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 were cloned into pCEP4 plasmid, replacing the cytomegalovirus promoter. The plasmids were transfected into HepG2 cells, and Hygromycin-resistant clones were pooled. Library screening was done at 20 μmol/L concentration in HepG2 serum-free media. Hits were 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 lines

HCT116, SW48, RKO, LoVo, COLO205, IEC6, A427, HCC15, NCI-H1703, A549, HepG2, Hep3b, Huh7 and fibroblasts

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

30 μM; 48 hrs

Applications

FH535 showed selective anti-proliferation effect on some cancer cells (HCT116, SW48, RKO, LoVo, COLO205, A427, HCC15, NCI-H1703, A549, HepG2, Hep3b and Huh7 cells) expressing high or active Wnt/β-catenin pathway.

Animal models

Nude mouse bearing pancreatic cancer xenografts

Dosage form

25 mg/kg; i.p.; every 2 days for 20 days

Applications

FH535 significantly repressed pancreatic cancer xenograft growth in vivo and showed better performance status with respect to body weight loss.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

FH535 is a small molecule inhibitor of Wnt/B-catenin with IC50 values of 15.4μM, 10.9μM, 9.3μM, respectively in LCSC, Huh7, PLC cell lines [1].

FH535 can inhibit the growth of colon, lung, and hepatocellular carcinoma line but not normal fibroblasts. It makes FH535 potentially be a promising therapeutic approach for cancer cells. It is reported that FH535 has ability to inhibit growth, migration, and invasion of TN breast cancer cell lines (MDA-MB-231 and HCC38) without affecting adhesive abilities of cells to type I collagen [2].

FH535 is also an inhibitor of peroxisome proliferator–activated receptor (PPAR). It plays a role as a dual PPARγ and PPARδ antagonist that is able to inhibit GRIP1 and β-catenin recruitment [3].

References:
[1] Roberto R. Galuppo, Roberto Gedaly, Paul Angulo, Michael F. et al. FH535 inhibits wnt/β-catenin signaling pathway in liver cancer stem cells and HCC cell lines. Hepatology. 2013, October: 466A.
[2] Joji Iida, Jesse Dorchak, John R. Lehman, Rebecca Clancy, Chunqing Luo, Yaqin Chen, Stella Somiari, Rachel E. Ellsworth, Hai Hu, Richard J. Mural, Craig D. Shriver. FH535 Inhibited Migration and Growth of Breast Cancer
Cells. PLOS ONE. 2012, 7(9): 1-11.
[3] Shlomo Handeli and Julian A. Simon. A small-molecule inhibitor of Tcf/β-catenin signaling down-regulates PPARγ and PPARδ activities. Molecular Cancer Therapeutics. 2008, 7:521-529.