Figure 3

Anti-angiogenic and anti-tube formation potential of TAM and CXB. (A) In vivo CAM assay. CAMs were implanted with sponges loaded with serum-free medium alone or supplemented with vascular endothelial growth factor (VEGF), TAM, CXB, or TAM plus CXB. (B) Inhibition of capillary-like tube formation in vitro (HUVECs assay). HUVECs were seeded (7.5 x 10³ cells/well) into a 96-well tissue culture plate coated with 50 μl Matrigel. Then, TAM and/or CXB were added. Cells were incubated in HUVEC growth medium in a 37°C, 5% CO₂ incubator. Tube formation was observed for 24 h and images were taken (magnification of 10×). (C) Number of blood vessels in CAM assay was counted as means ± SD of blood vessel count for four independent experiments P < 0.05. (D) Number of capillary-like structures in capillary-like tube formation assay was counted using light microscopy. Data are presented as means ± SD of four independent experiments. (E) Western blotting analysis of apoptotic and angiogenic markers in MCF7 and MDA-MB-231 cells treated with TAM, CXB, or both. β-Actin was used as an invariant control for equal loading. Representative blots from three independent experiments are shown. T + C, TAM plus CXB; UT, untreated cells.