| Registro completo |
| Provedor de dados: |
BJMBR
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| País: |
Brazil
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| Título: |
Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system
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| Autores: |
Chuang,K.A.
Lieu,C.H.
Tsai,W.J.
Wu,M.H.
Chen,Y.C.
Liao,J.F.
Wang,C.C.
Kuo,Y.C.
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| Data: |
2010-10-01
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| Ano: |
2010
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| Palavras-chave: |
Cancer
Plasmids
Reporter assay
Screening
Wnt
Β-catenin
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| Resumo: |
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.
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| Tipo: |
Info:eu-repo/semantics/article
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| Idioma: |
Inglês
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| Identificador: |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2010001000003
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| Editor: |
Associação Brasileira de Divulgação Científica
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| Relação: |
10.1590/S0100-879X2010007500091
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| Formato: |
text/html
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| Fonte: |
Brazilian Journal of Medical and Biological Research v.43 n.10 2010
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| Direitos: |
info:eu-repo/semantics/openAccess
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