FH535, a Wnt/β-catenin signaling and PPAR inhibitor, exhibits anti-tumor activities.

FH535抑制β-连环蛋白/Tcf介导的转录,同时还具有抑制共激活剂GRIP1和β-连环蛋白对PPARδ 和 PPAR的聚集作用。在表达较高的或活跃的Wnt/β-连环蛋白通路癌细胞中,FH535能够抑制细胞增殖。

High-throughput Library Screen: Three copies of the optimized or mutated Tcf-binding element from TOPFLASH or FOPFLASH driving a secreted alkaline phosphatase reporter gene are cloned into pCEP4 plasmid, replacing the cytomegalovirus promoter. The plasmids are transfected into HepG2 cells, and hygromycin-resistant clones are pooled. Library screening is done at 20 μmol/L concentration in HepG2 serum-free media. Hits are tested in the HCT116 cell line for inhibition of TOPFLASH luciferase activity but not for inhibition of a reporter activity controlled from β-actin promoter.

Cell viability is determined by the modified 3H-thymidine incorporation assay. Briefly, cells are plated in 96-well microplates for 24 h and treated in triplicate with various concentrations of the test compound. After 48 h of compound exposure, the cells are incubated for an additional 48 h in compound-free medium. The cells are then incubated in medium containing 3H-thymidine for 24 h, washed and mixed with the scintillant in the 96-well plate. Individual wells are counted with a 96-well scintillation counter and the LC50 is calculated.(Only for Reference)