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1.  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 activity in an NF-κB luciferase and MTT assay using macrophage immune (RAW264.7) cells. A standardized commercial ethanol extract of nettle leaves were also evaluated. The methanolic extract of the flowering portions displayed significant anti-inflammatory activity on par with the standard anti-inflammatory agent celastrol (1) but was moderately cytotoxic. Alternatively, the polar extracts (water, methanol, ethanol) of the roots, stems and leaves plant portions 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 ≥ 1 with minimal cytotoxicity to RAW264.7 cells. Collectively these results suggest that using lipophilic extracts of the roots, stems or leaves of stinging nettle may be more effective then traditional tinctures (water, methanol, ethanol) to undergo clinical evaluations for the treatment of inflammatory disorders including arthritis. A chemical investigation into the lipophillic extracts of stinging nettle to identify the bioactive compound(s) responsible for their observed anti-inflammatory activity is further warranted.
doi:10.1016/j.phymed.2012.09.016
PMCID: PMC3529973  PMID: 23092723
Urtica dioica (Stinging Nettle); Anti-inflammatory; Rheumatoid arthritis; Osteoarthritis; Nuclear factor-kB
2.  3,3′-Diindolylmethane Induces a G1 Arrest in Human Prostate Cancer Cells Irrespective of Androgen Receptor and p53 Status 
Biochemical pharmacology  2009;78(5):469-476.
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 antiproliferative effect on both LNCaP and DU145 cells, as it significantly inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM mediated G1 cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4 protein and increased expression of the cell cycle inhibitor p27Kip1 protein in LNCaP and DU145 cells. Promoter deletion studies with p27Kip1 reporter gene constructs showed that this DIM-mediated increase in p27Kip1 was dependent on the Sp1 transcription factor. Moreover, using a dominant negative inhibitor of p38 MAPK, we showed that the induction of p27Kip1 and subsequent G1 arrest by DIM involves 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.
doi:10.1016/j.bcp.2009.05.008
PMCID: PMC2706920  PMID: 19433067
3; 3′-Diinolylmethane; Prostate cancer; Cell cycle arrest; p27Kip1; p38 MAPK; Cancer
3.  3,3′-Diindolylmethane reduces levels of HIF-1α and HIF-1 activity in hypoxic cultured human cancer cells 
Biochemical pharmacology  2008;75(9):1858-1867.
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)-1α 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-1α in hypoxic cells by increasing the rate of the prolylhydroxylase- and proteosome-mediated degradation of HIF-1α, and by decreasing the rate of HIF-1α transcription. Using enzyme kinetics studies, we established that DIM interacts with the oligomycin-binding site on the F1 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-1α 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-1α, in hypoxic tumor cells.
doi:10.1016/j.bcp.2008.01.017
PMCID: PMC2387239  PMID: 18329003
3,3′-Diindolylmethane; angiogenesis; hypoxia-inducible factor; cancer; ATPase; prolylhydroxylase
4.  Myxobacteria vs. Sponge-Derived Alkaloids: The Bengamide Family Identified as Potent Immune Modulating Agents by Scrutiny of LC-MS/ELSD Libraries 
Bioorganic & medicinal chemistry  2012;20(14):4348-4355.
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.
doi:10.1016/j.bmc.2012.05.043
PMCID: PMC3417756  PMID: 22705020
5.  Natural Product Libraries to Accelerate the High Throughput Discovery of Therapeutic Leads± 
Journal of natural products  2011;74(12):2545-2555.
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 the latrunculins (1–4, 10), fijianolides (5, 8–9), mycothiazole (11), the aignopsanes (6–7) and sacrotride A (13). Compounds 1–4, 5, 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 1–2 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.
doi:10.1021/np200673b
PMCID: PMC3246535  PMID: 22129061
6.  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 
Carcinogenesis  2011;32(9):1315-1323.
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 G1 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 17β-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.
doi:10.1093/carcin/bgr116
PMCID: PMC3165124  PMID: 21693539
7.  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 
Biochemical pharmacology  2007;75(3):713-724.
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 melatonine 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.
doi:10.1016/j.bcp.2007.09.024
PMCID: PMC3422660  PMID: 18023427
I3C; synthetic derivatives; N-alkoxy constituents; breast cancer cells; cell cycle arrest
8.  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 
Chemico-biological interactions  2010;186(3):255-266.
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 IC50 of 52 μM, and 1-benzyl-I3C IC50 of 0.05 μM). 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.
doi:10.1016/j.cbi.2010.05.015
PMCID: PMC3422669  PMID: 20570586
Indole-3-carbinol; 1-benzyl-indole-3-carbinol; breast cancer cells; anti-proliferative signaling; disrupted estrogen receptor responsiveness; high potency indole derivatives
9.  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 anti-proliferative 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. Co-immunoprecipitation 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.
doi:10.1002/ijc.24210
PMCID: PMC3400502  PMID: 19173291
Indole-3-carbinol; breast cancer; migration; focal adhesions; RhoA; Rho kinase
10.  Azonazine, A Novel Dipeptide from a Hawaiian Marine Sediment-Derived Fungus, Aspergillus insulicola 
Organic letters  2010;12(20):4458-4461.
Azonazine, a unique hexacyclic dipeptide was isolated from a Hawaiian marine sediment-derived fungus eventually identified as, Aspergillus insulicola. Its absolute configurations, 2R, 10R, 11S, 19R were 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.
doi:10.1021/ol101396n
PMCID: PMC2953366  PMID: 20866076
11.  Indole-3-Carbinol Triggers Aryl Hydrocarbon Receptor-dependent Estrogen Receptor (ER)α Protein Degradation in Breast Cancer Cells Disrupting an ERα-GATA3 Transcriptional Cross-Regulatory Loop 
Molecular Biology of the Cell  2010;21(7):1166-1177.
We have established in human breast cancer cells that indole-3-carbinol (I3C), a promising anti-cancer phytochemical from Brassica vegetables, ablates ERα expression by stimulating the Rbx-1 E3 ligase mediated degradation of ERα protein and disruption of a cross-regulatory positive feedback loop involving ERα and the GATA3 transcription factor.
Estrogen receptor (ER)α 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 ERα 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 ERα protein. I3C inhibits endogenous binding of ERα with the 3′-enhancer region of GATA3 and disrupts endogenous GATA3 interactions with the ERα promoter, leading to a loss of GATA3 and ERα expression. Ectopic expression of GATA3 has no effect on I3C-induced ERα protein degradation but does prevent I3C inhibition of ERα 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 ERα, GATA3, and AhR, a combination found in a large percentage of breast cancers but not in other critical ERα target tissues essential to patient health.
doi:10.1091/mbc.E09-08-0689
PMCID: PMC2847521  PMID: 20130088
12.  3,3′-Diindolylmethane Stimulates Murine Immune Function In Vitro and In Vivo* 
3,3′-Diindolylmethane (DIM), a major condensation product of indole-3-carbinol (I3C), exhibits chemopreventive properties in animal models of cancer. Recent studies have shown that DIM stimulates interferon-gamma (IFN-γ) production and potentiates the IFN-γ signaling pathway in human breast cancer cells via a mechanism that includes increased expression of the IFN-γ receptor. The goal of this study was to test the hypothesis that DIM modulates the murine immune function. Specifically, the effects of DIM were evaluated in a panel of murine immune function tests that included splenocyte proliferation, reactive oxygen species (ROS) generation, cytokine production and resistance to viral infection. DIM was found to induce proliferation of splenocytes as well as augment mitogen- and IL-2-induced splenocyte proliferation. DIM also stimulated the production of ROS by murine peritoneal macrophage cultures. Oral administration of DIM, but not intraperitoneal injection (i.p.), induced elevation of serum cytokines in mice, including interleukin (IL)-6, granulocyte-colony stimulating factor (G-CSF), IL-12 and IFN-γ. Finally, in a model of enteric virus infection, oral DIM administration to mice enhanced both clearance of reovirus from the GI tract and the subsequent mucosal IgA response. Thus, DIM is a potent stimulator of immune function. This property might contribute to the cancer inhibitory effects of this indole.
doi:10.1016/j.jnutbio.2007.05.004
PMCID: PMC2387240  PMID: 17707631
3,3′-diindolylmethane; Immune stimulation; Cytokine; Lymphocyte proliferation; reactive oxygen species
13.  Liquiritigenin is a plant-derived highly selective estrogen receptor β 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 ERβ-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERβ-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 ERβ but not ERα. The ERβ-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 ERβ.
doi:10.1016/j.mce.2007.11.020
PMCID: PMC2277338  PMID: 18177995
14.  Drug and Cell Type-Specific Regulation of Genes with Different Classes of Estrogen Receptor β-Selective Agonists 
PLoS ONE  2009;4(7):e6271.
Estrogens produce biological effects by interacting with two estrogen receptors, ERα and ERβ. Drugs that selectively target ERα or ERβ might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERβ-selective compounds have been identified. One class of ERβ-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERβ much greater than ERα. A second class of ERβ-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERβ. Diarylpropionitrile represents a third class of ERβ-selective compounds because its selectivity is due to a combination of greater binding to ERβ and transcriptional activity. However, it is unclear if these three classes of ERβ-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERβ selectivity and pattern of gene expression of these three classes of ERβ-selective compounds compared to estradiol (E2), which is a non-selective ER agonist. U2OS cells stably transfected with ERα or ERβ were treated with E2 or the ERβ-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERβ-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERβ, which is consistent with the finding that most genes regulated by the ERβ-selective compounds were similar to each other and E2. However, there were some classes of genes differentially regulated by the ERβ agonists and E2. Two ERβ-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERβ. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERβ-selective agonists and E2, there were some genes regulated that were distinct from each other and E2, suggesting that different ERβ-selective agonists might produce distinct biological and clinical effects.
doi:10.1371/journal.pone.0006271
PMCID: PMC2707612  PMID: 19609440

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