Figure 3

Diindolylmethane (DIM) suppresses signal transducer and activator of transcription 3 (STAT3) activated by interleukin (IL)-6 or in cells overexpressing STAT3. Effect of DIM on STAT3 activation and apoptosis. (A) (i) SKOV-3, (B) (i) OVCAR-3 or (C) (i) TOV-21G cells were stimulated with 10 ng/ml IL-6 for 15 minutes after treatment with 75 μM DIM for 24 h. Whole cell lysates were resolved on 10% SDS-PAGE for the analysis of STAT3 phosphorylation at Tyr-705 and Ser-727, STAT3 expression, and cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP). Actin was used as a control for loading. Effect of IL-6 on apoptosis was also determined in (A) (ii) SKOV-3 and (B) (ii) OVCAR-3 cells. (D) Effect of STAT3 small hairpin (sh)RNA on apoptosis was also determined in SKOV-3 cells. (E) Effect of DIM on STAT3 activation, STAT3 expression, and apoptosis in SKOV-3 cells overexpressing STAT3. Cells were transfected with an empty vector or a plasmid expressing STAT3 for 24 h and treated with 75 μM DIM for another 24 h. (E) (i) Whole cell lysates were analyzed by western blotting. (E) (ii) Effect of STAT3 overexpression on DIM-induced apoptosis was determined by flow cytometry. (F) Effect of DIM on IL-6 secretion was determined in (F) (i) SKOV-3, (ii) OVCAR-3 and (iii) OVCAR 429 cells. The experiments were repeated three times and similar results were obtained. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.