DHPM derivatives inhibit angiogenesis both in vitro and in vivo.A, Tube formation analysis by HUVEC cells seeded onto the surface of the polymerized ECMatrix™ in 96-well plates after 11 h of treatment with 4 m, 4bt (dimethylenastron), 4p, 4bc, 4x and monastrol. Representative images of cells treated with 300 μM of each compound documented using an inverted light microscope at 20x magnification (left panel). Tube-like structures formed by HUVEC cells control (asterisks) or cells exposed to 30 μM, IC50 or 300 μM of 4 m, 4bt (dimethylenastron), 4p, 4bc, 4x and monastrol for 11 h, which have lost their ability to cluster in tube-like structures (arrows), were quantified by pattern recognition (right panel). Columns, means of scored tube formation; bars, SEM; **P < 0.01; ***P < 0.001 compared with untreated control. Seven frames per group of three independent experiments (n = 1) were quantified. B, Chorioallantoic membrane (CAM) tumor assay. MCF-7 and MDA-MB-231 cells control or treated with 500 ng of 4 m, 4bt (dimethylenastron), 4p, 4bc, 4x, monastrol or genistein (antiangiogenic control) were implanted into CAM and on the 17th day of development the quantification of total nets was performed by Wimasis Image Analysis (left panel). Columns, mean of total nets (n = 3–5); bars, SEM. A representative image of the most effective treatment for each cell compared with the respective controls can be observed (right panels).