Target protein interactions of indole‐3‐carbinol and the highly potent derivative 1‐benzyl‐I3C with the C‐terminal domain of human elastase uncouples cell cycle arrest from apoptotic signaling

Abstract

Elastase is the only currently identified target protein for indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin in cruciferous vegetables such as broccoli, cabbage, and Brussels sprouts that induces a cell cycle arrest and apoptosis of human breast cancer cells. In vitro elastase enzymatic assays demonstrated that I3C and at lower concentrations its more potent derivative 1-benzyl-indole-3-carbinol (1-benzyl-I3C) act as non-competitive allosteric inhibitors of elastase activity. Consistent with these results, in silico computational simulations have revealed the first predicted interactions of I3C and 1-benzyl-I3C with the crystal structure of human neutrophil elastase, and identified a potential binding cluster on an external surface of the protease outside of the catalytic site that implicates elastase as a target protein for both indolecarbinol compounds. The Δ205 carboxyterminal truncation of elastase, which disrupts the predicted indolecarbinol binding site, is enzymatically active and generates a novel I3C resistant enzyme. Expression of the wild type and Δ205 elastase in MDA-MB-231 human breast cancer cells demonstrated that the carboxyterminal domain of elastase is required for the I3C and 1-benzyl-I3C inhibition of enzymatic activity, accumulation of the unprocessed form of the CD40 elastase substrate (a tumor necrosis factor receptor family member), disruption of NFκB nuclear localization and transcriptional activity, and induction of a G1 cell cycle arrest. Surprisingly, expression of the Δ205 elastase molecule failed to reverse indolecarbinol stimulated apoptosis, establishing an elastase-dependent bifurcation point in anti-proliferative signaling that uncouples the cell cycle and apoptotic responses in human breast cancer cells.

The Antiproliferative Response of Indole-3-Carbinol in Human Melanoma Cells Is Triggered by an Interaction with NEDD4-1 and Disruption of Wild-Type PTEN Degradation

“…For use of the MG-132 proteasome inhibitor, G-361 and SK-MEL-20 melanoma cells were treated with or without 200 μM I3C for 48 hours, and the cell cultures incubated in presence or absence of 10 μM MG-132 for the last 5 hours of the treatment. The DNA content of propidium iodide stained nuclei from 48 hour I3C treated and untreated cells was determined by flow cytometry as previously described (21). …”

“…Western Blot analyses of samples electrophoretically fractionated on 8–10% acrylamide gels were carried out as previously described (20, 21). ECL Lightening reagents were used to visualize the primary antibody bound protein bands in nitrocellulose membranes and the results captured on ECL Autoradiography Film (GE Healthcare, Piscataway, NJ).…”

“…We recently established that I3C and its more potent and stable derivative 1-benzyl-I3C act as direct noncompetitive inhibitors of the proteolytic activity of neutrophil elastase, the first such identified target protein for I3C (19–21). The I3C inhibition of elastase enzymatic activity directly prevents cleavage of the CD40 member of the tumor necrosis factor receptor gene family, which causes CD40 signaling to switch from activating cell survival pathways to triggering anti-proliferative cascades in human breast cancer cells (20, 21).…”

“…The I3C inhibition of elastase enzymatic activity directly prevents cleavage of the CD40 member of the tumor necrosis factor receptor gene family, which causes CD40 signaling to switch from activating cell survival pathways to triggering anti-proliferative cascades in human breast cancer cells (20, 21). Relatively little is known about the responsiveness of human skin cancers to I3C beyond the observations that I3C increases the sensitivity to UV induced apoptosis and enhances cytotoxic responses in human melanoma (22, 23) and squamous cell carcinomas (24), respectively.…”

Metabolic Enzyme Sulfotransferase 1A1 Is the Trigger for N-Benzyl Indole Carbinol Tumor Growth Suppression

“…It is speculated to act in the estrogen pathway, although this claim is controversial. The N-BICs ( Figure 1A) differ from the parent I3C by virtue of having a benzyl group off of the indole nitrogen, rendering them more stable than the parent to acid (Aronchik et al, 2012;Nguyen et al, 2010).…”