title: Visualization of cyclooxygenase-2 using a 2,3-diarylsubstituted indole-based inhibitor and confocal laser induced cryofluorescence microscopy at 20K in melanoma cells in vitro.
abstract: This study aimed at visualization of cyclooxygenase-2 (COX-2) protein expression in melanoma cells by confocal laser induced cryofluorescence microscopy using 4-(3-(4-methoxyphenyl)-1H-indol-2-yl)benzene-sulfonamide (C1) representative for a novel class of autofluorescent 2,3-diarylsubstituted indole-based selective COX-2 inhibitors. COX-2 expression was measured in human melanoma cell lines A2058 and MelJuso by immunocytochemistry and immunoblotting. Cellular uptake experiments using varying C1 concentrations down to 0.1 nM (with/without molar excess of celecoxib as control) were performed at 37 °C. Cryofluorescence microscopy was conducted at 20 K. COX-2 protein expression was successfully visualized by C1 in A2058 cells. COX-2-negative MelJuso cells showed no specific accumulation of C1. Control experiments using celecoxib and, additionally, implemented fluorescence spectroscopy confirmed specificity of both cellular uptake and intracellular association of C1. Cryofluorescence microscopy in combination with spectroscopy allowed for visualization of COX-2 protein expression in melanoma cells in vitro using a selective COX-2 inhibitor at very low concentrations.
authors.affiliation: Institute of Radiopharmacy (From January 2013 Institute of Radiopharmaceutical Cancer Research), Helmholtz-Zentrum Dresden-Rossendorf, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Tondera
authors.forename: Markus
authors.initials: M
authors.lastname: Laube
authors.forename: Christin
authors.initials: C
authors.lastname: Wimmer
authors.forename: Torsten
authors.initials: T
authors.lastname: Kniess
authors.forename: Birgit
authors.initials: B
authors.lastname: Mosch
authors.forename: Kay
authors.initials: K
authors.lastname: Großmann
authors.forename: Jens
authors.initials: J
authors.lastname: Pietzsch
mesh_terms: D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000032:analysis*; D052246:Cyclooxygenase 2 Inhibitors / Q000737:chemistry*; D006801:Humans; D007211:Indoles / Q000737:chemistry*; D008545:Melanoma / Q000201:enzymology*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D013449:Sulfonamides / Q000737:chemistry*; D000096926:Benzenesulfonamides
chemical_list: D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D007211:Indoles; D013449:Sulfonamides; C581021:4-(3-(4-methoxyphenyl)-1H-indol-2-yl)benzenesulfonamide; D000096926:Benzenesulfonamides; C496540:PTGS2 protein, human