The kidney and lungs were excised from indicated mice, then fixed in 4% paraformaldehyde and stained with haematoxylin and eosin. anti-metastatic mechanisms. In this study, six CRC cell lines were used. We showed that YH-306 significantly inhibited the migration and invasion of CRC cells in a dose-dependent manner. In addition, YH-306 inhibited cell adhesion and protrusion formation of HCT116 and HT-29 CRC cells. Moreover, YH-306 potently suppressed uninhibited proliferation in all six CRC cell lines tested and induced cell apoptosis in four cell lines. Furthermore, YH-306 inhibited CRC colonization and suppressed CRC growth in a xenograft mouse model, as well as hepatic/pulmonary metastasis actin polymerization assay This assay was performed as described 11 with some modifications. In brief, purified pyrene-labelled actin was re-suspended and incubated in general actin buffer for 1?hr on ice to depolyermize any actin oligomers, followed by micro-centrifugation at 4C for 30?min. Exactly, 2?M of actin alone or 2?M of actin, 13?nM of Arp2/3 complexes and 100?nM of WASP protein VCA domain were incubated with DMSO (control) or 50?M YH-306 for 15?min. on ice before pyrene actin fluorescence was measured over time. Western blot analysis After the treatment of YH-306, cells were harvested and lysed in radio immunoprecipitation assay buffer containing protease/phosphotase inhibitors (Roche). Lysates were combined with sample loading buffer and heated at 100C for 10?min. Protein samples were eluted in sample buffer and subjected to SDS-PAGE. Measurement of YH-306 binding to Arp2/3 using biolayer interferometry ProteinCsmall molecules interactions were examined with an Octet QK (FortBio, Shanghai, China) by biolayer interferometry as described in previous studies 20C23. In brief, Arp2/3 protein complex was PEG-biotinylated with NHS-PEG4biotin (Thermo-Pierce), and buffer exchanged on PD-10 desalting columns. Then, biotinylated Arp2/3 protein complex was immobilized on streptavidin-coated fibre optic tips (FortBio). YH-306 or CK-636, the positive control, was diluted into optimized binding (-)-Talarozole buffer [25?mM Na HEPES (pH 8.0), 50?mM arginine-glutamate, and 150?mM NaCl]. Statistical analysis Results were statistically analysed using the Student’s screening more than 70 analogues. As shown in Figure?Figure1B,1B, YH-306 significantly inhibited the migration of two human CRC cell lines (HCT116 and HT-29) and one mouse CRC cell line (CT-26) in a wound healing migration assay. To confirm the effect of YH-306 on migration, a transwell migration assay was performed and we found that migration of CT-26 cells was significantly reduced in a dose-dependent manner after treatment of YH-306, as shown in Figure?Figure1C.1C. During metastasis, cancer cells need to pass through the basement membrane, and invade surrounding tissues to infiltrate distant organs 5. To assess the effect of YH-306 on this process, we used type I collagen and Matrigel as substrates. As shown in Figure?Figure1D,1D, YH-306 evidently prevented CT-26 cells from invading the type (-)-Talarozole I collagen- or Matrigel-coated membrane in a dose-dependent manner. YH-306 inhibits adhesion and spreading of CRC cells Cancer cell adhesion and cell spreading based on ECM components such as type I collagen or fibronectin are required for movement of metastatic cancer into new sites. Suppression of adhesion and spreading of CRC cells is therefore considered as a promising strategy for metastatic cancer therapy 15. To determine whether YH-306 inhibit CRC cell adhesion, we treated HCT116 and HT-29 seeded onto type I collagen or fibronectin with various concentrations of YH-306. As shown in Figure?Figure2A,2A, 50?M YH-306 significantly reduced HCT116 and HT-29 adhesion onto type I collagen or fibronectin. Quantitative data revealed that 50?M YH-306 inhibited 67% of HCT116 cell and 78% of HT-29 cell attachment to type I collagen, and attachment to fibronectin was also significantly reduced by YH-306. These results showed that YH-306 significantly inhibited HCT116 and HT-29 cells attachment to type I collagen or fibronectin in a dose-dependent manner. Furthermore, we tested the effect of YH-306 on (-)-Talarozole cell spreading, and results in Figure?Figure2B2B showed that YH-306 significantly suppressed cell spreading on type I collagen or fibronectin in a dose-dependent Rabbit Polyclonal to AP-2 manner. Cells treated with YH-306 retained a rounded morphology (Fig.?(Fig.2B)2B) and had defects in polarized extension (Fig.?(Fig.2C2C). Open in a separate window Fig 2 YH-306 inhibits cell adhesion and spreading of colorectal cancer cells..