Indole specificity of the I3C inhibition of elastase enzymatic activity. (A) Flow cytometry analysis of MDA-MB-231 cells treated for 72 h with the DMSO vehicle control, 100 μM I3C, 30 μM DIM, or 100 μM tryptophol. (B) MDA-MB-231 cells were treated with 100 μM I3C, 30 μM DIM, 100 μM tryptophol, or the DMSO vehicle control for 72 h. Half of the immunoprecipitated cyclin E was assessed for the associated CDK2 kinase activity using Histone H1 as the in vitro substrate, and the other half was analyzed for cyclin E form by Western blots. (C) The effects of indoles on human neutrophil elastase processing of cyclin E was analyzed by using an in vitro transcription–translation system as described. The indicated reaction mixtures contained the DMSO vehicle control, 50 μM I3C, 30 μM DIM, or 50 μM tryptophol.

siRNA ablation of elastase production mimics the effects of I3C on cyclin E protein processing and the cell-cycle arrest. (A) MDA-MB-231 breast cancer cells were transfected with or without human neutrophil elastase-specific siRNA or with scrambled siRNA sequences for 24 h, and resulting levels of cellular elastase were monitored by indirect immunofluorescence. DAPI staining of nuclear DNA was used as a control for the presence of cells in siRNA-treated and untreated samples. (B) siRNA (elastase or scrambled)-transfected and control-transfected cells were then treated with or without 100 μM I3C for 48 h, and electrophoretically fractionated total cell extracts were examined for the levels of the 50-kDa and the 35-/33-kDa cyclin E by Western blot analysis. The level of hsp90 was used a gel loading control. (C) siRNA (elastase or scrambled)-transfected and control-transfected cells were then treated with or without 100 μM I3C for 48 h, and cell proliferation was monitored by flow cytometry for DNA content of propidium iodide-stained nuclei.

I3C inhibits elastase enzymatic activity and elastase-dependent processing of cyclin E. (A) Schematic representation of full-length cyclin E, highlighting the elastase consensus cleavage site (AVCADP). To assess endogenous cyclin protein forms, MDA-MB-231 cells were treated with or without 100 μM I3C for 48 h, and cell extracts were electrophoretically fractionated and analyzed by Western blots for the presence of the 50-kDa and hyperactive 35-kDa forms of cyclin E. (B) For the in vitro assay of elastase processing of cyclin E protein, full-length cyclin E was transcribed and translated in vitro followed by digestion of the cyclin E protein product by human neutrophil elastase in the presence of the indicated concentrations of I3C or DMSO. The “No DNA” sample was a reaction mixture without added cyclin E cDNA. Western blot analysis of the entire reaction mixtures was performed by using cyclin E-specific antibodies. (C) The in vitro elastase processing of cyclin E was assessed in the presence of the DMSO vehicle control, 50 μM I3C, and the known elastase inhibitors 25 μM elastatinal or 5 μM methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (CMK). Western blot analysis of the entire reaction mixture was performed by using cyclin E-specific antibodies. (D) For zymography, equal amounts of purified human elastase were electrophoretically resolved on a 10% polyacrylamide gel containing elastin as a substrate. After renaturation with 2.5% Triton X-100, each lane was cut out and placed in developing buffer containing either DMSO or the indicated concentrations of I3C for 25 h at 37 °C. Gel slices were stained with Coomassie blue followed by an overnight destain. Clear bands represent proteolytic degradation of substrates. (E) Purified human elastase, chymotrypsin, trypsin, or thrombin were electrophoretically resolved in gels containing gelatin (for evaluation of elastase, chymotrypsin, or trypsin) or fibronectin (for evaluation of thrombin), the enzymes were renatured in 2.5% Triton X-100, and individual gel slabs were developed in either DMSO or 500 μM I3C for 25 h at 37 °C.

Kinetic analysis of the indole inhibition of elastase activity. (A) The in vitro elastase cyclin E processing assay was used to determine the effects of I3C on the K_m and V_max for human neutrophil elastase enzymatic activity. Human elastase, the indicated cyclin E substrate concentrations, and indicated concentration of I3C were all mixed to a total volume of 20 μL and incubated for 5 min at 37 °C followed by Western blot analysis of cyclin E. The reaction velocities were determined as a function of the loss of the 50-kDa cyclin E protein and were graphed versus the cyclin E concentration. (B) Lineweaver–Burk and Dixon plot analysis of the I3C inhibition of elastase processing of cyclin E using the in vitro transcription–translation reactions. Double reciprocal plotting of 1/velocity versus 1/substrate yields the Lineweaver–Burk plot. For the Dixon plots, the ratio 1/V is plotted against the different concentrations of I3C at 2 different substrate concentrations (0.6 and 1.2 ng), and the K_i values were graphically determined as the intercept with the abscissa. (C) Lineweaver–Burk and Dixon plot analysis of the I3C inhibition of human elastase hydrolysis of the chromogenic substrate methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (MetS).