RESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Furanos/antagonistas & inhibidores , Inflamación/tratamiento farmacológico , Pironas/antagonistas & inhibidores , Animales , Western Blotting , Línea Celular , Citocinas/antagonistas & inhibidores , Citocinas/genética , Humanos , Inmunomodulación , Concentración 50 Inhibidora , Ratones , Modelos Moleculares , Estructura Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Elastasa de Leucocito/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Femenino , Humanos , Indoles/química , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/química , Elastasa de Leucocito/genética , Modelos Moleculares , Mutación , FN-kappa B/análisis , FN-kappa B/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Verduras/químicaRESUMEN
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.
Asunto(s)
Alcaloides/química , Azepinas/química , Factores Inmunológicos/química , Myxococcales/química , Poríferos/química , Alcaloides/aislamiento & purificación , Alcaloides/farmacología , Animales , Azepinas/aislamiento & purificación , Azepinas/farmacología , Quimiocina CCL2/metabolismo , Cromatografía Líquida de Alta Presión , Células HCT116 , Humanos , Quinasa I-kappa B/metabolismo , Factores Inmunológicos/aislamiento & purificación , Factores Inmunológicos/farmacología , Interleucina-6/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Espectrometría de Masas , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/fisiología , Fase G1/efectos de los fármacos , Indoles/farmacología , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/fisiología , Telomerasa/genética , Línea Celular Tumoral , Quinasa 6 Dependiente de la Ciclina/genética , Regulación hacia Abajo , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Femenino , Regulación de la Expresión Génica , Humanos , Fosforilación , Factor de Transcripción Sp1/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Productos Biológicos , Técnicas Químicas Combinatorias , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Células HT29 , Células HeLa , Humanos , Biología Marina , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Poríferos/química , Sesquiterpenos/química , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/farmacología , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , Ciclina E/metabolismo , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Elastasa Pancreática/antagonistas & inhibidores , Brassica/química , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Dieta , Humanos , Oligopéptidos/farmacología , Elastasa Pancreática/genética , ARN Interferente Pequeño/genéticaRESUMEN
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.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Indoles/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular Tumoral , Activación Enzimática , Técnica del Anticuerpo Fluorescente , HumanosRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Fase G1/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/genética , Femenino , Humanos , Regiones Promotoras Genéticas , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Receptor beta de Estrógeno/agonistas , Flavanonas/farmacología , Glycyrrhiza/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Femenino , Flavanonas/química , Humanos , Ratones , Ratones Desnudos , Coactivador 2 del Receptor Nuclear/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Útero/citología , Útero/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
3,3'-Diindolylmethane (DIM), a major condensation product of indole-3-carbinol, exhibits chemopreventive properties in animal models of cancer. Recent studies have shown that DIM stimulates interferon-gamma (IFN-gamma) production and potentiates the IFN-gamma signaling pathway in human breast cancer cells via a mechanism that includes increased expression of the IFN-gamma 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 interleukin (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, induced elevation of serum cytokines in mice, including IL-6, granulocyte colony-stimulating factor (G-CSF), IL-12 and IFN-gamma. 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.
Asunto(s)
Anticarcinógenos/farmacología , Inmunidad/efectos de los fármacos , Indoles/farmacología , Animales , Anticuerpos Antivirales/análisis , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/biosíntesis , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inmunoglobulina A/efectos de los fármacos , Inmunoglobulina A/inmunología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Orthoreovirus de los Mamíferos/efectos de los fármacos , Orthoreovirus de los Mamíferos/inmunología , Especies Reactivas de Oxígeno/metabolismo , Infecciones por Reoviridae/inmunología , Bazo/citología , Bazo/inmunologíaRESUMEN
Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis in multiple organs. 3,3'-Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising antitumor agent. Previous studies in our laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in human breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that 3,3'-diindolylmethane is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20 micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular ATP level, and significantly stimulated mitochondrial reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580 and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase (JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3'-diindolylmethane-induced p21 overexpression, we showed that 3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21 up-regulation in human breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Indoles/farmacología , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosforilación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína de Retinoblastoma/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor (ER)-alpha (ERalpha) and ERbeta. Indole-3-carbinol (I3C) strongly down-regulated ERalpha protein and transcript levels, without altering the level of ERbeta protein, in both cell lines. In cells transfected with the ERalpha promoter linked to a luciferase gene reporter, I3C ablated ERalpha promoter activity. Propyl pyrazole triol (PPT) is a highly selective ERalpha agonist, whereas, 17beta-estradiol activates both ERalpha and ERbeta. I3C treatment inhibited the PPT- and 17beta-estradiol-induced proliferation of breast cancer cells, disrupted the PPT and 17beta-estradiol stimulation of estrogen response element (ERE)-driven reporter plasmid activity as well as of endogenous progesterone receptor transcripts. Using an in vitro ERE binding assay, I3C was shown to inhibit the level of functional ERalpha and stimulated the level of ERE binding ERbeta even though the protein levels of this receptor remained constant. In ERalpha-/ERbeta+ MDA-MB-231 breast cancer cells, I3C treatment stimulated a 6-fold increase in binding of ERbeta to the ERE. I3C also induced ERE- and activator protein 1-driven reporter plasmid activities in the absence of an ER agonist, suggesting that ERbeta is activated in indole-treated cells. Taken together, our results demonstrate that the expression and function of ERalpha and ERbeta can be uncoupled by I3C with a key cellular consequence being a significantly higher ERbeta:ERalpha ratio that is generally highly associated with antiproliferative status of human breast cancer cells.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/antagonistas & inhibidores , Indoles/farmacología , Neoplasias de la Mama/genética , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Estrógenos/farmacología , Humanos , Regiones Promotoras Genéticas , Receptores de Progesterona/metabolismo , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
3,3'-Diindolylmethane (DIM) is a promising anticancer agent derived from Brassica vegetables, but the mechanisms of DIM action are largely unknown. We have shown that DIM can upregulate the expression and stimulate the secretion of interferon-gamma (IFNgamma) in the human MCF-7 breast cancer cell line. This novel effect may provide important clues to explain the anticancer effects of DIM because it is well known that IFNgamma plays an important role in preventing the development of primary and transplanted tumors. Utilizing promoter deletions, we show here that the region between -108 and -36 bp in the IFNgamma promoter, which contains two conserved and essential regulatory elements, is required for DIM-induced IFNgamma expression. DIM activates both JNK and p38 pathways, induces the phosphorylation of c-Jun and ATF-2, and increases the binding of the homodimer or heterodimer of c-Jun/ATF-2 to the proximal AP-1.CREB-ATF-binding element. Moreover, studies with specific enzyme inhibitors showed that up-stream Ca2+-dependent kinase(s) is required for the inducing effects of DIM in MCF-7 cells. These results establish that DIM-induced IFNgamma expression in human breast tumor cells is mediated by activation of both JNK and p38 pathways, which is ultimately dependent on intracellular calcium signaling.
Asunto(s)
Neoplasias de la Mama/genética , Regulación de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Interferón gamma/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Factor de Transcripción Activador 2 , Secuencia de Bases , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Cartilla de ADN , Activación Enzimática , Humanos , MAP Quinasa Quinasa 4 , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Indole-3-carbinol (I3C), a dietary compound found naturally in cruciferous vegetables of the Brassica genus such as broccoli and brussels sprouts, induces a G1 growth arrest of human reproductive cancer cells. We previously reported that in LNCaP prostate cancer cells, I3C down-regulated cyclin-dependent kinase (CDK) 2 activity. In our current study, Western blotting and quantitative RT-PCR demonstrated that I3C treatment increased both the transcripts and protein levels of the CDK2 inhibitor p21(waf1/cip1) (p21). Transfection of luciferase reporter plasmids containing wild-type and mutated p21 promoter fragments revealed that I3C induced p21 gene transcription through a p53 DNA binding element. Oligonucleotide precipitation showed that I3C increased the level of activated p53 nuclear protein that is competent to bind its DNA target site on the p21 promoter. Ablation of p53 production using short interfering RNA (siRNA) prevented that the I3C induced G1 arrest and up-regulation of p21 expression. Western blots using p53 phospho-specific antibodies revealed that I3C treatment increased the levels of three phosphorylated forms of p53 (Ser15, Ser37, Ser392) that are known to contribute to p53 protein stability and greater transactivation potential. Taken together, our results establish that the I3C induced G1 arrest of human prostate cancer cells requires the induced production of the activated phosphorylated forms of p53, which stimulate transcription of the CDK2 inhibitor p21.
Asunto(s)
Anticarcinógenos/farmacología , Ciclo Celular/efectos de los fármacos , Indoles/farmacología , Neoplasias de la Próstata , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Luciferasas/genética , Masculino , Mutagénesis Sitio-Dirigida , Fosforilación , Regiones Promotoras Genéticas , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Transfección , Regulación hacia ArribaRESUMEN
5,6,11,12,17,18-Hexahydrocyclonona[1,2-b:4,5-b*:7,8-b**]triindole (CTr) is a major digestive product of indole-3-carbinol (I3C) from Brassica vegetables and exhibits strong estrogenic activities. CTr increases proliferation of estrogen-dependent breast tumor cells, binds with strong affinity for the estrogen receptor-alpha (ERalpha), and activates expression of estrogen (E(2))-dependent genes. To begin to examine the structural features that determine the biological activity of CTr, we prepared and studied the effects of two analogs, 9,18-dihydro-12H-[1,2,5]trithionino[3,4-b:6,7-b*:9,8-b**]triindole (S(3)CTr) and 5,6,11,12,17,18-hexahydro-5,11,17-trimethylcyclonona[1,2-b:4,5-b*:7,8-b**]triindole (Me(3)CTr). N-Methylation of CTr completely ablated the estrogenic activities of CTr. In the dose range in which CTr was clearly estrogenic, Me(3)CTr exhibited no detectable effect on cell growth, ERalpha binding to E(2), or ERalpha-responsive gene expression. S(3)CTr showed mixed ERalpha agonist activities. It bound to the ERalpha and activated receptor binding with DNA, weakly activated expression of transfected E(2)-responsive reporter gene constructs, and strongly inhibited the E(2)-induced activation of these reporter constructs. S(3)CTr activated aryl hydrocarbon receptor (AhR)-mediated pathways, consistent with the moderately strong binding affinity of S(3)CTr for the AhR. Comparisons of the conformational characteristics among CTr and its two analogs indicated that the estrogenic effects of CTr are highly sensitive to apparently minor structural modifications, and further supported the hypothesis for a central role of hydrogen bonding around the nitrogen atom in CTr binding to the ligand binding site of ERalpha.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/metabolismo , Indoles/farmacología , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Animales , Unión Competitiva/fisiología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , ADN de Neoplasias/química , ADN de Neoplasias/genética , Ensayo de Cambio de Movilidad Electroforética , Femenino , Humanos , Indoles/química , Ratones , Modelos Moleculares , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Relación Estructura-Actividad , TransfecciónRESUMEN
We investigated the mechanism of ligand-independent activation of the estrogen receptor (ER) by 3,3'-diindolylmethane (DIM), a promising anticancer agent derived from vegetables of the Brassica genus, in Ishikawa and HEC-1B human endometrial cancer cells. DIM stimulated the activity of an ER-responsive reporter by over 40-fold, equivalent to the maximum induction produced by estradiol (E2), whereas cotreatment of cells with the ER antagonist, ICI-182,780 (ICI), abolished the stimulatory effect of DIM. DIM also induced the expressions of the endogenous genes, TGF-alpha, alkaline phosphatase, and progesterone receptor similar to levels induced by E2. Induction of gene expression by DIM was inhibited by the protein synthesis inhibitor, cycloheximide. In addition, cotreatment of cells with the protein kinase A (PKA) inhibitor, H89, or the MAPK inhibitor, PD98059, reduced DIM activation of the ER by 75% and 50%, respectively. Simultaneous treatment of cells with both inhibitors completely abolished the effect of DIM. DIM stimulated MAPK activity and induced phosphorylation of the endogenous PKA target, cAMP response element binding protein (CREB), in a PKA-dependent manner. Expression of MCREB, a nonphosphorylatable CREB mutant, partially abolished activation of the ER by DIM. These results demonstrate that DIM is a mechanistically novel activator of the ER that requires PKA-dependent phosphorylation of CREB.
Asunto(s)
Anticarcinógenos/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Estradiol/análogos & derivados , Receptor alfa de Estrógeno/efectos de los fármacos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Cicloheximida/farmacología , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/metabolismo , Inhibidores Enzimáticos/farmacología , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Flavonoides/farmacología , Fulvestrant , Humanos , Isoquinolinas/farmacología , Ligandos , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , Mutación Puntual , Sulfonamidas/farmacología , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/metabolismo , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
3,3'-Diindolylmethane (DIM) is a major in vivo derivative of the putative anticancer agent indole-3-carbinol (I3C), which is present in vegetables of the Brassica genus. At concentrations above 10 microM, DIM inhibited DNA synthesis and cell proliferation in both estrogen receptor replete (MCF-7) and deficient (MDA-MB-231) human breast cancer cells in a concentration- and time-dependent manner. These antiproliferative effects were accompanied by characteristic indications of programmed cell death in both cell lines, including externalization of phosphatidylserine, chromatin condensation, and DNA fragmentation. Furthermore, Western and Northern blot analyses, as well as coimmunoprecipitation assays, revealed that in both MCF-7 and MDA-MB-231 cells, DIM treatment decreased total transcript and protein levels of the apoptosis inhibitory protein Bcl-2, and the amount of Bcl-2 bound to the pro-apoptotic protein Bax. DIM treatment also caused an increase in Bax protein levels, but did not affect the level of Bax that was bound to Bcl-2. As a functional test of the role of Bcl-2 down-regulation in the DIM-induced apoptotic response, ectopic expression of Bcl-2 in MCF-7 cells was shown to attenuate the apoptotic effect of DIM. These results demonstrate that DIM can induce apoptosis in breast cancer cells independent of estrogen receptor status by a process that is mediated by the modulated expression of the Bax/Bcl-2 family of apoptotic regulatory factors.
Asunto(s)
Anticarcinógenos/farmacología , Apoptosis , Indoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Expresión Génica , Humanos , Pruebas de Precipitina , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2RESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Citocinas/inmunología , Lignanos/farmacología , Macrófagos/efectos de los fármacos , Animales , Línea Celular , Citocinas/genética , Dinoprostona/inmunología , Proteínas I-kappa B/inmunología , Lipopolisacáridos , Macrófagos/inmunología , Ratones , Proteínas Quinasas Activadas por Mitógenos/inmunología , Inhibidor NF-kappaB alfa , FN-kappa B/inmunología , Nitritos/inmunología , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Indoles/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Factor de Crecimiento de Hepatocito/farmacología , Factor de Crecimiento de Hepatocito/fisiología , Humanos , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
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.