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Evaluation of anti-Wnt/?-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system.
| Content Provider | Scilit |
|---|---|
| Author | Chuang, K. A. Lieu, C. H. Tsai, W. J. Wu, M. H. Chen, Y. C. Liao, J. F. Wang, C. C. Kuo, Y. C. |
| Copyright Year | 2010 |
| Description | 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. |
| Related Links | http://www.scielo.br/pdf/bjmbr/v43n10/536.pdf |
| Ending Page | 941 |
| Page Count | 11 |
| Starting Page | 931 |
| ISSN | 0100879X |
| e-ISSN | 1414431X |
| DOI | 10.1590/s0100-879x2010007500091 |
| Journal | Brazilian Journal of Medical and Biological Research |
| Issue Number | 10 |
| Volume Number | 43 |
| Language | English |
| Publisher | FapUNIFESP (SciELO) |
| Publisher Date | 2010-10-01 |
| Access Restriction | Open |
| Subject Keyword | Brazilian Journal of Medical and Biological Research Biochemistry and Molecular Biology |
| Content Type | Text |
| Resource Type | Article |
| Subject | Neuroscience Cell Biology Physiology Medicine Biochemistry Biophysics Immunology Pharmacology, Toxicology and Pharmaceutics |
