In vitro activity: T-5224 is a novel and selective inhibitor of c-Fos/activator protein-1 (AP-1) which is a transcriptional factor that regulates the expression of various genes associated with tumor invasion and migration. T-5224 has potential use in cancer and rheumatoid arthritis therapy. T-5224 ameliorates liver injury and improves survival through decreasing production of proinflammatory cytokines and chemokines in endotoxemic mice. T-5224 has been investigated in phase II human clinical trials. T-5224 (0-80 μM) significantly inhibits the invasion, migration, and MMP activity of HSC-3-M3 cells in a dose-dependent manner. T-5224 inhibited the invasion and migration of HNSCC cells in vitro, and prevented lymph node metastasis in head and neck cancer in an animal model. T-5224 (0-80 μM) significantly inhibits the invasion, migration, and MMP activity of HSC-3-M3 cells in a dose-dependent manner. There is no significant influence on HSC-3-M3 amd OSC-19 cells proliferation

Kinase Assay:

Cell Assay: T-5224 is dissolved in DMSO and diluted in DMEM. HSC-3-M3 cells are starved for 24 h with DMEM containing 0.5% FBS. The top chamber of the cell invasion device is coated with 50 μL of 0.1 × basement membrane extract solution and incubataed overnight. HSC-3-M3 cells (5.0 × 104 cells/well) are added to the top chamber with DMEM containing 0.5% FBS mixed with 0-80 μM T-5224; DMEM with 10% FBS is added to the bottom chamber and incubated for 48 h. The bottom plate is read using a multilabel plate reader. The data are compared with the standard curve to determine the fraction of invaded cells