3,3'-Diindolylmethane Inhibits Activation of Akt through Inhibition of Hepatocyte Growth Factor Receptor c-Met Signaling.

Abstract

Abstract Background: 3,3'-Diindolylmethane (DIM), the major acid condensation product of indole-3-carbinol, is a promising anticancer compound that is bioavailable, non-toxic, and easily obtainable. Although DIM has strong anticancer activity against breast cancer cells in vitro and in vivo, the exact mechanism for this activity is not known. We and others have found that DIM inhibits activation of the Akt pathway in breast cancer cells. Akt is a serine threonine protein kinase involved in many important cellular functions including proliferation and cell survival. Akt is active when phosphorylated at serine-473 and threonine-308. In breast cancer cells, Akt can be activated by various stimuli, including signaling from growth factor receptors like the hepatocyte growth factor (HGF) receptor c-Met, which is often overexpressed in breast cancer. In this project, we show that c-Met is an upstream point of inhibition of the Akt pathway by DIM.Materials and Methods: When MDA-MB-231 breast cancer cells reached 70-80% confluency, we serum-starved them by reducing the fetal bovine serum concentration in the media from 10% to 1% for 20 hours and then treated with DIM or the vehicle control DMSO for 4 hours or the indicated time. When using recombinant growth factors to activate Akt or c-Met, we incubated cells in medium containing no fetal bovine serum and added the growth factors for 10 minutes after incubation with DIM. To analyze protein expression and protein phosphorylation we performed Western blotting. To analyze c-Met kinase activity we used a commercially available kit.Results: DIM does not inhibit activation of Akt downstream of either epidermal growth factor or insulin-like growth factor-1. However, DIM does inhibit Akt activation downstream of HGF and of fetal bovine serum. DIM inhibits tyrosine phosphorylation of c-Met at Y1349 and Y1234/1235. DIM does not affect total c-Met protein or mRNA levels and does not directly inhibit c-Met kinase activity.Discussion: While we have determined that DIM inhibits c-Met phosphorlyation, and therefore HGF signaling, the mechanism of this inhibition requires further study. We hypothesize that DIM induces serine phosphorylation of c-Met, since c-Met can be inhibited by reactive oxygen species (ROS)-induced phosphorylation of serine-985 by protein kinase C (PKC), and DIM increases ROS levels in cells.Components of the Akt pathway are frequent targets for mutations in breast cancer and c-Met and its ligand HGF are associated with lower survival and increased risk of distant metastases in breast cancer. This project represents an important step in determining the mechanism of action for DIM, a promising phytochemical for the treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6117.