Ida Aronchik

Indole‐3‐carbinol activates the ATM signaling pathway independent of DNA damage to stabilize p53 and induce G1 arrest of human mammary epithelial cells

The phytochemical indole‐3‐carbinol (I3C), from cruciferous vegetables such as broccoli, has been shown to elicit a potent anti‐proliferative response in human breast cancer cell lines. Treatment of the immortalized human mammary epithelial cell line MCF10A with I3C induced a G1 cell cycle arrest, elevated p53 tumor suppressor protein levels and stimulated expression of downstream transcriptional target, p21. I3C treatment also elevated p53 levels in several breast cancer cell lines that express mutant p53. I3C did not arrest MCF10A cells stably transfected with dominant‐negative p53, establishing a functional requirement for p53. Cell fractionation and immunolocalization studies revealed a large fraction of stabilized p53 protein in the nucleus of I3C‐treated MCF10A cells. With I3C treatment, phosphatidyl‐inositol‐3‐kinase family member ataxia telangiectasia‐mutated (ATM) was phosphorylated, as were its substrates p53, CHK2 and BRCA1. Phosphorylation of p53 at the N‐terminus has previously been shown to disrupt the interaction between p53 and its ubiquitin ligase, MDM2, and therefore stabilizing p53. Coimmunoprecipitation analysis revealed that I3C reduced by 4‐fold the level of MDM2 protein that associated with p53. The p53–MDM2 interaction and absence of p21 production were restored in cells treated with I3C and the ATM inhibitor wortmannin. Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double‐strand breaks. Taken together, our results demonstrate that I3C activates ATM signaling through a novel pathway to stimulate p53 phosphorylation and disruption of the p53–MDM2 interaction, which releases p53 to induce the p21 CDK inhibitor and a G1 cell cycle arrest.

DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells

Live‐cell detection of intracellular enzyme activity requires that substrates are cell‐permeable and that the generated products are easily detected and retained in cells. Our objective was to create a novel fluorogenic substrate that could be used for real‐time detection of apoptosis in living cells. We have synthesized a highly cell‐permeable caspase‐3 substrate, DEVD‐NucView488, by linking a fluorogenic DNA‐binding dye to the caspase‐3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD‐NucView488 detected caspase‐3 activation within a live‐cell population much earlier and with higher sensitivity compared with other apoptosis reagents that are currently available. Furthermore, cells incubated with DEVD‐NucView488 exhibited no toxicity and normal apoptotic progression. DEVD‐NucView488 is an ideal substrate for kinetic studies of caspase‐3 activation because it detects caspase‐3 activity in real‐time and also efficiently labels DNA in nuclei of caspase‐3‐activated cells for real‐time fluorescent visualization of apoptotic morphology. The strategy utilized in the design of this fluorogenic substrate can be applied in future endeavors to develop substrates for detecting real‐time intracellular enzyme activity.—Cen, H., Mao, F., Aronchik, I., Fuentes, R. J., Firestone, G. L. DEVD‐NucView488: a novel class of enzyme substrates for real‐time detection of caspase‐3 activity in live cells. FASEB J. 22, 2243–2252 (2008)

The dietary phytochemical indole-3-carbinol is a natural elastase enzymatic inhibitor that disrupts cyclin E protein processing

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, induces a G1 cell-cycle arrest of human breast cancer cells, although the direct cellular targets that mediate this process are unknown. Treatment of highly invasive MDA-MB-231 breast cancer cells with I3C shifted the stable accumulation of cyclin E protein from the hyperactive lower-molecular-mass 35-kDa form that is associated with cancer cell proliferation and poor clinical outcomes to the 50-kDa cyclin E form that typically is expressed in normal mammary tissue. An in vitro cyclin E processing assay, in combination with zymography, demonstrated that I3C, but not its natural dimer, 3,3′-diindolylmethane, disrupts proteolytic processing of the 50-kDa cyclin E into the lower-molecular-mass forms by direct inhibition of human neutrophil elastase enzymatic activity. Analysis of elastase enzyme kinetics using either cyclin E or N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanalide as substrates demonstrated that I3C acts as a noncompetitive inhibitor of elastase activity with an inhibitory constant of ≈12 μM. Finally, siRNA ablation of neutrophil elastase protein production in MDA-MB-231 cells mimicked the I3C-disrupted processing of the 50-kDa cyclin E protein and the indole-induced cell-cycle arrest. Taken together, our results demonstrate that elastase is the first identified specific target protein for I3C and that the direct I3C inhibition of elastase enzymatic activity implicates the potential use of this indole, or related compounds, in targeted therapies of human breast cancers where high elastase levels are correlated with poor prognosis.

Indole-3-carbinol inhibits MDA-MB-231 breast cancer cell motility and induces stress fibers and focal adhesion formation by activation of Rho kinase activity

Indole‐3‐carbinol (I3C), a phytochemical derived from cruciferous vegetables such as broccoli and Brussels sprouts, has potent antiproliferative effects in human breast cancer cells and has been shown to decrease metastatic spread of tumors in experimental animals. Using chemotaxis and fluorescent‐bead cell motility assays, we demonstrated that I3C significantly decreased the in vitro migration of MDA‐MB‐231 cells, a highly invasive breast cancer cell line. Immunofluorescence staining of the actin cytoskeleton revealed that concurrent with the loss of cell motility, I3C treatment significantly increased stress fiber formation. Furthermore, I3C induced the localization of the focal adhesion component vinculin and tyrosine‐phosphorylated proteins to the cell periphery, which implicates an indole‐dependent enhancement of focal adhesions within the outer boundary of the cells. Coimmunoprecipitation analysis of focal adhesion kinase demonstrated that I3C stimulated the dynamic formation of the focal adhesion protein complex without altering the total level of individual focal adhesion proteins. The RhoA‐Rho kinase pathway is involved in stress fiber and focal adhesion formation, and I3C treatment stimulated Rho kinase enzymatic activity and cofilin phosphorylation, which is a downstream target of Rho kinase signaling, but did not increase the level of active GTP‐bound RhoA. Exposure of MDA‐MB‐231 cells to the Rho kinase inhibitor Y‐27632, or expression of dominant negative RhoA ablated the I3C induced formation of stress fibers and of peripheral focal adhesions. Expression of constitutively active RhoA mimicked the I3C effects on both processes. Taken together, our data demonstrate that I3C induces stress fibers and peripheral focal adhesions in a Rho kinase‐dependent manner that leads to an inhibition of motility in human breast cancer cells.

Direct Inhibition of Elastase Activity by Indole-3-Carbinol Triggers a CD40-TRAF Regulatory Cascade That Disrupts NF-κB Transcriptional Activity in Human Breast Cancer Cells

Treatment of highly tumorigenic MDA-MB-231 human breast cancer cells with indole-3-carbinol (I3C) directly inhibited the extracellular elastase-dependent cleavage of membrane-associated CD40, a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 signaling has been implicated in regulating cell survival, apoptosis, and proliferation, as well as in sensitizing breast cancer cells to chemotherapy, and is therefore an important potential target of novel breast cancer treatments. The I3C-dependent accumulation of full-length unprocessed CD40 protein caused a shift in CD40 signaling through TNF receptor-associated factors (TRAF), including the TRAF1/TRAF2 positive regulators and TRAF3 negative regulator of NF-kappaB transcription factor activity. Because TRAF1 is a transcriptional target gene of NF-kappaB, I3C disrupted a positive feedback loop involving these critical cell survival components. siRNA ablation of elastase expression mimicked the I3C inhibition of CD40 protein processing and G(1) cell cycle arrest, whereas siRNA knockdown of TRAF3 and the NF-kappaB inhibitor IkappaB prevented the I3C-induced cell cycle arrest. In contrast, siRNA knockdown of PTEN had no effect on the I3C control of NF-kappaB activity, showing the importance of CD40 signaling in regulating this transcription factor. Our study provides the first direct in vitro evidence that I3C directly inhibits the elastase-mediated proteolytic processing of CD40, which alters downstream signaling to disrupt NF-kappaB-induced cell survival and proliferative responses. Furthermore, we have established a new I3C-mediated antiproliferative cascade that has significant therapeutic potential for treatment of human cancers associated with high levels of elastase and its CD40 membrane substrate.

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

Human melanoma cells displaying distinct PTEN genotypes were used to assess the cellular role of this important tumor-suppressor protein in the antiproliferative response induced by the chemopreventative agent indole-3-carbinol (I3C), a natural indolecarbinol compound derived from the breakdown of glucobrassicin produced in cruciferous vegetables such as broccoli and Brussels sprouts. I3C induced a G1-phase cell-cycle arrest and apoptosis by stabilization of PTEN in human melanoma cells that express wild-type PTEN, but not in cells with mutant or null PTEN genotypes. Importantly, normal human epidermal melanocytes were unaffected by I3C treatment. In wild-type PTEN-expressing melanoma xenografts, formed in athymic mice, I3C inhibited the in vivo tumor growth rate and increased PTEN protein levels in the residual tumors. Mechanistically, I3C disrupted the ubiquitination of PTEN by NEDD4-1 (NEDD4), which prevented the proteasome-mediated degradation of PTEN without altering its transcript levels. RNAi-mediated knockdown of PTEN prevented the I3C-induced apoptotic response, whereas knockdown of NEDD4-1 mimicked the I3C apoptotic response, stabilized PTEN protein levels, and downregulated phosphorylated AKT-1 levels. Co-knockdown of PTEN and NEDD4-1 revealed that I3C-regulated apoptotic signaling through NEDD4-1 requires the presence of the wild-type PTEN protein. Finally, in silico structural modeling, in combination with isothermal titration calorimetry analysis, demonstrated that I3C directly interacts with purified NEDD4-1 protein. Implications: This study identifies NEDD4-1 as a new I3C target protein, and that the I3C disruption of NEDD4-1 ubiquitination activity triggers the stabilization of the wild-type PTEN tumor suppressor to induce an antiproliferative response in melanoma. Mol Cancer Res; 12(11); 1621–34. ©2014 AACR.

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

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.

Indole-3-carbinol (I3C) analogues are potent small molecule inhibitors of NEDD4-1 ubiquitin ligase activity that disrupt proliferation of human melanoma cells

Graphical abstract Figure. No Caption available. ABSTRACT The HECT domain‐containing E3 ubiquitin ligase NEDD4‐1 (Neural precursor cell Expressed Developmentally Down regulated gene 4‐1) is frequently overexpressed in human cancers and displays oncogenic‐like properties through the ubiquitin‐dependent regulation of multiple protein substrates. However, little is known about small molecule enzymatic inhibitors of HECT domain‐containing ubiquitin ligases. We now demonstrate that indole‐3‐carbinol (I3C), a natural anti‐cancer phytochemical derived from cruciferous vegetables such as cabbage and broccoli, represents a new chemical scaffold of small molecule enzymatic inhibitors of NEDD4‐1. Using in vitro ubiquitination assays, I3C, its stable synthetic derivative 1‐benzyl‐I3C and five novel synthetic analogues were shown to directly inhibit NEDD4‐1 ubiquitination activity. Compared to I3C, which has an IC50 of 284 &mgr;M, 1‐benzyl‐I3C was a significantly more potent NEDD4‐1 enzymatic inhibitor with an IC50 of 12.3 &mgr;M. Compounds 2242 and 2243, the two indolecarbinol analogues with added methyl groups that results in a more nucleophilic benzene ring &pgr; system, further enhanced potency with IC50s of 2.71 &mgr;M and 7.59 &mgr;M, respectively. Protein thermal shift assays that assess small ligand binding, in combination with in silico binding simulations with the crystallographic structure of NEDD4‐1, showed that each of the indolecarbinol compounds bind to the purified catalytic HECT domain of NEDD4‐1. The indolecarbinol compounds inhibited human melanoma cell proliferation in a manner that generally correlated with their effectiveness as NEDD4‐1 enzymatic inhibitors. Taken together, we propose that I3C analogues represent a novel set of anti‐cancer compounds for treatment of human melanomas and other cancers that express indolecarbinol‐sensitive target enzymes.

Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells

Antiproliferative signaling of combinations of the nonsteroidal anti-inflammatory drug acetylsalicylic acid (aspirin) and indole-3-carbinol (I3C), a natural indolecarbinol compound derived from cruciferous vegetables, was investigated in human melanoma cells. Melanoma cell lines with distinct mutational profiles were sensitive to different extents to the antiproliferative response of aspirin, with oncogenic BRAF-expressing G361 cells and wild-type BRAF-expressing SK-MEL-30 cells being the most responsive. I3C triggered a strong proliferative arrest of G361 melanoma cells and caused only a modest decrease in the proliferation of SK-MEL-30 cells. In both cell lines, combinations of aspirin and I3C cooperatively arrested cell proliferation and induced a G1 cell cycle arrest, and nearly ablated protein and transcript levels of the melanocyte master regulator microphthalmia-associated transcription factor isoform M (MITF-M). In melanoma cells transfected with a −333/+120-bp MITF-M promoter-luciferase reporter plasmid, treatment with aspirin and I3C cooperatively disrupted MITF-M promoter activity, which accounted for the loss of MITF-M gene products. Mutational analysis revealed that the aspirin required the LEF1 binding site, whereas I3C required the BRN2 binding site to mediate their combined and individual effects on MITF-M promoter activity. Consistent with LEF1 being a downstream effector of Wnt signaling, aspirin, but not I3C, downregulated protein levels of the Wnt co-receptor LDL receptor-related protein-6 and β-catenin and upregulated the β-catenin destruction complex component Axin. Taken together, our results demonstrate that aspirin-regulated Wnt signaling and I3C-targeted signaling pathways converge at distinct DNA elements in the MITF-M promoter to cooperatively disrupt MITF-M expression and melanoma cell proliferation.