To identify small molecule compounds capable of inhibiting cancer cell invasion, a novel three-dimensional high-throughput invasion assay was used to screen the National Cancer Institutes Developmental Therapeutics Program compound library (Diversity Set II) against aggressive, androgen-independent PC3 human prostate cancer cells. with the SpectraMaxL (Molecular Devices). Transwell Migration. Transwell migration assays were performed as described previously (Dufour et al., 2008), except nuclei were stained in Hoechst/phosphate-buffered saline (PBS; 1:2000) for 20 minutes and imaged using a Nikon Eclipse TE2000-S equipped with a Vincristine sulfate Sutter Instruments SmartShutter System and a QiClick QImaging camera. Migrated cells were counted with the assistance of the Nikon Elements Basic Research Software analysis tools. Scratch Wound Migration Assay. Cells were grown to confluence in a 12-well plate and serum starved to induce cell cycle synchronization with or without TFP overnight under standard tissue culture conditions. A scratch wound was made in each well the Rabbit polyclonal to PNLIPRP2 following morning, and cells were washed twice with 1 PBS and supplemented with complete media containing drugs or vehicle. Cells were allowed Vincristine sulfate to migrate over 8 hours, with bright field images being taken at time 0 and time 8 hours. Area for time 0 and time 8 hours was calculated using the Nikon Elements Basic Research Software analysis tools and percent change was calculated. Two-Dimensional Dot Migration Assay. A collagen-cell mixture was dotted in a 96-well dish in a similar fashion to the three-dimensional invasion assay. After collagen solidification, cell-matrix dots were overlaid with complete media. Cells were allowed to migrate up to 8 hours. Cells were then stained in Hoechst/PBS (1:2000), and images were captured using the previously described microscope and camera system. Migration was then quantified by counting nuclei using the Nikon Elements Basic Research Software analysis tools. Gelatin Zymography. Gelatin zymography was performed as described (Zucker et al., 1995). After electrophoresis, the gels were incubated in Triton X-100 to replace SDS followed by incubation in a Tris-based buffer overnight at 37C. Staining was accomplished using Coomassie Brilliant Blue, and cleared areas were indicative of gelatinolytic activity. Immunoblotting and Immunofluorescent Staining. Immunoblotting was done according to previously published methods and developed on a BioRad ChemiDoc (Hercules, CA) (Cao et al., 1996). Immunofluorescent staining began by fixing treated cells in 4% paraformaldehyde in PBS at 4C, followed by permeabilization in 0.2% Triton X-100 at room temperature for 10 minutes. Blocking solution was composed of 3% bovine serum albumin/5% normal goat serum in PBS. After 1-hour blocking at room temperature, cells were exposed to antiCp-test was used to determine significant differences; any < 0.05 was considered significant. Results Identification of Compounds Capable of Inhibiting Cancer Cell Invasion. To identify small molecule compounds capable of inhibiting cancer cell invasion, a novel three-dimensional high-throughput invasion assay was used to screen the National Cancer Institutes Developmental Therapeutics Program compound library (Diversity Set II) against aggressive, androgen-independent PC3 human prostate cancer cells. This particular compound library includes 1974 compounds and covers a wide variety of chemical structures. After incubating compounds (10 tubulin were used as controls. (B) TFP treatment of Vincristine sulfate HT1080 cells reduces the transcriptional activity of Pulkoski-Gross, J. Li, Cao. Pulkoski-Gross, J. Li, Zheng, Y. Li, Ouyang. Pulkoski-Gross, J. Li, Rigas, Zucker, Cao. Pulkoski-Gross. Footnotes This work was supported in part by the Baldwin Breast Cancer Foundation and National Cancer Institute [1R01CA166936 to J.C.] and the United States Army Medical Research Acquisition Activity award [W81XWH10-1-0873 to B.R.]. dx.doi.org/10.1124/mol.114.096941..