title: Cell-based assay for low- and high-scale screening of the Wnt/β-catenin signaling modulators.
abstract: Deregulation of the Wnt/β-catenin signaling pathway is associated with many serious disorders, including cancer and Alzheimer's disease. The pivotal player is β-catenin, which avoids degradation after activation of the pathway and is translocated to the nucleus, where it interacts with TCF/LEF transcription factors and induces expression of genes involved in cell cycle and apoptosis regulation. The identification of small molecules that may affect Wnt/β-catenin signaling remains an important target during the development of novel therapies. We used the TCF/LEF lentiviral vector and the Wnt-independent H1703 cell line to develop a luciferase reporter-based cell assay for screening of the Wnt/β-catenin pathway modulators. Following the optimization of cell density, concentration of activator, and stimulation time, the reporter system was validated by demonstrating its specific and dose-dependent response to several established modulators of Wnt/β-catenin signaling such as Wnt3a, small interfering RNA (siRNA) against β-catenin, glycogen synthase kinase 3 (GSK-3), and β-catenin/TCF transcription complex inhibitors. Two pilot screens of inhibitors and activators of Wnt/β-catenin signaling identified potential novel modulators of this pathway. Our findings suggest that the H1703-7TFP assay constitutes a suitable model of low background and high sensitivity for the low- and high-scale screening of the Wnt/β-catenin pathway modulators.
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland; Department of Molecular Cancerogenesis, Medical University of Lodz, 90-419 Lodz, Poland. Electronic address: maria.genowefa.mazur@gmail.com.
authors: Maria
authors: M
authors: Mazur
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland; Postgraduate School of Molecular Medicine, 02-091 Warsaw, Poland.
authors: Anna
authors: A
authors: Bujak
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland; Postgraduate School of Molecular Medicine, 02-091 Warsaw, Poland.
authors: Mikolaj
authors: M
authors: Matloka
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland.
authors: Sylwia
authors: S
authors: Janowska
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland; Department of Medical Biotechnology, Medical University of Lodz, 90-419 Lodz, Poland.
authors: Pawel
authors: P
authors: Gunerka
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland.
authors: Lukasz
authors: L
authors: Bojarski
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland.
authors: Aleksandra
authors: A
authors: Stanczak
authors: Laboratory of Animal Models, Nencki Institute, 02-093 Warsaw, Poland.
authors: Agata
authors: A
authors: Klejman
authors: Department of Molecular Cancerogenesis, Medical University of Lodz, 90-419 Lodz, Poland.
authors: Andrzej
authors: A
authors: Bednarek
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland.
authors: Monika
authors: M
authors: Lamparska-Przybysz
authors: Innovative Drugs R&D Department, Celon Pharma, 05-092 Lomianki/Kielpin, Poland.
authors: Maciej
authors: M
authors: Wieczorek
mesh_terms: D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000379:methods; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051820:TCF Transcription Factors / Q000235:genetics / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects*; D060509:Wnt3A Protein / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
chemical_list: D051820:TCF Transcription Factors; C555510:WNT3A protein, human; D060509:Wnt3A Protein; D051176:beta Catenin; D038362:Glycogen Synthase Kinase 3
keywords: Alzheimer’s disease; GSK-3 inhibitor; High-throughput screening; Luciferase reporter cell line; Wnt/β-catenin signaling