Figure 4

Diindolylmethane (DIM) blocks cell migration and neovascularization by inhibiting hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). (A) SKOV-3, (B) OVCAR-3 or (C) TOV21G cells were plated, scratched with a pipette tip and incubated in the absence or presence of 50 μM DIM. Photographs were taken at 0 h and 24 h using an inverted microscope. The wound area in DIM treated and control cells were quantified by ImageJ software. Results are presented as means ± SD of triplicates. (D) DIM inhibits interleukin (IL)-6-induced vessel sprouting ex vivo. Representative photographs are presented. (E) Effect of DIM on proangiogenic proteins was analyzed by western blotting. SKOV-3 or OVCAR-3 cells were treated with 10 μM MG132 for 1 h and then exposed to 75 μM DIM for 6 h. (F) Cells were treated as above and then treated with IL-6 for 15 minutes. Representative blots of HIF-1α and VEGF are shown. Blots were further stripped and probed with actin. Experiments were performed independently three times. The Student's t test was used for statistical analysis to compare control and DIM treatment.