title: Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system.
abstract: Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.
authors.affiliation: Department of Biotechnology, National Yang-Ming University, Taipei, Taiwan.
authors.forename: K A
authors.initials: KA
authors.lastname: Chuang
authors.forename: C H
authors.initials: CH
authors.lastname: Lieu
authors.forename: W J
authors.initials: WJ
authors.lastname: Tsai
authors.forename: M H
authors.initials: MH
authors.lastname: Wu
authors.forename: Y C
authors.initials: YC
authors.lastname: Chen
authors.forename: J F
authors.initials: JF
authors.lastname: Liao
authors.forename: C C
authors.initials: CC
authors.lastname: Wang
authors.forename: Y C
authors.initials: YC
authors.lastname: Kuo
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000379:methods; D017930:Genes, Reporter / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000037:antagonists & inhibitors*; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D010091:Oximes / Q000037:antagonists & inhibitors*; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection / Q000379:methods; D051153:Wnt Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C483321:6-bromoindirubin-3'-oxime; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007211:Indoles; D010091:Oximes; C555510:WNT3A protein, human; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin; C069741:norcantharidin; D008156:Luciferases