| Indole-3-carbinol (I3C), a phytochemical derived from Brassica vegetables, such as broccoli and cabbage, exhibits potent anti-tumorigenic properties in human reproductive cancers. In human breast cancer cells, I3C inhibition of both intracellular and extracellular forms of its target protein neutrophil elastase ablates processing of the cell cycle component cyclin E and Tumor Necrosis Factor Receptor superfamily member CD40, respectively, which together induce a G1 cell cycle arrest. I3C inhibition of elastase activity causes the accumulation of full-length CD40 in the plasma membrane and amplification of downstream signaling, leading to downregulation of NFkappaB transcription factor activity and disruption of its nuclear localization. Through this pathway, I3C decreased expression of NFkappaB target genes critically involved in breast cancer cell-cycle progression, as well as TRAF1 expression, which disrupts a positive feedback loop between TRAF1 signaling and NFkappaB activity. SiRNA ablation of NFkappaB negative regulators TRAF3 and IkappaB resulted in loss of I3C induced cell-cycle arrest, indicating that these molecules are critically involved in the indole antiproliferative effect. Utilizing a series of in vitro enzymatic assays and computer-aided analyses, I3C was shown to inhibit elastase activity through interaction with the specific binding sites within the enzyme structure. The N-butoxy and N-benzyl derivatives of I3C inhibit elastase activity with higher efficacy than the parent indole. Computational modeling of molecular binding revealed the presence of an additional interaction site that is preferentially occupied by both derivatives and not by I3C, suggesting that an enhanced interaction with elastase accounts for the increased antiproliferative effects of the I3C derivatives.Finally, using a combination of biochemical assays and computer-aided modeling, the phosphatase PTEN was identified as another direct binding target for I3C. It is proposed that I3C binding to PTEN disrupts the phosphatase interaction with its ubiquitin ligase NEDD4-1 and thereby prevents PTEN proteasome mediated degradation. Using the human melanoma cell-line G-361, I3C was shown to stabilize membrane-localized PTEN protein, which caused a decreased phosphorylation of AKT and IKK kinases, resulting in the loss of nuclear-associated NFkappaB transcription factor activity. Through this signaling cascade, I3C induces a G1 cell cycle arrest, accompanied by downregulation of cell-cycle components cyclin D1 and CDK6 and upregulation of CDK inhibitors p21 and p27 protein levels, and stimulates apoptosis associated with caspase 3 and PARP activation. Taken together, this study implicates I3C as a potent and promising therapeutic agent for treatment of human reproductive cancers and melanoma. |
