DBPT induced G2/M-phase arrest at 5 M; however, it did not markedly affect apoptosis under the experimental conditions (Fig. M. Treating LNCaP and DU145 cells with DBPT led to a time-dependent cell-cycle arrest in the G2/M phase and increased levels of G2/M checkpoint proteins, such as cyclin B1, cdc25C, phosphorylated histone H3, and MPM-2. DBPT induced the phosphorylation of Bcl-xL and Bim, and induced apoptosis, as evidenced by cleavage of caspase and poly (ADP-ribose) polymerase. DBPT also effectively induced apoptosis in Bcl-2-overexpressing DU145 cells. Furthermore, DBPT decreased hypoxia-inducible factor 1 and vascular endothelial growth factor expression in LNCaP cells under both normoxia and hypoxia. CONCLUSIONS DBPT can suppress proliferation, induce apoptosis, and down-regulate pro-angiogenic molecules in prostate cancer cells, and might be useful in treating prostate cancer. (BD Pharmingen, San Diego, CA); hypoxia-inducible factor (HIF)-1 and HIF-1 (BD Biosciences, San Diego, CA); caspase-9, phosphorylated JNK, JNK, and phosphorylated c-Jun (Cell Signaling Technology, Beverly, MA); Bim (Calbiochem, La Jolla, CA); and -actin (Sigma-Aldrich, St. Louis, MO). Open in a separate window Fig. 1 Chemical structure of 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidione (DBPT) Cell Proliferation Assay The antiproliferative effects of DBPT on LNCaP and DU145 cells were determined by the XTT assay. Cells (5C10 103 cells in 100 l of culture medium/well) were seeded in 96-well flat-bottomed plates and treated the next day with DBPT at the indicated concentrations. After 72 h, cell viability was determined IDO-IN-4 by colorimetric assay with the tetrazolium dye XTT using a Cell Proliferation Kit II IDO-IN-4 (Roche Diagnostics, Indianapolis, IN) as described previously [7]. The experiments were performed at least three times for each cell line. Flow Cytometry Analysis For detection of cell-cycle progression and apoptosis, fixed cells were resuspended in phosphate-buffered saline containing 10 g/ml propidium iodide (PI; Roche Diagnostics) and 10 g/ml RNase A (Sigma-Aldrich) at 37C for 30 min. Cell-cycle analysis was performed using an Epics Profile II flow cytometer (Beckman Coulter, Fullerton, CA) with IDO-IN-4 MultiCycle software (Phoenix Flow Systems, San Diego, CA). The accumulation of IDO-IN-4 sub-G1 cells, an indicator of DNA fragmentation and apoptosis, was used to quantify apoptosis. Western Blot Analysis Whole-cell extracts and cytosolic extracts were prepared as described previously [8,9]. Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). Briefly, equal amounts of proteins (30C50 g) were subjected to electrophoresis under reducing conditions on 8C12.5% (w/v) polyacrylamide gels and then transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). The membranes were incubated with a primary antibody followed by a peroxidase-linked secondary antibody, which was detected using the ECL Western blotting system (Amersham). Plasmid Transfection The plasmid containing Bcl-2 cDNA was provided by Dr. Timothy J. McDonnell (The University of Texas M. D. Anderson Cancer Center) [10]. Plasmid transfection was performed using FuGENE6 reagent (Roche Diagnostics), and transfected cells were selected IDO-IN-4 in the presence of 500 g/ml G418. Enzyme-Linked Immunosorbent Assay for VEGF The VEGF concentration in media from DBPT-treated LNCaP cells was determined by using an enzyme-linked immunosorbent assay kit for VEGF protein (R&D Systems, Inc., Minneapolis, MN) according to the manufacturers protocol. The results were expressed as the concentration of VEGF (pg/ml) per microgram of protein in each well. RESULTS DBPT Inhibited Proliferation of Human Prostate Cancer Cells To test whether DBPT is active against prostate cancer cells, we determined the cell growth inhibitory effect of DBPT on cells from two human prostate cancer cell lines, LNCaP and DU145. The cells were treated with DBPT at various concentrations for 72 h and examined for cell viability by uvomorulin using an XTT assay. DBPT inhibited the growth of LNCaP and DU145 cells in a dose-dependent manner (Fig. 2A). The 50% inhibitory concentrations (IC50 values) in LNCaP.