RESUMEN
After birth, the development of secondary lymphoid tissues (SLTs) in the colon is dependent on the expression of the aryl hydrocarbon receptor (AhR) in immune cells as a response to the availability of AhR ligands. However, little is known about how AhR activity from intestinal epithelial cells (IECs) may influence the development of tertiary lymphoid tissues (TLTs). As organized structures that develop at sites of inflammation or infection during adulthood, TLTs serve as localized centers of adaptive immune responses, and their presence has been associated with the resolution of inflammation and tumorigenesis in the colon. Here, we investigated the effect of the conditional loss of AhR activity in IECs in the formation and immune cell composition of TLTs in a model of acute inflammation. In females, loss of AhR activity in IECs reduced the formation of TLTs without significantly changing disease outcomes or immune cell composition within TLTs. In males lacking AhR expression in IECs, increased disease activity index, lower expression of functional-IEC genes, increased number of TLTs, increased T-cell density, and lower B- to T-cell ratio were observed. These findings may represent an unfavorable prognosis when exposed to dextran sodium sulfate (DSS)-induced epithelial damage compared with females. Sex and loss of IEC AhR also resulted in changes in microbial populations in the gut. Collectively, these data suggest that the formation of TLTs in the colon is influenced by sex and AhR expression in IECs.NEW & NOTEWORTHY This is the first research of its kind to demonstrate a clear connection between biological sex and the development of tertiary lymphoid tissues (TLT) in the colon. In addition, the research finds that in a preclinical model of inflammatory bowel disease, the expression of the aryl hydrocarbon receptor (AhR) influences the development of these structures in a sex-specific manner.
Asunto(s)
Colon , Mucosa Intestinal , Receptores de Hidrocarburo de Aril , Estructuras Linfoides Terciarias , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Animales , Femenino , Colon/metabolismo , Colon/inmunología , Colon/patología , Masculino , Estructuras Linfoides Terciarias/patología , Estructuras Linfoides Terciarias/inmunología , Estructuras Linfoides Terciarias/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/inmunología , Ratones , Células Epiteliales/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
We recently reported that novel ring-substituted analogs of 3,3'-diindolylmethane (ring-DIMs) have anti-androgenic and growth inhibitory effects in androgen-dependent prostate cancer cells. The objectives of this study were to confirm the ability of 4,4'- and 7,7'-dibromo- and dichloro-substituted ring-DIMs to inhibit androgen-stimulated proliferation of androgen-dependent LNCaP human prostate cancer cells using a non-invasive, real-time monitoring technique. In addition, their ability to induce apoptotic and necrotic cell death in androgen-dependent as well as -independent (PC-3) prostate cancer cells was studied. Prostate cancer cells were treated with increasing concentrations of DIM and ring-DIMs (0.3-30 µM) and effects on cell proliferation were measured in real-time using an xCELLigence cellular analysis system. Chromatin condensation and loss of membrane integrity were determined by Hoechst and propidium iodide staining, respectively. Apoptotic protein markers were measured by immunoblotting and activation of caspases determined using selective fluorogenic substrates. Intra- and extracellular concentrations of DIM and ring-DIMs were assessed by electrospray ionization tandem mass spectrometry. Ring-DIMs inhibited androgen-stimulated LNCaP cell proliferation and induced apoptosis and necrosis in LNCaP and PC-3 cells with 2-4 fold greater potencies than DIM. DIM and the ring-DIMs increased caspases -3, -8 and -9 activity, elevated expression of Fas, FasL, DR4 and DR5 protein, and induced PARP cleavage in both cell lines. The cytotoxicity of the most potent ring-DIM, 4,4'-dibromoDIM, but not the other compounds was decreased by an inhibitor of caspase -3. The 4,4'-dibromoDIM was primarily found in the extracellular medium, whereas all other compounds were present to a much larger extent in the cell. In conclusion, ring-DIMs inhibited prostate cancer cell growth and induced cell death in LNCaP and PC-3 cells with greater potencies than DIM; they also structure-dependently activated different cell death pathways suggesting that these compounds have clinical potential as chemopreventive and chemotherapeutic agents in prostate cancer, regardless of hormone-dependency.
Asunto(s)
Andrógenos/farmacología , Apoptosis/efectos de los fármacos , Indoles/farmacología , Indoles/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dihidrotestosterona/farmacología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/química , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Masculino , Necrosis , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Receptores de Muerte Celular/genética , Receptores de Muerte Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
HOTAIR is a long intervening non-coding RNA (lincRNA) that associates with the Polycomb Repressive Complex 2 (PRC2) and overexpression is correlated with poor survival for breast, colon and liver cancer patients. In this study, we show that HOTAIR expression is increased in pancreatic tumors compared with non-tumor tissue and is associated with more aggressive tumors. Knockdown of HOTAIR (siHOTAIR) by RNA interference shows that HOTAIR has an important role in pancreatic cancer cell invasion, as reported in other cancer cell lines. In contrast, HOTAIR knockdown in Panc1 and L3.6pL pancreatic cancer cells that overexpress this lincRNA decreased cell proliferation, altered cell cycle progression and induced apoptosis, demonstrating an expanded function of HOTAIR in pancreatic cancer cells compared with other cancer cell lines. Results of gene array studies showed that there was minimal overlap between HOTAIR-regulated genes in pancreatic cells and breast cancer cells, and HOTAIR uniquely suppressed several interferon-related genes and gene sets related to cell cycle progression in pancreatic cancer cells and tumors. Analysis of selected genes suppressed by HOTAIR in Panc1 and L3.6pL cells showed by knockdown of EZH2 and chromatin immunoprecipitation assays that HOTAIR-mediated gene repression was both PRC2-dependent and -independent. HOTAIR knockdown in L3.6pL cells inhibited tumor growth in mouse xenograft model, further demonstrating the pro-oncogenic function of HOTAIR in pancreatic cancer.
Asunto(s)
Carcinoma Ductal Pancreático/diagnóstico , Transformación Celular Neoplásica/genética , Neoplasias Pancreáticas/diagnóstico , ARN Largo no Codificante/fisiología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/fisiología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Pronóstico , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/farmacología , Trasplante HeterólogoRESUMEN
BACKGROUND: Tolfenamic acid (TA) is an NSAID currently under investigation as an anticancer agent in humans. TA induces proteosome-dependent degradation of transcription factors Sp 1, 3, and 4. These proteins are known to be overexpressed in many human cancers. HYPOTHESIS: To evaluate the protein expression of Sps in canine tissue, and efficacy of TA against several canine tumor cell lines. METHODS: Six canine cell lines (2 osteosarcoma, 2 mammary carcinoma, 2 melanoma) were evaluated. Protein levels of Sp 1-4 and their downstream targets were evaluated using Western Blots. Cell survival and TUNEL assays were performed on cell lines, and Sp1 expression was evaluated on histologic samples from archived canine cases. ANIMALS: Six immortalized canine cancer cell lines derived from dogs were used. Archived tissue samples were also used. RESULTS: Sps were highly expressed in all 6 cell lines and variably expressed in histologic tissues. TA decreased expression of Sps 1-4 in all cell lines. All of the downstream targets of Sps were inhibited in the cell lines. Variable Sp1 expression was identified in all histologic samples examined. TA significantly inhibited cell survival in all cell lines in a dose dependant fashion. The number of cells undergoing apoptosis was significantly increased (P < .05) in all cell lines after exposure to TA in a dose-dependent fashion. CONCLUSIONS, AND CLINICAL IMPORTANCE: Tolfenamic acid is a potential anticancer NSAID and further investigation is needed to determine its usefulness in a clinical setting.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Enfermedades de los Perros/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Neoplasias/veterinaria , Factores de Transcripción Sp/metabolismo , ortoaminobenzoatos/farmacología , Animales , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/veterinaria , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/patología , Perros , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Melanoma/tratamiento farmacológico , Melanoma/patología , Melanoma/veterinaria , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Osteosarcoma/veterinaria , ARN Neoplásico/química , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Factores de Transcripción Sp/biosíntesis , Factores de Transcripción Sp/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The human POK family members are transcription factors with a POZ domain and zinc-fingers that act primarily as transcriptional repressors. Several members of this family are involved in oncogenesis and this prompted us to assess whether expression levels of individual POK family members are associated with clinical outcomes in cancer. We have observed that ZBTB4 (zinc-finger and BTB domain containing 4) is downregulated in breast cancer patients, and that its expression is significantly correlated with relapse-free survival. Further integrative analysis of mRNA and microRNA (miR) expression data from the NCI-60 cell lines revealed an inverse correlation between ZBTB4 and oncogenic miRs derived from the miR-17-92 cluster and its paralogs. The experimental results using MDA-MB-231 and MCF-7 human breast cancer cells confirm that miRNAs derived from these clusters, containing miR-17-5p, miR-20a, miR-106a, miR-106b and miR-93, negatively regulate ZBTB4 expression. Overexpression of ZBTB4 or restoration of ZBTB4 by using an antagomir inhibit growth and invasion of breast cancer cells, and this effect is due, in part, to ZBTB4-dependent repression of the specificity protein 1 (Sp1), Sp3 and Sp4 genes, and subsequent downregulation of several Sp-dependent oncogenes, in part, through competition between ZBTB4 and Sp transcription factors for GC-rich promoter sequences. These results confirm that ZBTB4 functions as a novel tumor-suppressor gene with prognostic significance for breast cancer survival, and the oncogenic miR-17-92/ZBTB4/Sp axis may be a potential therapeutic target.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Oncogenes , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Unión Competitiva , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Supervivencia sin Enfermedad , Regulación hacia Abajo , Femenino , Genes Reporteros , Humanos , Estimación de Kaplan-Meier , Luciferasas/biosíntesis , Luciferasas/genética , MicroARNs/metabolismo , Pronóstico , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Largo no Codificante , Proteínas Represoras/genética , Factores de Transcripción Sp/genética , Factores de Transcripción Sp/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
The orphan nuclear receptor TR3 (NR41A and Nur77) is overexpressed in most lung cancer patients and is a negative prognostic factor for patient survival. The function of TR3 was investigated in non-small-cell lung cancer A549 and H460 cells, and knockdown of TR3 by RNA interference (siTR3) inhibited cancer cell growth and induced apoptosis. The prosurvival activity of TR3 was due, in part, to formation of a p300/TR3/ specificity protein 1 complex bound to GC-rich promoter regions of survivin and other Sp-regulated genes (mechanism 1). However, in p53 wild-type A549 and H460 cells, siTR3 inhibited the mTORC1 pathway, and this was due to activation of p53 and induction of the p53-responsive gene sestrin 2, which subsequently activated the mTORC1 inhibitor AMP-activated protein kinase α (AMPKα) (mechanism 2). This demonstrates that the pro-oncogenic activity of TR3 in lung cancer cells was due to inhibition of p53 and activation of mTORC1. 1,1-Bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) is a recently discovered inhibitor of TR3, which mimics the effects of siTR3. DIM-C-pPhOH inhibited growth and induced apoptosis in lung cancer cells and lung tumors in murine orthotopic and metastatic models, and this was accompanied by decreased expression of survivin and inhibition of mTORC1 signaling, demonstrating that inactivators of TR3 represent a novel class of mTORC1 inhibitors.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Proteínas/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Anciano , Animales , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Indoles/farmacología , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Persona de Mediana Edad , Complejos Multiproteicos , Proteínas Nucleares/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/deficiencia , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Fenoles/farmacología , Pronóstico , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TORRESUMEN
Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that induces proliferation of MCF-7 breast cancer cells, and cotreatment with the phosphoinositide 3-kinase (PI3-K) inhibitor LY294002 and the antiestrogen ICI 182780 inhibits IGF-I-induced growth. The role of estrogen receptor alpha (ERalpha) in mediating responses induced by IGF-I was investigated in cells transfected with small inhibitory RNA for ERalpha (iERalpha). The results showed that IGF-I-dependent phosphorylation of Akt and mitogen-activated protein kinase, induction of G(1)-S-phase progression and enhanced expression of cyclin D1 and cyclin E were dependent on ERalpha. Moreover, these same IGF-I-induced responses were also inhibited by the antiestrogen ICI 182780 and this was in contrast to a previous report suggesting that ICI 182780 did not affect IGF-I-dependent activation of PI3-K or induction of cyclin D1 expression. ICI 182780 exhibits antimitogenic activity and iERalpha inhibits G(1)-S-phase progression and proliferation of MCF-7 cells treated with IGF-I, suggesting that the effects of the antiestrogen are primarily related to downregulation of ERalpha.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Secuencia de Bases , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclinas/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Femenino , Fulvestrant , Humanos , Proteínas Sustrato del Receptor de Insulina , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mitógenos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
The gonadal and placental paralogues of porcine aromatase cytochrome P450 (P450arom) were examined for novel catalytic properties to shed light on the evolutionary survival of duplicated copies of an enzyme critical to reproduction. Recombinant gonadal P450arom catalyzed the formation of a novel metabolite from testosterone, identified by gas chromatography/mass spectrometry and biochemical analyses as 1 beta-hydroxytestosterone (1 beta OH-T), in almost equal proportion to 17beta-estradiol (E(2)). This activity was absent in reactions with the porcine placental paralogue (or other orthologues) of P450arom and was minimal with androstenedione. Incubations with both porcine enzymes and with bovine and human P450arom demonstrated that 1 beta OH-T was not aromatizable, and 1 beta OH-T activated the androgen receptor of prostate cancer cells in vitro. Porcine testicular and follicular granulosa tissues synthesized 1 beta OH-T, which was also detected in testicular venous plasma. These results constitute the first of identification of a novel, perhaps potent, nonaromatizable metabolite of testosterone, whose synthesis (paradoxically) can be definitively ascribed to the activity of the gonadal paralogue of porcine P450arom. It probably represents an evolutionary gain of function associated with fixation and the survival of the genes after CYP19 duplication. Novel activities and adaptive functions may exist among other duplicated vertebrate aromatases.
Asunto(s)
Aromatasa/genética , Aromatasa/metabolismo , Duplicación de Gen , Animales , Bovinos , Estradiol/metabolismo , Evolución Molecular , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hidroxitestosteronas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Ovario/enzimología , Placenta/enzimología , Embarazo , Proteínas Recombinantes , Especificidad por Sustrato , Porcinos , Testículo/enzimología , Testosterona/metabolismo , TritioRESUMEN
1. The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) protect animals against chemically induced tumours. Putative chemoprotective mechanisms include modulated expression of hepatic biotransformation enzymes. However, few, if any, studies have used human primary cells as test models. 2. The present study investigated the effects of these phytochemicals on the expression of four carcinogenesis-relevant enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and 87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5 x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on GSTA1 transcription were seen. 4. The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.
Asunto(s)
Anticarcinógenos/farmacología , Carcinógenos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Hepatocitos/efectos de los fármacos , Anticarcinógenos/metabolismo , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Curcumina/metabolismo , Curcumina/farmacología , Citocromo P-450 CYP1A1/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/efectos de los fármacos , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Enzimas/efectos de los fármacos , Flavanonas/metabolismo , Flavanonas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Transferasa , Hepatocitos/fisiología , Humanos , Inactivación Metabólica , Indoles/metabolismo , Indoles/farmacología , Isotiocianatos/metabolismo , Isotiocianatos/farmacología , NAD(P)H Deshidrogenasa (Quinona)/efectos de los fármacos , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Plantas/química , Sulfóxidos , Tiocianatos/metabolismo , Tiocianatos/farmacologíaRESUMEN
Transferrin (Tf) is an iron transport protein expressed in MCF-7 human breast cancer cells. In nuclear run-on assays, 17beta-estradiol (E2) increased the rate of Tf gene expression approximately 3-fold within 1 h after treatment and reporter gene activity was also induced in MCF-7 cells transfected with a construct containing a -3600 to +39 Tf gene promoter insert. Deletion and mutation analysis identified an E2-responsive promoter region between -811 and -762, which was GC-rich (80%) and contained two nonconsensus estrogen response elements (EREs). E2-responsiveness of this region was associated with a GGACA(N)(3)TGGCC motif (-803 to -791) which bound human estrogen receptor alpha (hERalpha) in gel mobility shift assays. In Drosophila Schneider SL-2 cells, the -811 to -752 was E2-responsive after cotransfection with hERalpha expression plasmid plus E2, whereas Sp1 protein did not induce transactivation. These studies confirm that E2 induces Tf gene expression through a nonconsensus distal ERE.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Estrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica , Transferrina/genética , Ensayo de Cambio de Movilidad Electroforética , Receptor alfa de Estrógeno , Humanos , Mutación , Regiones Promotoras Genéticas/genética , Receptores de Estrógenos/metabolismo , Elementos de Respuesta/genética , Transfección , Células Tumorales CultivadasRESUMEN
The rat creatine kinase B (CKB) gene is induced by estrogen in the uterus, and constructs containing rat CKB gene promoter inserts are highly estrogen-responsive in cell culture. Analysis of the upstream -568 to -523 region of the promoter in HeLa cells has identified an imperfect palindromic estrogen response element (ERE) that is required for hormone inducibility. Analysis of the CKB gene promoter in MCF-7 breast cancer cells confirmed that pCKB7 (containing the -568 to -523 promoter insert) was estrogen-responsive in transient transfection studies. However, mutation and deletion analysis of this region of the promoter showed that two GC-rich sites and the concensus ERE were functional cis-elements that bound estrogen receptor alpha (ERalpha)/Sp1 and ERalpha proteins, respectively. The role of these elements was confirmed in gel mobility shift and chromatin immunoprecipitation assays and transfection studies in MDA-MB-231 and Schneider Drosophila SL-2 cells. These results show that transcriptional activation of CKB by estrogen is dependent, in part, on ERalpha/Sp1 action which is cell context-dependent.
Asunto(s)
Creatina Quinasa/genética , Estradiol/farmacología , Secuencia Rica en GC , Regiones Promotoras Genéticas , Activación Transcripcional/efectos de los fármacos , Animales , Secuencia de Bases , Neoplasias de la Mama , Línea Celular , Forma BB de la Creatina-Quinasa , Análisis Mutacional de ADN , Drosophila , Ensayo de Cambio de Movilidad Electroforética/métodos , Receptor alfa de Estrógeno , Femenino , Humanos , Isoenzimas , Datos de Secuencia Molecular , Receptores de Estrógenos/genética , Eliminación de Secuencia , Factor de Transcripción Sp1/genética , Células Tumorales CultivadasRESUMEN
Resveratrol decreases basal and induced CYP1A1 mRNA/protein levels in both in vitro and in vivo models, and some studies suggest that resveratrol acts as an aryl hydrocarbon receptor (AhR) antagonist. Treatment of T47D or MCF-7 cells with 10 microM resveratrol inhibited induction of CYP1A1 mRNA and CYP1A1-dependent activity after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as previously reported. In contrast, resveratrol did not inhibit TCDD-induced reporter gene activity in cells transfected with an Ah-responsive construct containing a human CYP1A1 gene promoter insert, whereas 3'-methoxy-4'-nitroflavone, a "pure" AhR antagonist, inhibited this response. Resveratrol induced transformation of the rat cytosolic AhR and, after treatment of T47D and MCF-7 cells with resveratrol, a transformed nuclear AhR complex was observed. In contrast to 3'-methoxy-4'-nitroflavone, resveratrol did not block TCDD-induced AhR transformation in vitro or nuclear uptake of the AhR complex in breast cancer cells. Thus, the action of resveratrol on the AhR was consistent with that of an AhR agonist; however, resveratrol did not exhibit functional AhR agonist or antagonist activities in breast cancer cells. Actinomycin D chase experiments in T47D cells showed that resveratrol and dehydroepiandrosterone both increased the rate of CYP1A1 mRNA degradation, whereas resveratrol did not affect CYP1A1-dependent activity in cells pretreated with TCDD for 18 hr. These data suggest that resveratrol inhibits CYP1A1 via an AhR-independent post-transcriptional pathway.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Citocromo P-450 CYP1A1/genética , Silenciador del Gen/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , Estilbenos/farmacología , Neoplasias de la Mama/patología , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/metabolismo , Estabilidad de Enzimas/efectos de los fármacos , Humanos , Procesamiento Postranscripcional del ARN , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Resveratrol , Células Tumorales CultivadasRESUMEN
Structure-dependent estrogen receptor alpha (ER alpha) agonist and antagonist activities of synthetic and natural estrogenic compounds were investigated in human HepG2, MDA-MB-231 and U2 cancer cell lines. Compounds used in this study include 4'-hydroxytamoxifen, ICI 182,780, bisphenol-A (BPA), 2',4',6'-trichloro-4-biphenylol (3Cl-PCB-OH), 2',3',4',5'-tetrachloro-4-biphenylol (4Cl-PCB-OH), p-t-octylphenol, p-nonylphenol, naringenin, kepone, resveratrol, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). Cells were transfected with a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) and either wild-type estrogen receptor alpha (ER-wt) or variants expressing activation function-1 (ER-AF1) or AF-2 (ER-AF2). The ER agonist activities of the synthetic mono and dihydroxy aromatic compounds are comparable in all three-cell lines, whereas the activities of naringenin, kepone and resveratrol are dependent on cell context and expression of wild-type or variant forms of ER alpha. In contrast, the ER antagonist activities for these compounds were highly complex and, with the exception of 3Cl-PCB-OH, all compounds inhibited E2-induced wild-type or variant ER action. Results of this in vitro study suggest that the estrogenic and antiestrogenic activity of structurally diverse synthetic and natural estrogenic compounds is complex, and this is consistent with published data that often give contradictory results for these compounds.
Asunto(s)
Estrógenos/metabolismo , Polimorfismo Genético , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Tamoxifeno/análogos & derivados , Compuestos de Bencidrilo , División Celular , Relación Dosis-Respuesta a Droga , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno , Estrógenos no Esteroides/farmacología , Fulvestrant , Humanos , Fenoles/farmacología , Bifenilos Policlorados/farmacología , Receptores de Estrógenos/agonistas , Elementos de Respuesta , Tamoxifeno/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
The reproductive and developmental effects of 17beta-estradiol (E2) and methoxychlor (MXC) observed in treated rodents appear to be linked to some unique but also overlapping patterns of gene expression. The MXC metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) was previously shown to have selective agonist activity through estrogen receptor alpha (ERalpha) and antagonist activity through ERbeta and androgen receptor (AR). To discover gene families regulated by HPTE and E2, and to characterize similarities and differences in patterns of gene expression induced by these selective ER ligands, we analyzed tissues from mice treated for 3 days with a combined treatment of E2 and HPTE (E2 + HPTE), or the antiandrogen flutamide (FLU). RNA from uteri and ovaries was analyzed with cDNA microarrays and real-time RT-PCR. Results indicate that HPTE and E2 acted similarly to regulate most gene families in the uterus, which expresses predominantly ERalpha. However, in both the uterus and the ovary, there were a few genes that displayed differential patterns of gene regulation by E2 or HPTE treatment, presumably through ERbeta, AR, or other unidentified pathways. In the uterus, progesterone receptor, ERalpha, AR, insulin-like growth factor 1, insulin-like growth factor binding protein 5, and clusterin mRNAs were significantly reduced with both E2 or HPTE treatments, whereas cathepsin B was induced. Conversely, in the ovary, induction of cathepsin B by E2 was reversed after cotreatment with HPTE, and ERbeta expression was induced similarly by HPTE and FLU but not by E2. In addition, E2 uniquely regulated glutathione peroxidase 3, glutathione S-transferase, and cytochrome P450 17alpha-hydroxylase, with no effect of HPTE or FLU treatments. This analysis demonstrated several gene families that appear to be regulated in a ligand-specific pattern, which may explain the unique but overlapping reproductive tissue pathologies following exposure to E2 and MXC.
Asunto(s)
Estradiol/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Genitales Femeninos/efectos de los fármacos , Metoxicloro/metabolismo , Receptores Androgénicos/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Cartilla de ADN/química , Combinación de Medicamentos , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Flutamida/toxicidad , Genitales Femeninos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ovario/efectos de los fármacos , Ovario/metabolismo , Fenoles/toxicidad , ARN Mensajero/metabolismo , Receptores Androgénicos/genética , Receptores de Estrógenos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Útero/efectos de los fármacos , Útero/metabolismoRESUMEN
Diindolylmethane (DIM) is formed by acid catalyzed dimerization of the phytochemical indole-3-carbinol, and both compounds inhibit formation and/or growth of mammary tumors in rodents. In this study, we have investigated the aryl hydrocarbon receptor (AhR) agonist activity and inhibitory AhR-estrogen receptor crosstalk induced by the following methyl-substituted DIMs: 1,1'-dimethyl-, 2,2'-dimethyl-, 5,5'-dimethyl-, 6,6'-dimethyl-, and 7,7'-dimethylDIM and 1,1',2,2'-tetramethylDIM. The six compounds bound to the rat cytosolic AhR in a transformation assay but, at concentrations < or = 10 microM, exhibited minimal to non-detectable AhR agonist or antagonist activities associated with CYP1A1 induction. In contrast, the methyl-substituted DIMs inhibited estrogen-induced T47D human breast cancer cell growth and the four most active compounds (1,1'-, 2,2'-, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM) inhibited one or more estrogen-induced responses in the 21-day-old female B6C3F1 mice at a dose of 100 mg/kg/day (X3). Induction of hepatic CYP1A1-dependent activity was not observed at this high dose. The antitumorigenic activity of these compounds was examined in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor model in which the DIM analogs were orally administered (by gavage in corn oil) at a dose of 1 mg/kg/day (X10). 1,1'-DimethylDIM, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM significantly inhibited mammary tumor growth, and this was not accompanied by changes in organ/body weights or histopathology. These studies demonstrate that methyl-substituted DIMs are selective AhR modulators (SAhRMs) with potential for clinical treatment of breast cancer.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Indoles/farmacología , Neoplasias Mamarias Experimentales/patología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Animales , Anticarcinógenos/química , Femenino , Humanos , Indoles/química , Ratones , Ratones Endogámicos , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/metabolismo , Especificidad de la Especie , Células Tumorales Cultivadas/efectos de los fármacos , Útero/efectos de los fármacosRESUMEN
Cyclin D1 gene expression is induced by 17beta-estradiol (E2) in human breast cancer cells and is important for progression of cells through the G(1) phase of the cell cycle. The mechanism of activation of cyclin D1 is mitogen- and cell context-dependent, and this study describes the role of multiple promoter elements required for induction of cyclin D1 by E2 in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Transcriptional activation of cyclin D1 by E2 was dependent, in part, on a proximal cAMP-response element at -66, and this was linked to induction of protein kinase A-dependent pathways. These results contrasted to a recent report showing that induction of cyclin D1 by E2 in ER-positive MCF-7 and HeLa cells was due to up-regulation of c-jun and subsequent interaction of c-Jun-ATF-2 with the CRE. Moreover, further examination of the proximal region of the cyclin D1 promoter showed that three GC-rich Sp1-binding sites at -143 to -110 were also E2-responsive, and interaction of ERalpha and Sp1 proteins at these sites was confirmed by electromobility shift and chromatin immunoprecipitation assays. Thus, induction of cyclin D1 by E2 in ZR-75 cells is regulated through nuclear ERalpha/Sp1 and epigenetic protein kinase A activation pathways, and our results suggest that this mechanism may be cell context-dependent even among ER-positive breast cancer cell lines.
Asunto(s)
Neoplasias de la Mama/genética , Ciclina D1/genética , Elementos de Facilitación Genéticos/fisiología , Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Femenino , Humanos , Regiones Promotoras Genéticas , Elementos de Respuesta , Células Tumorales CultivadasRESUMEN
Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.
Asunto(s)
Adenosina Desaminasa/metabolismo , Neoplasias de la Mama/enzimología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas Serina-Treonina Quinasas , Receptor Cross-Talk , Receptores de Estrógenos/metabolismo , Adenosina Desaminasa/genética , Secuencia de Bases , Northern Blotting , Western Blotting , Neoplasias de la Mama/patología , Cartilla de ADN , Activación Enzimática , Receptor alfa de Estrógeno , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Células Tumorales CultivadasRESUMEN
Tamoxifen (TAM) is a highly effective selective estrogen receptor (ER) modulator used extensively for the treatment and prevention of breast cancer. However, prolonged treatment of women with TAM may be a risk factor for endometrial cancer, and research in our laboratory is focused on the development of selective aryl hydrocarbon receptor modulators that can be used in combination with TAM to improve its efficacy in the breast and inhibit TAM-induced endometrial effects. This study investigated the effects of the selective aryl hydrocarbon receptor modulators 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF) alone and in combination with TAM in the carcinogen-induced mammary tumor model and in the ovariectomized uterotropic assay using female Sprague Dawley rats. The lowest effective dose of 6-MCDF that inhibited tumor growth was 50 microg/kg/day, and TAM was antitumorigenic at a dose of 100 microg/kg/day. In animals cotreated with TAM + 6-MCDF at doses of 100, 50, or 25 microg/kg/day of each compound, complete inhibition of mammary tumor growth was observed at all doses, and the results are consistent with a more than additive antitumorigenic response for the low dose group (25 + 25 microg/kg) and additive interactions at the 50 and 100 microg/kg doses. In a separate experiment, 6-MCDF (800 microg/kg) inhibited TAM-induced peroxidase activity and progesterone receptor binding in the ovariectomized rat uterus but did not affect TAM-induced bone growth in ovariectomized rats. This study also investigated the effects of TAM and 6-MCDF alone and in combination on ERalpha protein levels in MCF-7 human breast cancer cells as a model for studying interactions between these compounds. The results show that 6-MCDF decreased TAM-induced ERalpha levels in the absence or presence of 17beta-estradiol through proteasome activation, and these interactions may contribute to the observed combined antitumorigenic effects of these compounds.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzofuranos/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Receptores de Hidrocarburo de Aril/análisis , Tamoxifeno/farmacología , Animales , Antineoplásicos Hormonales/administración & dosificación , Benzofuranos/administración & dosificación , Desarrollo Óseo/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Sinergismo Farmacológico , Moduladores de los Receptores de Estrógeno/administración & dosificación , Femenino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Complejos Multienzimáticos/metabolismo , Complejo de la Endopetidasa Proteasomal , Ratas , Ratas Sprague-Dawley , Tamoxifeno/administración & dosificación , Útero/efectos de los fármacosRESUMEN
Estrogen receptor-alpha (ER alpha) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. The classic mechanism of ER alpha action is associated with estrogen-induced formation of a nuclear ER alpha homodimer, binding to 5'-regulatory estrogen response elements (EREs) in target gene promoters, interaction with other nuclear proteins, and general transcription factors to activate gene expression. ER alpha also interacts with Sp1 protein to transactivate genes through binding Sp1(N)xERE or Sp1(N)xERE half-site (1/2) motifs where both ER alpha and Sp1 bind DNA elements. Activation through Sp1(N)xERE1/2 requires interactions of both proteins with their cognate DNA elements as well as additional nuclear factors to form a functional ER alpha/Sp1-DNA complex. Recent studies also show that ER alpha and Sp1 physically interact and ER alpha preferentially binds to the C-terminal DNA-binding domain of Sp1 protein. Moreover, ER alpha/Sp1 can activate transcription from a consensus GC-rich Sp1 binding site in transient transfection studies in MCF-7 human breast cancer cells, and this response is also observed with ER alpha variants that do not contain the DNA-binding domain. Several genes that are induced by estrogens in MCF-7 cells are activated through one or more GC-rich sites in their regulatory regions and these include the cathepsin D, E2F1, bcl-2, c-fos, adenosine deaminase, insulinlike growth factor binding protein 4, and retinoic acid receptor alpha 1 genes. ER alpha/Sp1 and ER beta/Sp1 action is dependent on ligand structure and cell context and ER beta/Sp1 is primarily associated with decreased ligand-dependent gene expression. ER alpha/Sp1, like ER alpha/AP1, represents a pathway for hormone activation of genes in which the receptor does not bind DNA, and results of ongoing studies suggest that ER alpha/Sp1 plays an important role in transcriptional activation of multiple growth regulatory genes in breast cancer cells.
Asunto(s)
Estradiol/fisiología , Receptores de Estrógenos/fisiología , Factor de Transcripción Sp1/fisiología , Activación Transcripcional/fisiología , Regulación de la Expresión Génica , HumanosRESUMEN
The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic helix-loop-helix transcription factor that forms heterodimers with the aryl hydrocarbon receptor (AhR) or hypoxia inducible factor-1alpha to activate transcription via xenobiotic response element or hypoxia response element, respectively. Thus, it plays a major role in two key biochemical pathways involved in tumor growth. We previously showed that estrogen receptor (ER)-negative breast cancer cell lines expressed a splice variant of ARNT that was associated with Ah nonresponsiveness. We have now used a sensitive PCR method to analyze the expression of the variant in a series of 92 breast cancers to assess interactions with the ER and prognosis. The splice variant could be detected in all of the cases examined, with high ratios of variant:full-length ARNT (> or =10) characterized in 10 cases. When the patient group was split into quartiles by increasing splice variant ratios, there was an inverse relationship of ER status to ARNT splice-variant ratios (P = 0.01, chi(2)). Univariate analysis showed that cases with high ARNT splice-variant ratios > or =10 had a worse relapse-free and overall survival (P > or = 0.03; hazard ratio, 2.7; and P = 0.006; hazard ratio, 3.9, respectively). In multivariate analysis for relapse-free and overall survival, ARNT splice-variant ratio was the strongest independent factor and, although inversely related to ER, remained a separate risk factor. At least two potential mechanisms could explain this phenomenon: the loss of aryl hydrocarbon receptor-mediated antiestrogenic activity or the blockade of a proapoptotic pathway induced by hypoxia. Because several enzymes involved in drug resistance are induced through a xenobiotic response element, the tumors presenting high ARNT splice-variant ratios may be specifically targeted by drugs normally degraded or inactivated. This study shows the biological importance of ARNT splice variants in the behavior of human breast cancer and suggests that the breast cell lines in which the splice variant was discovered may be useful models for further investigation.