Evaluation of anti-Wnt/ß-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system.
Braz J Med Biol Res
; 43(10): 931-41, 2010 Oct.
Article
em En
| MEDLINE
| ID: mdl-20835687
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Oximas
/
Transdução de Sinais
/
Compostos Bicíclicos Heterocíclicos com Pontes
/
Proteínas Wnt
/
Beta Catenina
/
Indóis
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Braz J Med Biol Res
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Taiwan