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

FH535 inhibits β-catenin/Tcf-mediated transcription and also suppresses the aggregation of co-activator GRIP1 and β-catenin with PPARδ and PPARγ. In cancer cells with elevated or active Wnt/β-catenin pathways, FH535 can inhibit cell proliferation.

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)