Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system.

\ Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/beta-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator beta-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/beta-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X beta-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 GSK3 beta inhibitor (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO; 5 mu M) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 mu M) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 mu M NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing H-3-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 mu M) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant beta-catenin signaling cells and decreased beta-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/beta-catenin signaling inhibitor NCTD.