Diindolylmethane (DIM) potentiates the effect of cisplatin in ovarian cancer cells. (A) SKOV-3 cells were exposed to 20 or 50 μM DIM for 24 h, followed by exposure to 10 μM cisplatin for another 24 h. Survival of control and treated cells was evaluated by sulforhodamine B assay. (B) Combination effect of cisplatin and DIM on the signal transducer and activator of transcription 3 (STAT3) pathway. Representative blots of control and treated cells were examined for Tyr-705 and Ser-727 STAT3, STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase 3. Blots were stripped and probed with actin. (C) Effect of combination treatment on nuclear localization of STAT3. Nuclear fractions from cells treated with or without 50 μM DIM or 10 μM cisplatin or both were subjected to western blotting. Representative blots of Tyr-705 STAT3 and STAT3 are shown. Lamin B was used as loading control. (D) Luciferase activity in combination treatment was measured in SKOV-3. Whole cell lysates were collected and firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. (E) Aortic rings (1 mm) were harvested from Sprague-Dawley rats, immersed in matrigel, and treated with interleukin (IL)-6 (50 ng/ml) in the absence or presence of cisplatin (10 μM) with or without DIM (50 μM) for 4 days, and then photographed under a microscope (4 ×). Representative photographs are shown. (F) DIM inhibits the invasion of SKOV-3 cells. Invasion assay was performed using Boyden's chamber (BD Sciences) according to manufacturer's instructions. (G) DIM inhibits vascular epithelial growth factor (VEGF) secretion. Cells were plated, stimulated with VEGF, and treated with DIM for 24 h. Media was collected and assayed for VEGF by ELISA (Invitrogen) kit according to manufacturer's instructions. All experiments were performed independently three times. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.