RESUMEN
Metastasis is the primary cause of death in cancer patients. CXCR4/CXCL12 chemokine axis provides directional cues for breast cancer cells to metastasize to specific organs. Despite their potential clinical importance, how CXCR4 expression in breast cancer cells is regulated at the molecular level is not well understood. We identified an isoform of C/EBPß, liver-enriched inhibitory protein (LIP), as a previously unrecognized transcriptional regulator of CXCR4 in breast cancer cells. LIP up-regulated the transcription of CXCR4 through direct interaction with the CXCR4 promoter. The increase in CXCR4 mRNA was paralleled by an increased cell surface expression of the CXCR4, which in turn promoted CXCR4-mediated breast cancer cell migration. A significant positive correlation between LIP and CXCR4 expression was observed in stage III and IV human breast carcinoma specimens. Neuregulin 1 (or NRG1, hereafter referred to as heregulin) increased CXCR4 expression in breast cancer cells, and this coincided with increased LIP binding on the CXCR4 promoter. These findings may have important implications for understanding the molecular basis of CXCR4-mediated breast cancer cell metastasis and could potentially allow us to develop novel strategies to reduce morbidity and mortality in patients with metastatic breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Receptores CXCR4/genética , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Ratones , Estadificación de Neoplasias , Neurregulina-1/farmacología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Receptores CXCR4/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Factor de Transcripción YY1/metabolismoRESUMEN
Colorectal cancer is the third leading cause of cancer-related mortality in the world; death usually results from uncontrolled metastatic disease. Previously, we developed a novel strategy of TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) in combination with hyperthermia to treat hepatic colorectal metastases. However, previous studies suggest a potential hepatocyte cytotoxicity with TRAIL. Unlike TRAIL, anti-human TRAIL receptor antibody induces apoptosis without hepatocyte toxicity. In this study, we evaluated the anti-tumor efficacy of humanized anti-death receptor 4 (DR4) antibody mapatumumab (Mapa) by comparing it with TRAIL in combination with hyperthermia. TRAIL, which binds to both DR4 and death receptor 5 (DR5), was approximately tenfold more effective than Mapa in inducing apoptosis. However, hyperthermia enhances apoptosis induced by either agent. We observed that the synergistic effect was mediated through elevation of reactive oxygen species, c-Jun N-terminal kinase activation, Bax oligomerization, and translocalization to the mitochondria, loss of mitochondrial membrane potential, release of cytochrome c to cytosol, activation of caspases, and increase in poly(ADP-ribose) polymerase cleavage. We believe that the successful outcome of this study will support the application of Mapa in combination with hyperthermia to colorectal hepatic metastases.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Anticuerpos Monoclonales Humanizados , Apoptosis , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Terapia Combinada , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Hipertermia Inducida , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/terapia , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Here, we demonstrate that troglitazone (Rezulin), a peroxisome proliferator-activated receptor agonist, acted in synergy with heregulin to induce massive cell death in breast cancer cells. Although the combination of heregulin and troglitazone (HRG/TGZ) induced both apoptosis and necrosis, the main mode of cell death was caspase-independent and occurred via necrosis. This combination increased generation of superoxide in mitochondria, which in turn destabilized mitochondria potential. Pretreatment with N-acetyl-l-cysteine and catalase expression ameliorated cell death induced by the combination treatment, indicating a role of oxidative stress in mediating HRG/TGZ-induced cell death. Notably, pretreatment with pyruvate significantly prevented the cell death, suggesting a potential mechanistic link between metabolic stress and HRG/TGZ-induced cell death. The activation of the HRG signaling axis has been considered as a poor prognostic factor in breast cancer and confers resistance to gefitinib (Iressa) and tamoxifen. However, our data presented here paradoxically suggest that HRG expression can actually be beneficial when it comes to treating breast cancer with peroxisome proliferator-activated receptor-γ ligands. Taken together, the combination of HRG and TGZ may provide a basis for the development of a novel strategy in the treatment of apoptosis-resistant and/or hormone-refractory breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Cromanos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neurregulina-1/farmacología , PPAR gamma/agonistas , Tiazolidinedionas/farmacología , Antineoplásicos/agonistas , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromanos/agonistas , Sinergismo Farmacológico , Femenino , Humanos , Necrosis , Neurregulina-1/agonistas , Estrés Oxidativo/efectos de los fármacos , PPAR gamma/genética , PPAR gamma/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazolidinedionas/agonistas , TroglitazonaRESUMEN
The aim of this study was to investigate the effect of garlic constituent diallyl trisulfide (DATS) on the cell-death signaling pathway in a human breast cell line (MDA-MB-231). We observed that DATS (10-100 µM) treatment resulted in dose- and time-dependent cytotoxicity. Treatment of MDA-MB-231 cells with a cytotoxicity inducing concentration of DATS (50-80 µM) resulted in an increase in the intracellular level of reactive oxygen species (ROS). Data from assay with MitoSOX(TM) Red reagent suggest that mitochondria are the main source of ROS generation during DATS treatment. DATS-induced oxidative stress was detected through glutaredoxin (GRX), a redox-sensing molecule, and subsequently GRX was dissociated from apoptosis signal-regulating kinase 1 (ASK1). Dissociation of GRX from ASK1 resulted in the activation of ASK1. ASK1 activated a downstream signal transduction JNK (c-Jun N-terminal kinase)-Bim pathway. SP600125, a JNK inhibitor, inhibited DATS-induced Bim phosphorylation and protected cells from DATS-induced cytotoxicity. Our results indicate that the cytotoxicity caused by DATS is mediated by the generation of ROS and subsequent activation of the ASK1-JNK-Bim signal transduction pathway in human breast carcinoma MDA-MB-231 cells.
Asunto(s)
Compuestos Alílicos/toxicidad , Antineoplásicos/toxicidad , Antioxidantes/toxicidad , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Sulfuros/toxicidad , Compuestos Alílicos/farmacología , Antineoplásicos/farmacología , Antioxidantes/farmacología , Apoptosis , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ajo/química , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Sulfuros/farmacologíaRESUMEN
Approximately 25% of patients with colorectal cancer develop metastases to the liver, and surgery is currently the best treatment available. But there are several patients who are unresectable, and isolated hepatic perfusion (IHP) offers a different approach in helping to treat these patients. IHP is a method used for isolating the liver and delivering high doses of chemotherapeutic agents. The efficacy of IHP has been improved by combining hyperthermia not only with chemotherapeutics but with other deliverable agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we used human colorectal cancer CX-1 cells and treated them with hyperthermia and TRAIL, causing cytotoxicity. We were able to demonstrate that the numbers of live cells were significantly reduced with hyperthermia and 10 ng/ml of TRAIL combined. We also showed that the effect of hyperthermia on TRAIL in our studies was enhancement of the apoptotic pathway by the promotion of JNK and Bim(EL) activity as well as PARP cleavage. We have also used our CX-1 cells to generate tumors in Balb/c nude mice. With intratumoral injections of TRAIL combined with hyperthermia at 42 degrees C, we were able to show a delayed onset of tumor growth in our xenograft model.
Asunto(s)
Neoplasias Colorrectales/prevención & control , Hipertermia Inducida , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antracenos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Terapia Combinada , Calor , Humanos , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Carga Tumoral/efectos de los fármacosRESUMEN
Stimulation of adipogenesis in mouse preadipocytes requires C/EBPbeta as well as activation of the MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/glycogen synthase kinase 3 (GSK3) site regulates adiponectin gene expression during the C/EBPbeta-facilitated differentiation of mouse fibroblasts into adipocytes. First, we show that exposure of 3T3-L1 preadipocytes to insulin, dexamethasone (DEX), and isobutylmethylxanthine (MIX) leads to the phosphorylation of C/EBPbeta at threonine 188. Pretreating the cells with a MEK1-specific inhibitor (U0126) significantly attenuates this activity. Similarly, these effectors activate the phosphorylation of T188 within an ectopic C/EBPbeta overexpressed in Swiss mouse fibroblasts, and this event involves both MEK1 and GSK3 activity. We further show that expression of C/EBPbeta (p34kD LAP isoform) in Swiss mouse fibroblasts exposed to DEX, MIX, and insulin induces expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and some adiponectin but that it does not activate expression of FABP4/aP2. In fact, complete conversion of these fibroblasts into lipid-laden adipocytes, which includes activation of FABP4 and adiponectin expression, requires their exposure to a potent PPARgamma ligand such as troglitazone. Expression of a mutant C/EBPbeta in which threonine 188 has been modified to alanine (C/EBPbeta T188A) can induce PPARgamma production in the mouse fibroblasts, but it is incapable of stimulating adiponectin expression in the absence or presence of troglitazone. Interestingly, replacement of T188 with aspartic acid creates a C/EBPbeta molecule (C/EBPbeta T188D) that possesses adipogenic activity similar to that of the wild-type molecule. The absence of adiponectin expression correlates with a reduced amount of C/EBPalpha in the adipocytes expressing the T188A mutant suggesting that C/EBPalpha is required for expression of adiponectin. In fact, ectopic expression of PPARgamma in C/EBPalpha-deficient fibroblasts (NIH 3T3 cells) produces a modest amount of adiponectin, whereas expression of both PPARgamma and C/EBPalpha in NIH 3T3 cells facilitates production of abundant quantities of adiponectin. These data demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/GSK3 site is required for both C/EBPalpha and adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes.
Asunto(s)
Adipocitos/citología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/fisiología , Péptidos y Proteínas de Señalización Intercelular , Proteínas/metabolismo , Adiponectina , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Fibroblastos/citología , Regulación de la Expresión Génica/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Metabolismo de los Lípidos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Treonina/metabolismoRESUMEN
The Wnt/beta-catenin signalling pathway appears to operate to maintain the undifferentiated state of preadipocytes by inhibiting adipogenic gene expression. To define the mechanisms regulating suppression of Wnt/beta-catenin signalling, we analysed the beta-catenin expression in response to activation of transcription factors that regulate adipogenesis. The results show an extensive down-regulation of nuclear beta-catenin that occurs during the first few days of differentiation of 3T3-L1 preadipocytes and coincides with the induction of the adipogenic transcription factors, C/EBPbeta (CCAAT-enhancer-binding protein) and PPARgamma (peroxisome-proliferator-activated receptor). To assess the role of each of these factors in this process, we conditionally overexpressed C/EBPbeta in Swiss mouse fibroblasts using the TET-off system. Abundant expression of C/EBPbeta alone had minimal effect on beta-catenin expression, whereas expression of C/EBPbeta, in the presence of dexamethasone, induced PPARgamma expression and caused a measurable decrease in beta-catenin. In addition, exposure of cells expressing both C/EBPbeta and PPARgamma to a potent PPARgamma ligand resulted in an even greater decrease in beta-catenin by mechanisms that involve the proteasome. Our studies also suggest a reciprocal relationship between PPARgamma activity and beta-catenin expression, since ectopic production of Wnt-1 in preadipocytes blocked the induction of PPARgamma gene expression. Moreover, by suppressing beta-catenin expression, ectopic expression of PPARgamma in Wnt-1-expressing preadipocytes rescued the block in adipogenesis after their exposure to the PPARgamma ligand, troglitazone.
Asunto(s)
Adipocitos/metabolismo , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/fisiología , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/fisiología , Proteínas de Pez Cebra , Adipocitos/citología , Animales , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Diferenciación Celular , Línea Celular , Cisteína Endopeptidasas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Ratones , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Complejo de la Endopetidasa Proteasomal , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Células Madre/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Wnt , Proteína Wnt1 , beta CateninaRESUMEN
We demonstrate that exposure of post-confluent 3T3-L1 preadipocytes to insulin, isobutylmethylxanthine (MIX), dexamethasone (DEX), and fetal bovine serum induces a rapid but transient activation of MEK1 as indicated by extensive phosphorylation of ERK1 and ERK2 during the initial 2 h of adipogenesis. Inhibition of this activity by treating the cells with a MEK1-specific inhibitor (U0126 or PD98059) prior to the induction of differentiation significantly attenuated the expression of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, perilipin, and adipocyte-specific fatty acid-binding protein (aP2). Treating the preadipocytes with troglitazone, a potent PPARgamma ligand, could circumvent the inhibition of adipogenic gene expression by U0126. Fibroblast growth factor-2 (FGF-2), in the presence of dexamethasone, isobutylmethylxanthine, and insulin, induces a prolonged activation of the MEK/ERK signaling pathway, which lasts for at least 12 h post-induction, and this activity is less sensitive to the MEK inhibitors. Consequently, preadipocytes treated with U0126 in the presence of fibroblast growth factor-2 (FGF-2) express normal post-induction levels of MEK activity, and, in so doing, are capable of undergoing adipogenesis. We further show that activation of MEK1 significantly enhances the transactivation of the C/EBPalpha minimal promoter during the early phase of the differentiation process. Our results suggest that activation of the MEK/ERK signaling pathway during the initial 12 h of adipogenesis enhances the activity of factors that regulate both C/EBPalpha and PPARgamma expression.