That could indicate the activation of Wnt-signaling, which is inhibited in Alzheimer's disease. Both of the compounds have potential for further studies because of the good binding to PKC and the beneficial effects on neurite outgrowth and Wnt signaling.
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