Anti-inflammatory effects of chicanine on murine macrophage by down-regulating LPS-induced inflammatory cytokines in IκBα/MAPK/ERK signaling pathways.

Schisandra chinensis Baill is a Chinese traditional medicine with multiple pharmacological activities. In this study, chicanine, one of the major lignan compounds of S. chinesis, was investigated for suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages (RAW 264.7 cells). Chicanine was found to have anti-inflammatory properties with the inhibition of nitric oxide (NO) and Prostaglandin E (2) (PGE2) production and nuclear factor-κB (NF-κB) signaling in LPS-stimulated RAW 264.7 cells with no cytotoxic effects. Treatment of RAW 264.7 cells with chicanine down-regulated LPS-induced expression of pro-inflammatory cytokines including TNFα, IL-1ß, MCP-1, G-CSF, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). These inhibitory effects were found with the blockage of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and also IκB-α phosphorylation. These results indicated that anti-inflammatory actions of chicanine in macrophages involved inhibition of LPS-induced TLR4-IκBα/MAPK/ERK signaling pathways.

3,3'-diindolylmethane rapidly and selectively inhibits hepatocyte growth factor/c-Met signaling in breast cancer cells.

3,3'-Diindolylmethane (DIM), an indole derivative from vegetables of the Brassica genus, has antiproliferative activity in breast cancer cells. Part of this activity is thought to be due to DIM inhibition of Akt signaling, but an upstream mechanism of DIM-induced Akt inhibition has not been described. The goals of this study were to investigate the kinetics of inhibition of Akt by physiologically relevant concentrations of DIM and to identify an upstream factor that mediates this effect. Here we report that DIM (5-25 µM) inhibited Akt activation from 30 min to 24h in tumorigenic MDA-MB-231 cells but did not inhibit Akt activation in non-tumorigenic preneoplastic MCF10AT cells. DIM inhibited hepatocyte growth factor (HGF)-induced Akt activation by up to 46%, cell migration by 66% and cell proliferation by up to 54%, but did not inhibit induction of Akt by epidermal growth factor or insulin-like growth factor-1. DIM decreased phosphorylation of the HGF receptor, c-Met, at tyrosines 1234 and 1235, indicating decreased activation of the receptor. This decrease was reversed by pretreatment with inhibitors of p38 or calcineurin. Our results demonstrate the important role of HGF and c-Met in DIM's anti-proliferative effect on breast cancer cells and suggest that DIM could have preventive or clinical value as an inhibitor of c-Met signaling.

(+)-Altholactone exhibits broad spectrum immune modulating activity by inhibiting the activation of pro-inflammatory cytokines in RAW 264.7 cell lines.

An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflammatory activity in a nuclear factor-kappa B (NF-κB) luciferase and MTT assay using transfected macrophage immune (Raw264.7) cells. A high-throughput LC/MS-ELSD based library approach of the extract in combination with the NF-κB and MTT assays revealed the styryl lactone (+)-altholactone (2) was responsible for the activity. Compound 2, its acetylated derivate (+)-3-O-acetylaltholactone (3), and the major compound of this class, (+)-goniothalmin (1), were further evaluated to determine their anti-inflammatory potential in the NF-κB assay. Concentration-response studies of 1-3 indicated that only 2 possessed NF-κB based anti-inflammatory activity. Compound 2 reduced the LPS-induced NO production, phosphorylation of IκBα, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) using Western blot analysis. Further studies using qPCR indicated 2 reduced the expression of eight pro-inflammatory cytokines/enzymes (0.8-5.0µM) which included: COX-2, iNOS, IP-10, IL-1ß, MCP-1, GCS-F, IL-6 and IFN-ß. These results indicated that 2 displays broad spectrum immune modulating activity by functioning as an anti-inflammatory agent against LPS-induced NF-κB signaling. Conversely the selective cytotoxicity and in vivo anti-tumor and anti-inflammatory activity previously reported for 1 do not appear to arise from a mechanism that is linked to the NF-κB immune mediated pathway.

Lipophilic stinging nettle extracts possess potent anti-inflammatory activity, are not cytotoxic and may be superior to traditional tinctures for treating inflammatory disorders.

Extracts of four plant portions (roots, stems, leaves and flowers) of Urtica dioica (the stinging nettle) were prepared using accelerated solvent extraction (ASE) involving water, hexanes, methanol and dichloromethane. The extracts were evaluated for their anti-inflammatory and cytotoxic activities in an NF-κB luciferase and MTT assay using macrophage immune (RAW264.7) cells. A standardized commercial ethanol extract of nettle leaves was also evaluated. The methanolic extract of the flowering portions displayed significant anti-inflammatory activity on par with a standard compound celastrol (1) but were moderately cytotoxic. Alternatively, the polar extracts (water, methanol, ethanol) of the roots, stems and leaves displayed moderate to weak anti-inflammatory activity, while the methanol and especially the water soluble extracts exhibited noticeable cytotoxicity. In contrast, the lipophilic dichloromethane extracts of the roots, stems and leaves exhibited potent anti-inflammatory effects greater than or equal to 1 with minimal cytotoxicity to RAW264.7 cells. Collectively these results suggest that using lipophilic extracts of stinging nettle may be more effective than traditional tinctures (water, methanol, ethanol) in clinical evaluations for the treatment of inflammatory disorders especially arthritis. A chemical investigation into the lipophilic extracts of stinging nettle to identify the bioactive compound(s) responsible for their observed anti-inflammatory activity is further warranted.

Myxobacteria versus sponge-derived alkaloids: the bengamide family identified as potent immune modulating agents by scrutiny of LC-MS/ELSD libraries.

A nuclear factor-κB (NF-κB) luciferase assay has been employed to identify the bengamides, previously known for their anti-tumor activity, as a new class of immune modulators. A unique element of this study was that the bengamide analogs were isolated from two disparate sources, Myxococcus virescens (bacterium) and Jaspis coriacea (sponge). Comparative LC-MS/ELSD and NMR analysis facilitated the isolation of M. viriscens derived samples of bengamide E (8) and two congeners, bengamide E' (13) and F' (14) each isolated as an insperable mixture of diastereomers. Additional compounds drawn from the UC, Santa Cruz repository allowed expansion of the structure activity relationship (SAR) studies. The activity patterns observed for bengamide A (6), B (7), E (8), F (9), LAF 389 (12) and 13-14 gave rise to the following observations and conclusions. Compounds 6 and 7 display potent inhibition of NF-κB (at 80 and 90 nM, respectively) without cytotoxicity to RAW264.7 macrophage immune cells. Western blot and qPCR analysis indicated that 6 and 7 reduce the phosphorylation of IκBα and the LPS-induced expression of the pro-inflammatory cytokines/chemokines TNFα, IL-6 and MCP-1 but do not effect NO production or the expression of iNOS. These results suggest that the bengamides may serve as therapeutic leads for the treatment of diseases involving inflammation, that their anti-tumor activity can in part be attributed to their ability to serve as immune modulating agents, and that their therapeutic potential against cancer merits further consideration.

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.

Natural product libraries to accelerate the high-throughput discovery of therapeutic leads.

A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.

Indole-3-carbinol downregulation of telomerase gene expression requires the inhibition of estrogen receptor-alpha and Sp1 transcription factor interactions within the hTERT promoter and mediates the G1 cell cycle arrest of human breast cancer cells.

Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G(1) cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element-Sp1 composite element within the hTERT promoter. I3C inhibited 17ß-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1-DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.

Azonazine, a novel dipeptide from a Hawaiian marine sediment-derived fungus, Aspergillus insulicola.

Azonazine, a unique hexacyclic dipeptide, was isolated from a Hawaiian marine sediment-derived fungus eventually identified as Aspergillus insulicola. Its absolute configuration, 2R,10R,11S,19R, was established using NMR, HRESIMS, and CD data plus insights derived from molecular models. A possible route for its biogenesis is proposed, and biological properties were explored against cancer cell lines and in an NFκB inhibition assay.

1-Benzyl-indole-3-carbinol is a novel indole-3-carbinol derivative with significantly enhanced potency of anti-proliferative and anti-estrogenic properties in human breast cancer cells.

Indole-3-carbinol (I3C), a natural autolysis product of a gluccosinolate present in Brassica vegetables such as broccoli and cabbage, has anti-proliferative and anti-estrogenic activities in human breast cancer cells. A new and significantly more potent I3C analogue, 1-benzyl-I3C was synthesized, and in comparison to I3C, this novel derivative displayed an approximate 1000-fold enhanced potency in suppressing the growth of both estrogen responsive (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cells (I3C IC(50) of 52 microM, and 1-benzyl-I3C IC(50) of 0.05 microM). At significantly lower concentrations, 1-benzyl-I3C induced a robust G1 cell cycle arrest and elicited the key I3C-specific effects on expression and activity of G1-acting cell cycle genes including the disruption of endogenous interactions of the Sp1 transcription factor with the CDK6 promoter. Furthermore, in estrogen responsive MCF-7 cells, with enhanced potency 1-benzyl-I3C down-regulated production of estrogen receptor-alpha protein, acts with tamoxifen to arrest breast cancer cell growth more effectively than either compound alone, and inhibited the in vivo growth of human breast cancer cell-derived tumor xenografts in athymic mice. Our results implicate 1-benzyl-I3C as a novel, potent inhibitor of human breast cancer proliferation and estrogen responsiveness that could potentially be developed into a promising therapeutic agent for the treatment of indole-sensitive cancers.

Direct inhibition of elastase activity by indole-3-carbinol triggers a CD40-TRAF regulatory cascade that disrupts NF-kappaB 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.

Indole-3-carbinol triggers aryl hydrocarbon receptor-dependent estrogen receptor (ER)alpha protein degradation in breast cancer cells disrupting an ERalpha-GATA3 transcriptional cross-regulatory loop.

Estrogen receptor (ER)alpha is a critical target of therapeutic strategies to control the proliferation of hormone-dependent breast cancers. Preferred clinical options have significant adverse side effects that can lead to treatment resistance due to the persistence of active estrogen receptors. We have established the cellular mechanism by which indole-3-carbinol (I3C), a promising anticancer phytochemical from Brassica vegetables, ablates ERalpha expression, and we have uncovered a critical role for the GATA3 transcription factor in this indole-regulated cascade. I3C-dependent activation of the aryl hydrocarbon receptor (AhR) initiates Rbx-1 E3 ligase-mediated ubiquitination and proteasomal degradation of ERalpha protein. I3C inhibits endogenous binding of ERalpha with the 3'-enhancer region of GATA3 and disrupts endogenous GATA3 interactions with the ERalpha promoter, leading to a loss of GATA3 and ERalpha expression. Ectopic expression of GATA3 has no effect on I3C-induced ERalpha protein degradation but does prevent I3C inhibition of ERalpha promoter activity, demonstrating the importance of GATA3 in this I3C-triggered cascade. Our preclinical results implicate I3C as a novel anticancer agent in human cancers that coexpress ERalpha, GATA3, and AhR, a combination found in a large percentage of breast cancers but not in other critical ERalpha target tissues essential to patient health.

Selective activation of estrogen receptor-beta target genes by 3,3'-diindolylmethane.

3,3'-Diindolylmethane (DIM) is a natural compound found in cruciferous vegetables that has antiproliferative and estrogenic activity. However, it is not clear whether the estrogenic effects are mediated through estrogen receptor (ER)alpha, ERbeta, or both ER subtypes. We investigated whether DIM has ER subtype selectivity on gene transcription. DIM stimulated ERbeta but not ERalpha activation of an estrogen response element upstream of the luciferase reporter gene. DIM also selectively activated multiple endogenous genes through ERbeta. DIM did not bind to ERbeta, indicating that it activates genes by a ligand-independent mechanism. DIM causes ERbeta to bind regulatory elements and recruit the steroid receptor coactivator (SRC)-2 coactivator, which leads to the activation of ER target genes. Silencing of SRC-2 inhibited the activation of ER target genes, demonstrating that SRC-2 is required for transcriptional activation by DIM. Our results demonstrate that DIM is a new class of ERbeta-selective compounds, because it does not bind to ERbeta, but instead it selectively recruits ERbeta and coactivators to target genes.

Drug and cell type-specific regulation of genes with different classes of estrogen receptor beta-selective agonists.

Estrogens produce biological effects by interacting with two estrogen receptors, ERalpha and ERbeta. Drugs that selectively target ERalpha or ERbeta might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERbeta-selective compounds have been identified. One class of ERbeta-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERbeta much greater than ERalpha. A second class of ERbeta-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERbeta. Diarylpropionitrile represents a third class of ERbeta-selective compounds because its selectivity is due to a combination of greater binding to ERbeta and transcriptional activity. However, it is unclear if these three classes of ERbeta-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERbeta selectivity and pattern of gene expression of these three classes of ERbeta-selective compounds compared to estradiol (E(2)), which is a non-selective ER agonist. U2OS cells stably transfected with ERalpha or ERbeta were treated with E(2) or the ERbeta-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERbeta-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERbeta, which is consistent with the finding that most genes regulated by the ERbeta-selective compounds were similar to each other and E(2). However, there were some classes of genes differentially regulated by the ERbeta agonists and E(2). Two ERbeta-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERbeta. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERbeta-selective agonists and E(2), there were some genes regulated that were distinct from each other and E(2), suggesting that different ERbeta-selective agonists might produce distinct biological and clinical effects.

3,3'-Diindolylmethane induces a G(1) arrest in human prostate cancer cells irrespective of androgen receptor and p53 status.

3,3'-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived from Brassica vegetables. In this study we characterized the effect of DIM on cell cycle regulation in both androgen-dependent LNCaP and androgen receptor negative p53 mutant DU145 human prostate cancer cells. DIM had an anti-proliferative effect on both LNCaP and DU145 cells, as it significantly inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM-mediated G(1) cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4 protein and increased the expression of the cell cycle inhibitor p27(Kip1) protein in LNCaP and DU145 cells. Promoter deletion studies with p27(Kip1) reporter gene constructs showed that this DIM-mediated increase in p27(Kip1) was dependent on the Sp1 transcription factor. Moreover, using a dominant negative inhibitor of p38 MAPK, we showed that the induction of p27(Kip1) and subsequent G(1) arrest by DIM involve activation of the p38 MAPK pathway in the DU145 cells. Taken together, our results indicate that DIM is able to stop the cell cycle progression of human prostate cancer cells regardless of their androgen-dependence and p53 status, by differentially modulating cell cycle regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle regulatory effect in DU145 cells.

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.

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 G(1) 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 approximately 12 microM. 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.

3,3'-diindolylmethane reduces levels of HIF-1alpha and HIF-1 activity in hypoxic cultured human cancer cells.

3,3'-diindolylmethane (DIM) is a chemopreventive and chemotherapeutic phytochemical derived from the metabolism of indoles found at high concentrations in cruciferous vegetables. We have previously shown that DIM exhibits anti-angiogenic properties in cultured vascular endothelial cells and in Matrigel plug assays in rodents. In the present study, we demonstrate that DIM reduces the level of hypoxia-inducible factor (HIF)-1alpha in hypoxic tumor cell lines, as well as HIF-1 transcriptional activity as measured by a reporter assay. Moreover, DIM inhibited the expression of HIF-1-responsive endogenous genes, resulting in the reduced expression of key hypoxia responsive factors, VEGF, furin, enolase-1, glucose transporter-1 and phosphofructokinase. DIM reduced the level of HIF-1alpha in hypoxic cells by increasing the rate of the prolylhydroxylase- and proteasome-mediated degradation of HIF-1alpha, and by decreasing the rate of HIF-1alpha transcription. Using enzyme kinetics studies, we established that DIM interacts with the oligomycin-binding site on the F0 transmembrane component of mitochondrial F1F0-ATPase. The contributions of the resulting increases in levels of ROS and O2 in hypoxic cells to the inhibitory effects of DIM on HIF-1alpha expression are discussed. These studies are the first to show that DIM can decrease the accumulation and activity of the key angiogenesis regulatory factor, HIF-1alpha, in hypoxic tumor cells.

Liquiritigenin is a plant-derived highly selective estrogen receptor beta agonist.

After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

N-Alkoxy derivatization of indole-3-carbinol increases the efficacy of the G1 cell cycle arrest and of I3C-specific regulation of cell cycle gene transcription and activity in human breast cancer cells.

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts, induces a G1 cell cycle arrest of human breast cancer cells. Structure-activity relationships of I3C that mediate this anti-proliferative response were investigated using synthetic and natural I3C derivatives that contain substitutions at the indole nitrogen. Nitrogen substitutions included N-alkoxy substituents of one to four carbons in length, which inhibit dehydration and the formation of the reactive indolenine. Analysis of growth and cell cycle arrest of indole-treated human breast cancer cells revealed a striking increase in efficacy of the N-alkoxy I3C derivatives that is significantly enhanced by the presence of increasing carbon lengths of the N-alkoxy substituents. Compared to I3C, the half maximal growth arrest responses occurred at 23-fold lower indole concentration for N-methoxy I3C, 50-fold lower concentration for N-ethoxy I3C, 217-fold lower concentration for N-propoxy I3C, and 470-fold lower concentration for N-butoxy I3C. At these lower concentrations, each of the N-alkoxy substituted compounds induced the characteristic I3C response in that CDK6 gene expression, CDK6 promoter activity, and CDK2 specific enzymatic activity for its retinoblastoma protein substrate were strongly down-regulated. 3-Methoxymethylindole and 3-ethoxymethylindole were approximately as bioactive as I3C, whereas both tryptophol and melatonin failed to induce the cell cycle arrest, showing the importance of the C-3 hydroxy methyl substituent on the indole ring. Taken together, our study establishes the first I3C structure-activity relationship for cytostatic activities, and implicates I3C-based N-alkoxy derivatives as a novel class of potentially more potent experimental therapeutics for breast cancer.