Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells result from an estrogen receptor-mediated increase in transforming growth factor-alpha expression

3,3 ' -Diindolylmethane (DIM), a major in vivo product of indole-3-carbinol (13C), is a promising anticancer agent derived from vegetables of the Bras sica genus including broccoli, Brussels sprouts and cabbage. We report here that DIM has a potent cytostatic effect in cultured human Ishikawa endomet rial cancer cells. A combination of northern blot and quantitative PCR anal yses revealed that DIM induced the level of TGF-alpha transcripts by simila r to4-fold within 24 h of indole treatment. DIM also induced a 4-fold incre ase in the activity of the estrogen response marker, alkaline phosphatase ( AP). Co-treatment of cells with the estrogen receptor (ER) antagonist ICI, or with the inhibitor of PKA-mediated activation of the ER, H89, ablated th e DIM induction of both TGF-alpha expression and AP activity. Furthermore, DIM increased the maximum stimulatory effect of estrogen on TGF-alpha expre ssion. Co-treatment with the protein synthesis inhibitor, cycloheximide, ab olished the inductive effects of DIM, indicating differences in the mechani stic requirements of DIM and estrogen. DIM treatment also stimulated levels of secreted TGF-alpha protein by > 10-fold. The ectopic addition of TGF-al pha inhibited the growth of Ishikawa cells, whereas incubation with a TGF-a lpha antibody partially reversed the growth inhibitory effects of DIM. Take n together, these results extend our previous findings of the ligand indepe ndent estrogen receptor agonist activity of DIM, and uncover an essential r ole for the stimulation in TGF-alpha expression and the TGF-alpha activated signal transduction pathway in the potent cytostatic effects of DIM in end ometrial cancer cells.