Journal: Toxins

Article Title: Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines

doi: 10.3390/toxins16020080

Figure Lengend Snippet: Subtypes of nAChR studied in this work: muscle-type heteropentameric α1β1δε receptor and neuronal α7 and α9 homopentameric receptors. Orthosteric binding sites for agonists and competitive antagonists are shown as red triangles. Orthosteric agonists choline, acetylcholine, and epibatidine are shown as chemical formulae; peptide neurotoxins azemiopsin, [A10L]PnIA, and RgIA are shown by their respective sequences (braces denote disulfide bonds); and small protein α-bungarotoxin is shown by ribbon representation of spatial structure. Azemiopsin is the neurotoxic peptide that has been identified in Azemiops feae viper venom. [A10L]PnIA is the modified α-conotoxin that has been identified in the venom of marine snail Conus pennaceus , and α-conotoxin RgIA has been identified in Conus regius venom. The peptide toxins used in this study were chemically synthesized in house and described elsewhere. Arrows denote activation of the receptor, T-shaped arrows denote receptor inhibition.

Article Snippet: The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently.

Techniques: Binding Assay, Modification, Synthesized, Activation Assay, Inhibition

Journal: Toxins

Article Title: Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines

doi: 10.3390/toxins16020080

Figure Lengend Snippet: Registration of transient fluorescent signals from the cytoplasmic calcium indicator Fluo-4 AM in U87MG cells grown on FBS-supplemented medium and exposed to nAChR ligands. ( A ) The microphotographs show the change in indicator fluorescence intensity before and after application of acetylcholine (ACh). ( B ) Application of ACh caused an increase in intracellular Ca 2+ levels, which registered as an increase in fluorescence intensity (fluorescence units, fl.u.). ( C , D ) No significant effect of the inhibitors azemiopsin or RgIA on the ACh-stimulated change in Ca 2+ levels was detected, indicating the non-involvement of the muscle-type and α9 nAChRs. ( E ) The α7 nAChR antagonist [A10L]PnIA prevented an ACh-stimulated increase in intracellular Ca 2+ levels. Each curve shows the change in intracellular Ca 2+ concentration in a single cell. The arrow indicates the time of agonist application.

Article Snippet: The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently.

Techniques: Fluorescence, Concentration Assay

Journal: Toxins

Article Title: Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines

doi: 10.3390/toxins16020080

Figure Lengend Snippet: Effects of nAChR ligands on the intracellular Ca 2+ concentration in GBM. Cells were loaded with Fluo-4 fluorescent calcium indicator. The left diagram of each panel shows the acetylcholine-evoked Ca 2+ increase in cells grown on FBS-supplemented medium; the right diagram of the panel demonstrates the responses of the cells in serum-free (NS-21/EGF/bFGF-supplemented) medium. The data are presented for U87MG model cell line ( A ) as well as the GBM cell lines 011 ( B ), 019 ( C ), 022 ( E ), and 067 ( F ). The transient signal amplitude of the calcium indicator is measured in conventional units, c.u. For the violin plots, the black horizontal dotted lines display the mean as well as the 25th and 75th percentiles. Panel ( D ) shows the concentration-dependent ion currents in the whole-cell patch-clamp experiment for cell culture 019, grown in FBS-supplemented medium, in response to the mixture of α7 nAChR-selective agonist PNU 282987 and positive modulator PNU 120596. p -values were computed using the Kruskal–Wallis test and pairwise Dunn’s test. Asterisks indicate significant differences at * p < 0.05 and ** p < 0.01; ns, p > 0.05.

Article Snippet: The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently.

Techniques: Concentration Assay, Patch Clamp, Cell Culture

Journal: Toxins

Article Title: Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines

doi: 10.3390/toxins16020080

Figure Lengend Snippet: Influence of nAChR ligands on cell proliferation in FBS-containing ( A , C , E , G , I ) and serum-free ( B , D , F , H , J ) medium assessed by AlamarBlue assay. Values were normalized to control without any ligand. The data are presented for GBM cell lines 011 ( A , B ), 019 ( C , D ), 022 ( E , F ), and 067 ( G , H ), as well as for U87MG ( I , J ). The concentration of azemiopsin and [A10L]PnIA was 1 μM. Data are represented as the mean ± SD; for all experiments n = 5. p -values were computed using one-way ANOVA followed by Dunnett’s test. Asterisks indicate significant differences at * p < 0.05 and ** p < 0.01.

Article Snippet: The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently.

Techniques: Alamar Blue Assay, Concentration Assay

Journal: Toxins

Article Title: Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines

doi: 10.3390/toxins16020080

Figure Lengend Snippet: The effects of nAChR antagonists on the proliferation of GBM cells under different cultivation conditions assessed by AlamarBlue assay. Values were normalized to control without ligands. The data are presented for the U87MG model cell line ( A ) as well as GBM cell lines 011 ( B ), 019 ( C ), 022 ( D ), and 067 ( E ). The concentration of azemiopsin, RgIA, and [A10L]PnIA was 1 μM. Data are represented as the mean ± SD; for all experiments n = 5. p -values were computed using one-way ANOVA followed by Dunnett’s test. Asterisks indicate significant differences at * p < 0.05, ** p < 0.01 and **** p ≤ 0.0001.

Article Snippet: The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently.

Techniques: Alamar Blue Assay, Concentration Assay

Journal: Toxins

Article Title: Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines

doi: 10.3390/toxins16020080

Figure Lengend Snippet: The effect of nAChR stimulation with choline alone and in combination with different antagonists on the proliferation of GBM cells in serum-free medium. AlamarBlue assay was used. Values were normalized to control without ligands. The data are presented for the U87MG model cell line ( A ) as well as GBM cell lines 011 ( B ), 019 ( C ), 022 ( D ), and 067 ( E ). The concentration of epibatidine, azemiopsin, RgIA, and [A10L]PnIA was 1 μM. Data are represented as the mean ± SD; for all experiments n = 5. p -values were computed using one-way ANOVA followed by Dunnett’s and Tukey’s test. Asterisks indicate significant differences at * p < 0.05, ** p < 0.01, and *** p ≤ 0.001.

Article Snippet: The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently.

Techniques: Alamar Blue Assay, Concentration Assay