RESUMEN
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 percent of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38 percent 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 percent, 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.
Asunto(s)
Humanos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Indoles/antagonistas & inhibidores , Oximas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/antagonistas & inhibidores , beta Catenina/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Genes Reporteros/fisiología , Células Jurkat , Luciferasas/metabolismo , Plásmidos/efectos de los fármacos , Plásmidos/genética , Transfección/métodos , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/ß-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator ß-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/ß-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X ß-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 GSK3ß 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 ß-catenin signaling cells and decreased ß-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/ß-catenin signaling inhibitor NCTD.