RESUMEN
SerpinB3 is a hypoxia- and hypoxia-inducible factor-2α-dependent cystein protease inhibitor that is up-regulated in hepatocellular carcinoma and in parenchymal cells during chronic liver diseases (CLD). SerpinB3 up-regulation in CLD patients has been reported to correlate with the extent of liver fibrosis and the production of transforming growth factor-ß1, but the actual role of SerpinB3 in hepatic fibrogenesis is still poorly characterized. In the present study we analyzed the pro-fibrogenic action of SerpinB3 in cell cultures and in two different murine models of liver fibrosis. "In vitro" experiments revealed that SerpinB3 addition to either primary cultures of human activated myofibroblast-like hepatic stellate cells (HSC/MFs) or human stellate cell line (LX2 cells) strongly up-regulated the expression of genes involved in fibrogenesis and promoted oriented migration, but not cell proliferation. Chronic liver injury by CCl4 administration or by feeding a methionine/choline deficient diet to transgenic mice over-expressing human SerpinB3 in hepatocytes confirmed that SerpinB3 over-expression significantly increased the mRNA levels of pro-fibrogenic genes, collagen deposition and αSMA-positive HSC/MFs as compared to wild-type mice, without affecting parenchymal damage. The present study provides for the first time evidence that hepatocyte release of SerpinB3 during CLD can contribute to liver fibrogenesis by acting on HSC/MFs.
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Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Serpinas/metabolismo , Animales , Línea Celular , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Células Hep G2 , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Serpinas/genética , Serpinas/farmacologíaRESUMEN
Liver fibrogenesis is a dynamic and highly integrated molecular, tissue and cellular process that during the course of a chronic liver disease (CLD) leads progressively to an excess deposition of extracellular matrix (ECM) components in an attempt to limit the consequences of chronic parenchymal injury. Irrespective of etiology, liver fibrogenesis is sustained and modulated by an intense cross talk occurring between different hepatic cell populations that involves the synthesis and release of several mediators, including growth factors, cytokines, chemokines, reactive oxygen species, adipokines, vasoactive agents and plasma proteins. In this scenario a major pro-fibrogenic role is played by a heterogeneous population of α-smooth muscle actin (α-SMA) positive cells defined as hepatic myofibroblasts (MFs). Hepatic MFs are highly proliferative and contractile cells, primarily responsible for excess deposition of ECM components and involved in ECM altered remodeling observed in CLDs. MFs also represent a unique and critical cellular crossroad able to integrate incoming paracrine or autocrine signals, released from all hepatic cell populations involved or available in the microenvironment, as well as to synthetize and release mediators which sustain and perpetuate fibrogenesis, chronic inflammatory response and neo-angiogenesis. This review has been designed to offer critical knowledge on hepatic MFs, including terminology, essential definitions and characterization of MFs, with a focus on the origin of these cells (mainly from hepatic stellate cells and portal fibroblasts or, to a lesser extent, bone marrow-derived cells), the process of activation and the functional responses that these cells can operate in the fibrogenic progression of CLDs.
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Cirrosis Hepática/patología , Miofibroblastos/patología , Progresión de la Enfermedad , Humanos , Cirrosis Hepática/metabolismo , Miofibroblastos/metabolismoRESUMEN
Pathologic angiogenesis appears to be intrinsically associated with the fibrogenic progression of chronic liver diseases, which eventually leads to the development of cirrhosis and related complications, including hepatocellular carcinoma. Several laboratories have suggested that this association is relevant for chronic liver disease progression, with angiogenesis proposed to sustain fibrogenesis. This minireview offers a synthesis of relevant findings and opinions that have emerged in the last few years relating liver angiogenesis to fibrogenesis. We discuss liver angiogenesis in normal and pathophysiologic conditions with a focus on the role of hypoxia and hypoxia-inducible factors and assess the evidence supporting a clear relationship between angiogenesis and fibrogenesis. A section is dedicated to the critical interactions between liver sinusoidal endothelial cells and either quiescent hepatic stellate cells or myofibroblast-like stellate cells. Finally, we introduce the unusual, dual (profibrogenic and proangiogenic) role of hepatic myofibroblasts and emerging evidence supporting a role for specific mediators like vasohibin and microparticles and microvesicles.
RESUMEN
BACKGROUND: Signals from the tumor microenvironment (hypoxia, growth factors) are known to induce an invasive phenotype. Cyclooxygenase-2 (COX2) overexpression, involved in colorectal carcinoma (CRC) progression, is also associated with epidermal growth factor receptor (EGFR) up-regulation. The present study investigated whether inhibition of COX2 may affect, under normoxia and hypoxia, EGF-induced cell proliferation and invasiveness by using immunoblotting, trypan blue assay, Boyden chamber assay and zymography. RESULTS: The proliferative and invasive activity of HT-29 cells was enhanced under hypoxia. COX2 expression was increased after epidermal growth factor (EGF) stimulation under both hypoxia and normoxia, expression that was efficiently reduced by the COX2 inhibitor NS398. Under normoxia, NS398 reduced signalling pathways induced by EGF [phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT), extracellular-signal-regulated kinases (ERKs)], while under hypoxia, EGF stimulation and NS398 treatment was associated with HIF-1α expression. Under both conditions, NS398 was able to inhibit cell invasiveness and matrix-metalloproteinase-2 release. CONCLUSION: COX2 inhibition can contribute to reducing cell aggressiveness through interfering with EGF- and hypoxia-mediated signaling.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/farmacología , Receptores ErbB/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Nitrobencenos/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Hipoxia de la Célula , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Transición Epitelial-Mesenquimal , Células HT29 , HumanosRESUMEN
Liver fibrogenesis is a dynamic and highly integrated molecular, tissue and cellular process, potentially reversible, that drives the progression of chronic liver diseases (CLD) towards liver cirrhosis and hepatic failure. Hepatic myofibroblasts (MFs), the pro-fibrogenic effector cells, originate mainly from activation of hepatic stellate cells and portal fibroblasts being characterized by a proliferative and survival attitude. MFs also contract in response to vasoactive agents, sustain angiogenesis and recruit and modulate activity of cells of innate or adaptive immunity. Chronic activation of wound healing and oxidative stress as well as derangement of epithelial-mesenchymal interactions are "major" pro-fibrogenic mechanisms, whatever the etiology. However, literature has outlined a complex network of pro-fibrogenic factors and mediators proposed to modulate CLD progression, with some of them being at present highly debated in the field, including the role of epithelial to mesenchymal transition and Hedgehog signaling pathways. Hypoxia and angiogenesis as well as inflammasomes are recently emerged as ubiquitous pro-inflammatory and pro-fibrogenic determinants whereas adipokines are mostly involved in CLD related to metabolic disturbances (metabolic syndrome and/or obesity and type 2 diabetes). Finally, autophagy as well as natural killer and natural killer-T cells have been recently proposed to significantly affect fibrogenic CLD progression.
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Cirrosis Hepática/inmunología , Cirrosis Hepática/patología , Hígado/citología , Hígado/patología , Animales , Enfermedad Crónica , Progresión de la Enfermedad , Matriz Extracelular/inmunología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Hígado/inmunología , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Miofibroblastos/inmunología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Estrés OxidativoRESUMEN
INTRODUCTION: Cyclooxygenase-2 (COX-2) is overexpressed in several malignancies and is implicated in breast cancer progression. OBJECTIVES: We investigated whether changes in COX-2 expression may affect epithelial-to-mesenchymal transition (EMT) and then invasive potential of human breast cancer cells, in relationship with hypoxia. COX-2-null MCF-7 human breast cancer cells, MCF-7 cells transiently expressing COX-2 and COX-2-expressing MDA-MB-231 cells were employed. RESULTS: COX-2 overexpression resulted in downregulation of E-cadherin and ß-catenin, upregulation of vimentin, N-cadherin and SNAI1, suggesting EMT occurrence. COX-2-overexpressing MCF-7 cells were also characterized by increased invasiveness and release of matrix-metalloproteinase-9. The above-mentioned characteristics, homologous to those detected in highly invasive MDA-MB-231 cells, were reverted by treatment of COX-2-overexpressing MCF-7 cells with celecoxib, a COX-2-specific inhibitor, partly through the inhibition of COX-2-related intracellular generation of reactive oxygen species. Hypoxia further exacerbated COX-2 expression, EMT changes and invasive ability in both COX-2-overexpressing MCF-7 cells and MDA-MB-231 cells. Finally, immunohistochemistry performed on samples from normal and neoplastic human breast tissues revealed that COX-2-positive malignant cells were also positive for EMT-related antigens, hypoxia-inducible factor (HIF)-2α and the oxidative stress marker heme oxygenase. CONCLUSIONS: These findings support the existence of a direct link between COX-2 overexpression, EMT and invasiveness in human breast cancer cells, emphasizing the role of hypoxic microenvironment.
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Neoplasias de la Mama/metabolismo , Ciclooxigenasa 2/metabolismo , Transición Epitelial-Mesenquimal , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cadherinas/metabolismo , Línea Celular Tumoral , Ciclooxigenasa 2/genética , ADN Complementario/genética , Dinoprostona/metabolismo , Femenino , Hemo-Oxigenasa 1/metabolismo , Humanos , Hipoxia/metabolismo , Células MCF-7 , Invasividad Neoplásica , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismoRESUMEN
Fibrogenic progression of chronic liver diseases (CLDs) towards the end-point of cirrhosis is currently regarded, whatever the aetiology, as a dynamic and highly integrated cellular response to chronic liver injury. Liver fibrogenesis (i.e., the process) is sustained by hepatic populations of highly proliferative, pro-fibrogenic and contractile myofibroblast-like cells (MFs) that mainly originate from hepatic stellate cells (HSC) or, to a less extent, from portal fibroblasts or bone marrow-derived cells. As is well known, liver fibrosis (i.e., the result) is accompanied by perpetuation of liver injury, chronic hepatitis and persisting activation of tissue repair mechanisms, leading eventually to excess deposition of extracellular matrix (ECM) components. In this scenario, hypoxic areas represent a very common and major feature of fibrotic and cirrhotic liver during the progression of CLDs. Cells exposed to hypoxia respond by means of heterodimeric hypoxia-inducible factors (HIFs) that translocate into the nucleus and binds to a specific core sequence defined hypoxia-responsive element (HRE), present in the promoter on several genes which are considered as hypoxia-regulated target genes. HIFs transcription factors can activate a complex genetic program designed to sustain several changes necessary to efficiently counteract the decrease in oxygen tension. Accordingly, hypoxia, through up-regulation of angiogenesis, is currently believed to significantly contribute to fibrogenic progression of CLDs, mostly by affecting the pro-fibrogenic and pro-angiogenic behaviour of hepatic MFs. In addition, experimental and clinical evidence generated in the last decade also indicates that angiogenesis and fibrogenesis in CLDs may also be sustained by HIF-dependent but hypoxia-independent mediators.
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Hipoxia de la Célula/fisiología , Hepatopatías/metabolismo , Hepatopatías/patología , Neovascularización Patológica/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Enfermedad Crónica , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Miofibroblastos/metabolismo , Neovascularización Patológica/patología , Proteínas RepresorasRESUMEN
Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been reported to exert chemopreventive and antitumor effects on colon cancer, one of the most common solid epithelial malignancy worldwide. The aim of this study was to elucidate whether celecoxib may be able to affect epithelial-mesenchymal transition (EMT), a critical process involved in cancer cell invasiveness and metastasis and then proposed to be relevant for cancer progression. Human HT-29 colon cancer cells were exposed to carefully controlled hypoxic conditions and/or epidermal growth factor (EGF) and then investigated for EMT changes and signal transduction pathways involved by using morphological, molecular, and cell biology techniques. Celecoxib inhibited basal and EGF-stimulated proliferation, hypoxia-related HIF-1α recruitment/stabilization as well as hypoxia- and EGF-dependent activation of ERK and PI3K. Interestingly, celecoxib prevented EMT-related changes, as shown by modifications of ß-catenin intracellular localization or vimentin and E-cadherin levels, as well as HT-29 invasiveness induced by hypoxia, EGF, or hypoxia plus EGF. Finally, experiments performed on SW-480 colon cancer cells (i.e., cells lacking COX-2) exposed to hypoxia, used here as a stimulus able to induce EMT and invasiveness, revealed that in these cells celecoxib was ineffective. Results of the present study indicate that celecoxib has the potential to negatively affect induction of EMT and increased invasiveness of colon cancer cells as elicited by different signals originating from tumor microenvironment (i.e., hypoxia and EGF). Moreover, these effects are likely be related to the pharmacological inhibitory effect exerted on COX-2 activity.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Factor de Crecimiento Epidérmico/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Pirazoles/farmacología , Sulfonamidas/farmacología , Cadherinas/metabolismo , Celecoxib , Hipoxia de la Célula , Proliferación Celular , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HT29 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Vimentina/metabolismo , beta Catenina/metabolismoRESUMEN
Rofecoxib is a specific COX-2 inhibitor able to exert antiproliferative activity against colorectal cancer cells. It was withdrawn from the market after the demonstration of an increased risk of cardiovascular complications after prolonged use. Nevertheless, it remains an interesting compound for laboratory research as an experimental COX-2 inhibitor. In this study, the antiproliferative activity of a novel dinitro-oxy-substituted analogue of rofecoxib (NO-rofe), potentially less cardiotoxic, has been investigated in vitro on human colon cancer cells and compared with the action of the parent drug. Due to the fact that COX-2 inhibition is the main characteristic of coxibs, we performed all experiments in COX-2-overexpressing (HT-29) and COX-2-negative (SW-480) human colon cancer cells, to elucidate whether the observed effects were dependent on COX-2 inhibition. Moreover, experiments were performed in order to evaluate whether COX-2 pharmacological inhibition may affect beta-catenin/E-cadherin signaling pathway. NO-rofe exerted a significant antiproliferative activity on COX-2 positive HT-29 human colon cancer cells, being less effective on the COX-2 negative SW-480 human colon cancer cell line. In particular, the rofecoxib analogue retained similar potencies with respect to COX-2 inhibition but was much more active than rofecoxib in inhibiting the growth of human colon cancer cells in vitro. In addition, this novel compound resulted in the induction of membrane ß-catenin/E-cadherin expression, a feature that may significantly contribute to its antiproliferative activity.
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4-Butirolactona/análogos & derivados , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa 2/farmacología , Nitratos/farmacología , 4-Butirolactona/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The inducible COX-2 enzyme is over-expressed in human breast cancer and its over-expression generally correlates with angiogenesis, deregulation of apoptosis and worse prognosis. This observation may explain the beneficial effect of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors on breast cancer treatment. Here, we evaluated the antiproliferative activity of celecoxib, a selective COX-2 inhibitor, and its nitro-oxy derivative on human breast cancer cells characterized by low and high COX-2 expression, respectively. In ERα(+) MCF-7 cells celecoxib and its derivative induce a strong inhibition of cell growth, inhibition that is associated with the reduction of ERα expression and activation. These effects may be directly associated with ERK and Akt suppression and with PP2A and PTEN induction. In this cell line the drugs exert only weak effect on COX-2 level while they are able to reduce aromatase expression. On the contrary, in ERα(-) MDA-MB-231 cells, both drugs induce a marked inhibition of COX-2, inhibition that is associated with the reduction of aromatase expression and of cell proliferation. In both cell lines the effects of the drugs are associated with the suppression of cell invasion.
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Neoplasias de la Mama/patología , Carcinoma/patología , Proliferación Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa 2/farmacología , Pirazoles/farmacología , Sulfonamidas/farmacología , Antineoplásicos/farmacología , Celecoxib , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Biológicos , Invasividad NeoplásicaRESUMEN
It has been shown previously that a novel nitrooxy derivative of celecoxib exerts antiproliferative and pro-apoptotic effects in human colon cancer cells. The aim of this study was to elucidate whether these biological properties depend on COX-2 inhibition and/or NO release. Therefore, the derivative was decomposed into the parent compound celecoxib and the NO donor benzyl nitrate and the biological role of each was tested in COX-2-positive (HT-29) and -negative (SW-480) colon cancer cells. The main findings were that the nitro-oxy derivative behaved like celecoxib in HT-29 cells in terms of COX-2 and ERK/MAPK inhibition, as well as induction of apoptosis, while the benzyl nitrate had no such effects. Interestingly, the beta-catenin system was activated by the nitro-oxy derivative as well as by benzyl nitrate alone more potently than by the parent compound celecoxib, suggesting a possible regulatory role for NO. In SW480 cells, these activities were substantially less pronounced, suggesting the presence of COX-2-dependent mechanisms in the modulation of these parameters.
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Compuestos de Bencilo/farmacología , Neoplasias del Colon/tratamiento farmacológico , Ciclooxigenasa 2/metabolismo , Nitratos/farmacología , Óxido Nítrico/metabolismo , Pirazoles/farmacología , Sulfonamidas/farmacología , Apoptosis/efectos de los fármacos , Celecoxib , Procesos de Crecimiento Celular/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Inhibidores de la Ciclooxigenasa 2/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HT29 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , beta Catenina/metabolismoRESUMEN
OBJECTIVE: COX-2 is implicated in carcinogenesis and tumour progression in many cancers, including breast cancer. Recently, it has been reported that human breast carcinomas aberrantly express COX-2, and that raised tissue levels of COX-2 may have prognostic value. Patients expressing high levels of COX-2 can develop local recurrence, and have reduced disease-free and disease-related overall survival. The aim of this study was to investigate COX-2 expression in human ductal and lobular breast cancers and its possible association with clinicopathological features and prognostic molecular markers. RESEARCH DESIGN AND METHODS: Cytoplasmic COX-2 expression was detected by means of immunohistochemistry in a series of 91 breast carcinomas with ductal (n = 60) and lobular (n = 31) patterns. COX-2 expression was investigated by multivariate analyses and compared with clinicopathological features. RESULTS AND CONCLUSIONS: COX-2 immune positivity and percentage of positive cells correlated significantly with the size, grading, extent of primary tumour and vascular invasion of carcinoma but not with biological parameters (estrogen receptor, progesterone receptor and human EGF receptor 2). The findings of the present study suggest that COX-2 overexpression in lobular and ductal breast cancers, which correlates with traditional clinico-pathological parameters, may be considered as a negative prognostic marker.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Ciclooxigenasa 2/metabolismo , Regulación Neoplásica de la Expresión Génica , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/patología , Carcinoma Ductal de Mama/secundario , Carcinoma Lobular/diagnóstico , Carcinoma Lobular/patología , Carcinoma Lobular/secundario , Femenino , Humanos , Inmunohistoquímica , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Metástasis Linfática , Persona de Mediana Edad , Estadificación de Neoplasias , Neovascularización Patológica , Pronóstico , Carga Tumoral , Regulación hacia ArribaRESUMEN
Celecoxib is a non-steroidal anti-inflammatory drug (NSAID) developed as a selective inhibitor of cyclooxygenase-2 (COX-2). Despite the associated cardiovascular toxicity risk, celecoxib has been found to be effective in reducing cancer risk in animal and human studies. In the present study the antiproliferative activity of novel nitro-oxy-methyl substituted analogues of celecoxib (NO-cel), potentially less cardiotoxic, has been investigated in vitro on human colon cancer cells and compared with action of the parent drug. Moreover, experiments were performed in order to evaluate whether COX-2 pharmacological inhibition may affect beta-catenin/E-cadherin signalling pathway. All the tested analogues of celecoxib exerted a significant antiproliferative activity on COX-2 positive HT-29 human colon cancer cells, being less effective on the COX-2 negative SW-480 human colon cancer cell line. In particular, the analogue displaying two nitro-oxy functions fully mimicked the known inhibitory properties of celecoxib, including inhibition of COX-2, as well as of ERK/MAPK and beta-catenin signalling pathways. Interestingly, the latter compound also elicited a strong reorganization of the beta-catenin/E-cadherin complex, which has been suggested to be relevant for colon carcinogenesis. On these premises, NO-cel analogues of celecoxib can represent promising colon cancer chemopreventive agents potentially able to affect colon cancer development.
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Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Pirazoles/química , Pirazoles/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Cadherinas/metabolismo , Celecoxib , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/uso terapéutico , Humanos , Pirazoles/uso terapéutico , Sulfonamidas/uso terapéutico , beta Catenina/metabolismoRESUMEN
Epidemiological studies suggest that dietary PUFA may influence breast cancer progression. n-3 PUFA are generally known to exert antitumour effects, whereas reports relative to n-6 PUFA anti-carcinogen effects are controversial. Arachidonic acid (AA; 20:4n-6) and its metabolites have been shown to inhibit the growth of human breast cancer cell lines, even if the downstream mechanisms by which AA may influence carcinogenesis remain unresolved. We explored the molecular basis for AA influence on proliferation, signal transduction and apoptosis in two human breast cancer cell lines, MCF-7 and MDA-MB-231. In both cell lines AA inhibited cell growth in a dose-dependent manner, even if MDA-MB-231 was somewhat more growth-inhibited than MCF-7. AA decreased extracellular signal-regulated protein kinase 1/2 phosphorylation level, and positively modulated PPARgamma and PPARalpha expression, with only a slight effect against PPARbeta/delta. In addition, AA increased Bak (an apoptosis-regulating protein) expression and reduced procaspase-3 and -9 levels only in MDA-MB-231 cells, thus indicating that the growth inhibitory effect can be correlated with apoptosis induction. In both cell lines the use of a specific antagonist made it possible to establish a relationship between AA growth inhibitory effect and PPARalpha involvement. AA decreases cell proliferation most likely by inducing apoptosis in MDA-MB-231 cells, while in the MCF-7 cell line the growth inhibitory activity can be attributed to the inhibition of the signal transduction pathway involved in cell proliferation. In both cases, the results here presented suggest PPARalpha as a possible contributor to the growth inhibitory effect of AA.
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Ácido Araquidónico/farmacología , Neoplasias de la Mama/patología , PPAR alfa/farmacología , Análisis de Varianza , Apoptosis/efectos de los fármacos , Western Blotting/métodos , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , PPAR alfa/genética , PPAR alfa/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Conjugated linoleic acid (CLA), a naturally occurring substance in food sources, occurs as mixtures of positional and geometrical isomers of octadecadienoate (18:2), and may inhibit colon tumorigenesis. It has been hypothesized that CLA can modulate cell proliferation and differentiation through the activation of peroxisome proliferator-activated receptors (PPARs), among which PPARgamma is involved in growth inhibition of transformed cells. The aim of the present study was to investigate whether the antiproliferative effects of CLA are mediated by its interaction with PPARgamma and APC/beta-catenin signalling pathway in human colon cancer cells. In CLA-treated caco-2 cells we found a remarkable increase in the expression of PPARgamma, which translocated into the nucleus, while PPARalpha and beta/delta protein levels were not affected. GW259662, a well known PPARgamma antagonist, blocked the increase in PPARgamma protein rate and abrogated some biological effects of CLA, as it restored the proliferative capability of the cells and ERK1/2 phosphorylation level. We demonstrated that CLA treatment determined the down-regulation of APC and c-myc proteins, but in this case the administration of the antagonist was not able to revert CLA effects. Furthermore, CLA induced a reorganization of E-cadherin and beta-catenin, as well as a redistribution of actin and tubulin filaments. Our data suggest that CLA may regulate PPARgamma expression by selectively acting as an agonist; however, the discrepancies in PPARgamma antagonist efficacy suggest the involvement of other pathways, independent of PPARgamma, in CLA antiproliferative activity.
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Proliferación Celular/efectos de los fármacos , Genes APC , Ácido Linoleico/farmacología , PPAR gamma/fisiología , beta Catenina/fisiología , Western Blotting , Células CACO-2 , Cadherinas/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , beta Catenina/metabolismoRESUMEN
Conjugated linoleic acid (CLA) is a naturally occurring fatty acid, which has been shown to exert beneficial effects against breast carcinogenesis. It has been reported that CLA could modulate cellular proliferation and differentiation through the activation of peroxisome proliferator-activated receptors (PPARs). Among different PPAR isotypes, PPAR gamma is involved in growth inhibition of transformed cells. Ligands of PPAR gamma are considered as potential anticancer drugs, so CLA was tested for its ability to induce PPAR gamma expression in MCF-7 breast cancer cells. The effects of CLA and of a specific synthetic PPAR gamma antagonist were evaluated on cell growth as well as on parameters responsible for cell growth regulation. We demonstrated here that CLA stimulated the expression of PPAR gamma to levels up to control and caused PPAR gamma translocation into the nucleus. Furthermore, the overexpression of PPAR gamma positively correlates with the inhibition of cell proliferation and with the modulation of ERK signaling induced by CLA; in all cases the administration of the antagonist reverted CLA effects. The PPAR-signaling pathway is connected with the beta-catenin/E-cadherin pathway, thus we evaluated CLA effects on the expression and cellular distribution of these proteins, which are involved in cell adhesion and responsible for invasive behavior. The treatment with CLA determined the up-regulation and the redistribution of beta-catenin and E-cadherin and the antagonist reverted only the effect on beta-catenin. These studies indicate that CLA regulates PPAR gamma expression by selectively acting as an agonist and may influence cell-cell adhesion and invasiveness of MCF-7 cells.
Asunto(s)
Cadherinas/metabolismo , Proliferación Celular/efectos de los fármacos , Ácidos Linoleicos Conjugados/farmacología , PPAR gamma/metabolismo , beta Catenina/metabolismo , Actinas/metabolismo , Western Blotting , Línea Celular Tumoral , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inmunoprecipitación , Líquido Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microscopía Fluorescente , PPAR gamma/antagonistas & inhibidores , Transporte de Proteínas/efectos de los fármacos , Tubulina (Proteína)/metabolismoRESUMEN
Conjugated linoleic acid (CLA) is a naturally occurring compound found in dairy and beef products. In recent years, it has received considerable attention because several studies showed a lower incidence of certain cancers in animals fed CLA-supplemented diets. In vitro studies further showed growth inhibitory activity on tumor cell proliferation, the CLA being effective above all against colon cancer cells. The aim of the present work was to investigate the growth inhibitory effect of CLA on Caco-2 cell line. Under our experimental conditions, CLA repressed Caco-2 cell proliferation, and the growth-inhibitory action increased by repeating treatments. However, in Caco-2 cells, CLA was unable to induce apoptosis, as revealed by cell-cycle analysis and Western blot studies. To determine the mechanism by which CLA inhibits cell growth, we studied its effect on extracellular-regulated kinase signaling. Conjugated linoleic acid reduced expression levels of Raf-1 and phosphorylation of ERK1/2, which was accompanied by a decrease in the expression of the downstream transcription factor c-myc. Our data suggest that CLA is dependent, at least in part, on the ERK kinase pathway for its ability to inhibit the growth of Caco-2 cancer cells.
Asunto(s)
Células CACO-2/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Ácidos Linoleicos Conjugados/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , L-Lactato Deshidrogenasa/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Proteínas Proto-Oncogénicas c-raf/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Proteína Destructora del Antagonista Homólogo bcl-2/efectos de los fármacos , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismoRESUMEN
Conjugated linoleic acid (CLA) has protective properties in breast cancer. Here, we studied the mechanisms underlying the effects of CLA on MCF-7 breast cancer cell proliferation, especially in correlation with the involvement of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and protein phosphatase 2A (PP2A). CLA inhibits MCF-7 cell growth in a concentration- and time-dependent manner, without triggering apoptosis. In assessing expression levels of proteins that play obligatory roles in the ERK cascade, we evidenced that CLA down-regulated Raf-1 and decreased levels of phospho-ERK1/2, as well as c-myc expression. Increase in PP2A expression rates were additionally observed after CLA treatment of MCF-7 cells. The above effects, as well as CLA-induced inhibition of cell growth, were reversed by okadaic acid, a specific inhibitor of PP2A. Thus, PP2A likely participates in deactivation of ERK1/2, and its up-regulation may represent a novel mechanism for CLA-induced inhibition of cell proliferation.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ácidos Linoleicos Conjugados/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Neoplasias de la Mama/prevención & control , Carcinógenos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Ácidos Linoleicos Conjugados/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ácido Ocadaico/farmacología , Proteína Fosfatasa 2 , Regulación hacia Arriba/fisiologíaRESUMEN
In the present study, we evidence how in breast cancer cells low doses of Taxol for 18 h determined the upregulation of p53 and p21 waf expression concomitantly with a decrease of the anti-apoptotic Bcl-2. P53 and its gene product, the mdm2 protein, in treated cells exhibits a prevalent nuclear compartmentalization, thus potentiating p53 transactivatory properties. Indeed, the most important finding of this study consists with the evidence that Taxol at lower concentrations is able to produce the activation of p21 promoter via p53. Prolonged exposure of MCF-7 cells to Taxol (48 h) resulted in an increased co-association between p21 and PCNA compared to control and this well fits with the simultaneous block of cell cycle into the G2/M phase.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Paclitaxel/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Fase G2/efectos de los fármacos , Humanos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factores de TiempoRESUMEN
We investigated the molecular mechanisms involved in the anti-proliferative activity exerted by conjugated linoleic acid (CLA) on the estrogen unresponsive MDA-MB-231 human breast cancer cell line. The effects on cell proliferation, cell cycle progression and induction of apoptosis were examined. CLA caused the reduction of cell proliferation along with the accumulation of cells in the S phase of the cycle. The occurrence of apoptosis in these cells was indicated by flow cytometry data and further confirmed by the onset of cells with morphological features typical of apoptosis. ERK1/2 reduction and upregulation of pro-apoptotic protein Bak were induced. These events were associated with: (a) reduced levels of the anti-apoptotic protein Bcl-x(L), (b) the translocation of cytochrome c from the mitochondria to the cytosol, (c) the cleavage of pro-caspase-9 and pro-caspase-3. From the above data, we are induced to think that CLA may trigger apoptosis in the estrogen unresponsive MDA-MB-231 cell line via mechanisms involving above all the mitochondrial pathway.