RESUMEN
The mammalian target of rapamycin inhibitor (mTOR-I) Rapamycin, a drug widely used in kidney transplantation, exerts important anti-cancer effects, particularly in Kaposi's Sarcoma (KS), through several biological interactions. In this in vivo and in vitro study, we explored whether the activation of the autophagic pathway through the low-affinity receptor for nerve growth factor, p75NTR , may have a pivotal role in the anti-cancer effect exerted by Rapamycin in S. Our Kimmunohistochemistry results revealed a significant hyper-activation of the autophagic pathway in KS lesions. In vitro experiments on KS cell lines showed that Rapamycin exposure reduced cell viability by increasing the autophagic process, in the absence of apoptosis, through the transcriptional activation of p75NTR via EGR1. Interestingly, p75NTR gene silencing prevented the increase of the autophagic process and the reduction of cell viability. Moreover, p75NTR activation promoted the upregulation of phosphatase and tensin homolog (PTEN), a tumour suppressor that modulates the PI3K/Akt/mTOR pathway. In conclusion, our in vitro data demonstrated, for the first time, that in Kaposi's sarcoma, autophagy triggered by Rapamycin through p75NTR represented a major mechanism by which mTOR inhibitors may induce tumour regression. Additionally, it suggested that p75NTR protein analysis could be proposed as a new potential biomarker to predict response to Rapamycin in kidney transplant recipients affected by Kaposi's sarcoma.
Asunto(s)
Sarcoma de Kaposi , Sirolimus , Apoptosis , Autofagia , Humanos , Fosfatidilinositol 3-Quinasas , Sarcoma de Kaposi/patología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Most of the common drugs used to treat the cervical cancer, which main etiological factor is the HPV infection, cause side effects and intrinsic/acquired resistance to chemotherapy. In this study we investigated whether an olive leaf extract (OLE), rich in polyphenols, was able to exert anti-tumor effects in human cervical cancer cells (HeLa). MTT assay results showed a reduction of HeLa cells viability OLE-induced, concomitantly with a gene and protein down-regulation of Cyclin-D1 and an up-regulation of p21, triggering intrinsic apoptosis. OLE reduced NFkB nuclear translocation, which constitutive activation, stimulated by HPV-oncoproteins, promotes cancer progression and functional studies revealed that OLE activated p21Cip/WAF1 in a transcriptional-dependent-manner, by reducing the nuclear recruitment of NFkB on its responsive elements. Furthermore, OLE treatment counteracted epithelial-to-mesenchymal-transition and inhibited anchorage-dependent and -independent cell growth EGF-induced. Finally, MTT assay results revealed that OLE plus Cisplatin strengthened the reduction of cells viability Cisplatin-induced, as OLE inhibited NFkB, AkT and MAPK pathways, all involved in Cisplatin chemoresistance. In conclusion, we demonstrated that in HeLa cells OLE exerts pro-apoptotic effects, elucidating the molecular mechanism and that OLE could mitigate Cisplatin chemoresistance. Further studies are needed to explore the potential coadiuvant use of OLE for cervical cancer treatment.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Olea/química , Extractos Vegetales/farmacología , Polifenoles/farmacología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Quinasas p21 Activadas/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Regulación hacia Arriba/efectos de los fármacos , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
INTRODUCTION: Cytomegalovirus (CMV) infection represents a common cause of morbidity and mortality in kidney transplant recipients (KTR). The NF-kB signaling pathway is highly involved in the pathogenesis of CMV infection. The -94ins/delATTG functional polymorphism in the promoter of NFKB1 has been associated with low intracellular levels of the protein and high incidence of inflammatory and autoimmune disease. In this study, we evaluated the association of this NFKB1 polymorphism with the risk of CMV infection. METHODS: CMV infection was defined as virus isolation or detection of viral antigens or nucleic acid in any body fluid or tissue specimen. Using Cox regression and survival analysis, we analyzed the association between the polymorphism and CMV infection as well as recurrence in the first 12 months after transplantation. RESULTS: We analyzed the -94ins/delATTG NFKB1 polymorphism of 189 KTRs. The 65% of CMV infections occurred in ins/ins group. Survival free from CMV infection was 54.7% for ins/ins group and 79.4% for deletion carriers one year after transplantation (P < 0.0001). At multivariate regression, deletion carriers showed a lower risk of CMV infection and recurrence with respect to ins/ins KTRs (HR = 0.224 P = 0.0002; HR = 0.307, P = 0.012, respectively). CONCLUSIONS: In conclusion, pretransplantation screening for NFKB1 -94ins/delATTG polymorphism may predict CMV infection and improve the management of patients at higher risk of infection in the post-transplant period.
Asunto(s)
Infecciones por Citomegalovirus/diagnóstico , Trasplante de Riñón/efectos adversos , Subunidad p50 de NF-kappa B/genética , Complicaciones Posoperatorias/diagnóstico , Regiones Promotoras Genéticas/genética , Adulto , Biomarcadores/análisis , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/epidemiología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Femenino , Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Humanos , Mutación INDEL , Incidencia , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/genética , Complicaciones Posoperatorias/virología , Valor Predictivo de las Pruebas , Cuidados Preoperatorios/métodos , PronósticoRESUMEN
BACKGROUND: Clinical studies have demonstrated that, after renal transplantation (TX), testosterone deficiency (TD) at the time of the procedure is independently associated with lower survival of the patient and graft. However, data between TD and the functional CAG polymorphism of the androgen receptor promoter (AR) are discordant. We investigated the prevalence of TD and its association with body composition, biochemical parameters, the Aging Males' Symptoms rating scale (AMS) domains and AR polymorphism. METHODS: In 112 TX patients, we assessed the AMS, biochemical/hormonal (FSH/LH/TT) anthropometric/bioimpedance analysis parameters, and AR CAG polymorphism of AR by gene sequencing. RESULTS: Median values of total testosterone (TT) were 340 ng/dl and 52% of TX patients were affected by TD. Significant correlations between TT and FSH and FSH and LH (p = 0.005, p < 0.0001, respectively) were found. TD patients had lower estimated glomerular filtration rate (eGFR) and hemoglobin (Hb) (p = 0.034, p = 0.022 respectively) and showed higher values of C-reactive protein (p = 0.023) and fat tissue index/adipose tissue mass (p = 0.034 and p = 0.021, respectively), and lower values of serum albumin (p = 0.003) and high-density lipoprotein-cholesterol (p = 0.038) levels. Significant differences were found in the number of patients on mammalian target of rapamycin inhibitors immunosuppressant therapy (p = 0.045). Logistic regression analysis did not show any correlation between age, AMS scores, TT or CAG repeat length, gonadotropins, time of the transplant, and dialysis. CONCLUSIONS: Our results suggest that in TX recipients an appropriate sexual hormonal evaluation should be performed, as we found a high prevalence of TD. However, further studies are needed to clarify the association between TD and patient and graft survival.
Asunto(s)
Composición Corporal , Hipogonadismo/sangre , Trasplante de Riñón , Insuficiencia Renal Crónica/cirugía , Testosterona/deficiencia , Receptores de Trasplantes , Adulto , Anciano , Biomarcadores/sangre , Hormona Folículo Estimulante/sangre , Humanos , Hipogonadismo/epidemiología , Hipogonadismo/genética , Hipogonadismo/fisiopatología , Italia/epidemiología , Trasplante de Riñón/efectos adversos , Hormona Luteinizante/sangre , Persona de Mediana Edad , Polimorfismo Genético , Prevalencia , Receptores Androgénicos/genética , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/epidemiología , Factores de Riesgo , Testosterona/sangre , Resultado del TratamientoRESUMEN
Subclinical rejection (SCR) has been variably associated with reduced graft survival, development and progression of interstitial fibrosis/tubular atrophy and chronic allograft nephropathy, but data are controversial concerning SCR treatment in terms of graft survival improvement. In this single-center retrospective study, we enrolled 174 adult kidney transplant recipients with a protocol biopsy performed at 30 days after transplantation to evaluate the incidence rate and risk factors for early SCR and its impact on 10-year graft survival. Five patients showed primary non function and were excluded. Among 159/169 (94.08 %) patients with stable graft function who underwent protocol biopsy, 17 (10.7 %) showed signs of SCR and were treated with low-dose intravenous (i.v.) steroids. Ten patients showed functional impairment, 8 (4.73 %) resulting as acute rejection. At multivariate analysis, donor age [odds ratio (OR) 1.04, 95 % confidence interval (CI) 1.01-1.09], and delayed graft function (DGF) (OR 1.08, 95 % CI 1.03-1.12) were significantly associated with SCR. The 10-year graft survival rate in the SCR group was similar to that in the normal-findings group (76.5 vs. 74.9 % respectively; p = 0.61). At multivariate Cox regression, acute [hazard ratio (HR) 5.22, 95 % CI 1.70-16.01], but not sub-clinical, rejection was independently associated with long-term graft failure. In conclusion, early protocol biopsy is a useful and safe tool to detect early SCR which seems not to affect the long-term survival. We suggest that this could be, probably, linked to early SCR treatment with low dose i.v. steroids.
Asunto(s)
Corticoesteroides/uso terapéutico , Rechazo de Injerto/tratamiento farmacológico , Supervivencia de Injerto , Trasplante de Riñón/efectos adversos , Adulto , Funcionamiento Retardado del Injerto/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios RetrospectivosRESUMEN
The pathogenetic role of adiponectin (ADPN) in kidney failure is not yet elucidated, since in vitro and in vivo studies have demonstrated that ADPN exerts both anti-inflammatory and pro-inflammatory effects. Starting from our previous findings demonstrating that HK-2 cells express and secrete ADPN, in this study we investigated the autocrine role of ADPN in tubular inflammatory damage induced by lipopolysaccharide (LPS) and the underlying molecular mechanisms. Firstly, we observed that short-term exposure to LPS enhanced ADPN protein expression as well as the adiponectin receptor ADIPOR1 mRNA content together with its signaling pathway downstream, pAMPK/pERK/pJNK, whose up-regulation status was reversed when ADPN gene knockdown occurred. Interestingly, in the same experimental conditions, we observed that ADPN mediated the nuclear translocation of the transcription factors nuclear factor kappa B (NFkB) and pcFos/pcJun (activator protein 1, AP-1), both induced by the pJNK pathway and involved in tumor necrosis factor (TNF)-α transactivation. Indeed, by transient transfection assay, we observed that the LPS-induced increase of TNF-α promoter activity was abrogated in cells pretreated with the inhibitors of NFkB and AP-1. Collectively our results suggest that in HK-2 cells, ADPN produced upon LPS stimulus could worsen the inflammatory damage in an autocrine-dependent manner.
Asunto(s)
Adiponectina/metabolismo , Comunicación Autocrina/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Lipopolisacáridos/toxicidad , Nefritis/inducido químicamente , Proteínas Quinasas Activadas por AMP/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Adiponectina/genética , Sitios de Unión , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Nefritis/genética , Nefritis/metabolismo , Nefritis/patología , Fosforilación , Regiones Promotoras Genéticas , Interferencia de ARN , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
Clinical studies showed that renal expression and serum levels of nerve growth factor (NGF) are increased in renal diseases characterized by progressive fibrosis, a pathologic process in which TGF-ß1 mediates most of the key events leading to tubular epithelial-mesenchymal transition (EMT). However, the pathogenic role of high NGF levels has not yet been elucidated. In this study, we found that in tubular renal cells, HK-2, NGF transcriptionally up-regulated TGF-ß1 expression and secretion and enhanced cell motility by activating EMT markers via its receptors, TrkA and p75(NTR). Interestingly, we observed that TGF-ß1-SMAD pathway activation and the up-regulation of EMT markers NGF-induced were both prevented when knockdown of TGF-ß1 gene occurred and that the pretreatment with an antibody anti-NGF reversed the nuclear translocation of pSMAD3/SMAD4 complex. Collectively, our results demonstrated that NGF promotes renal fibrosis via TGF-ß1-signaling activation, suggesting that in kidney diseases high NGF serum levels could contribute to worsen renal fibrosis.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Fibrosis/patología , Túbulos Renales/fisiología , Factor de Crecimiento Nervioso/metabolismo , Insuficiencia Renal Crónica/patología , Factor de Crecimiento Transformador beta1/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Anticuerpos/inmunología , Línea Celular , Movimiento Celular , Células Epiteliales/citología , Humanos , Factor de Crecimiento Nervioso/sangre , Factor de Crecimiento Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteína smad3/antagonistas & inhibidores , Proteína smad3/inmunología , Proteína smad3/metabolismo , Proteína Smad4/antagonistas & inhibidores , Proteína Smad4/inmunología , Proteína Smad4/metabolismo , Transcripción Genética/genética , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
BACKGROUND: Data on serum soluble Klotho levels in chronic kidney disease are contradictory and even less is known after renal transplantation. Experimental studies demonstrated that recombinant human erythropoietin (rhEPO) treatment mitigates Klotho reduction caused by renal damage. Therefore, this study aimed to determine serum Klotho levels in a cohort of kidney transplant recipients (KTR) and to evaluate whether rhEPO treatment can modulate, in vivo and in vitro, soluble Klotho. METHODS: 117 KTR and 22 healthy subjects (HS) were enrolled. In 17 KTR, rhEPO was discontinued for 5 weeks and Klotho levels were compared to 34 propensity score-matched controls. Moreover, we evaluated Klotho mRNA expression and protein secretion in HK-2 tubular cells treated with cyclosporin A (CyA) and rhEPO, alone or in combination. RESULTS: Serum Klotho levels in KTR were significantly higher than in HS (0.68 vs. 0.37, p = 0.002) and significantly associated with estimated glomerular filtration rate (r = -0.378, p = 0.003) and fibroblast growth factor 23 (r = -0.307, p < 0.0001). After 5 weeks of rhEPO discontinuation, treated KTR showed a sharper reduction of Klotho levels than controls (-0.56 vs. -0.11 ng/ml, p < 0.0001). In HK-2 cells CyA treatment induced a Klotho down-regulation that was mitigated by rhEPO pre-treatment. In the same experimental conditions, our results revealed that cells treated with CyA + rhEPO secreted higher soluble Klotho levels than those exposed to CyA or rhEPO alone. CONCLUSIONS: Our results demonstrate that KTR have higher serum Klotho levels than HS and that rhEPO treatment modulates these concentrations, suggesting a link between rhEPO and soluble Klotho in KTR.
Asunto(s)
Eritropoyetina/uso terapéutico , Glucuronidasa/sangre , Hematínicos/uso terapéutico , Trasplante de Riñón , Adulto , Anciano , Biomarcadores/sangre , Estudios de Casos y Controles , Línea Celular , Estudios Transversales , Ciclosporina/farmacología , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Regulación de la Expresión Génica , Tasa de Filtración Glomerular/efectos de los fármacos , Glucuronidasa/genética , Humanos , Inmunosupresores/farmacología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Proteínas Klotho , Masculino , Persona de Mediana Edad , Puntaje de Propensión , ARN Mensajero/metabolismo , Proteínas Recombinantes/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Several studies have demonstrated that thyroid hormone T3 promotes cancer cell growth, even though the molecular mechanism involved in such processes still needs to be elucidated. In this study we demonstrated that T3 induced proliferation in papillary thyroid carcinoma cell lines concomitantly with an up-regulation of cyclin D1 expression, that is a critical mitogen-regulated cell-cycle control element. Our data revealed that T3 enhanced the recruitment of the TRß1/Oct-1 complex on Octamer-transcription factor-1 site within cyclin D1 promoter, leading to its transactivation. In addition, silencing of TRß1 or Oct-1 expression by RNA interference reversed both increased cell proliferation and up-regulation of cyclin D1, underlying the important role of both transcriptional factors in mediating these effects. Finally, T3-induced increase in cell growth was abrogated after knocking down cyclin D1 expression. All these findings highlight a new molecular mechanism by which T3 promotes thyroid cancer cell growth.
Asunto(s)
Carcinoma/metabolismo , Carcinoma/patología , Ciclina D1/metabolismo , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Receptores beta de Hormona Tiroidea/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Triyodotironina/farmacología , Carcinoma Papilar , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Biológicos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Cáncer Papilar Tiroideo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Nerve growth factor is a neurotrophin that promotes cell growth, differentiation, survival and death through two different receptors: TrkA(NTR) and p75(NTR). Nerve growth factor serum concentrations increase during many inflammatory and autoimmune diseases, glomerulonephritis, chronic kidney disease, end-stage renal disease and, particularly, in renal transplant. Considering that nerve growth factor exerts beneficial effects in the treatment of major central and peripheral neurodegenerative diseases, skin and corneal ulcers, we asked whether nerve growth factor could also exert a role in Cyclosporine A-induced graft nephrotoxicity. Our hypothesis was raised from basic evidence indicating that Cyclosporine A-inhibition of calcineurin-NFAT pathway increases nerve growth factor expression levels. Therefore, we investigated the involvement of nerve growth factor and its receptors in the damage exerted by Cyclosporine A in tubular renal cells, HK-2. Our results showed that in HK-2 cells combined treatment with Cyclosporine A + nerve growth factor induced a significant reduction in cell vitality concomitant with a down-regulation of Cyclin D1 and up-regulation of p21 levels respect to cells treated with Cyclosporine A alone. Moreover functional experiments showed that the co-treatment significantly up-regulated human p21promoter activity by involvement of the Sp1 transcription factor, whose nuclear content was negatively regulated by activated NFATc1. In addition we observed that the combined exposure to Cyclosporine A + nerve growth factor promoted an up-regulation of p75 (NTR) and its target genes, p53 and BAD leading to the activation of intrinsic apoptosis. Finally, the chemical inhibition of p75(NTR) down-regulated the intrinsic apoptotic signal. We describe two new mechanisms by which nerve growth factor promotes growth arrest and apoptosis in tubular renal cells exposed to Cyclosporine A.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclina D1/antagonistas & inhibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Sinergismo Farmacológico , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Factores de Transcripción NFATC/agonistas , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Receptores de Factor de Crecimiento Nervioso/agonistas , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Proteína p53 Supresora de Tumor/agonistas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína Letal Asociada a bcl/agonistas , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
BACKGROUND: Adiponectin (ADPN) is predominantly produced by adipose tissue, and high ADPN levels have been detected in patients affected by proteinuric glomerulonephritis. In this study we investigate whether human tubular epithelial cells express and secrete ADPN. METHODS: In human proximal tubular epithelial cells, HK-2, ADPN mRNA was evaluated by real-time PCR assay, while protein expression levels were measured by Western blot analysis and immunofluorescence assay. Moreover, renal ADPN distribution was assessed by immunohistochemical analysis of kidney biopsy samples from healthy patients. Finally, by ELISA, we measured ADPN concentrations in culture media of HK-2 cells treated with 10 µg/mL lipopolysaccharide (LPS). RESULTS: Our analyses revealed that HK-2 cells express ADPN both in terms of mRNA and protein. These results were confirmed by the observed cytoplasmatic HK-2 intense immunoreactivity for ADPN antibody and by immunohistochemical analysis showing a diffuse ADPN distribution in normal kidney tissue. Furthermore, we observed that tubular cells secrete ADPN in the basal condition and, more interestingly, that this secretion significantly increases (p<0.05) upon LPS treatment in a time-dependent manner. Finally, immunohistochemical analysis of kidney biopsy samples obtained from patients affected by membranous and rapidly progressive glomerulonephritis showed a similar pattern of ADPN staining to that observed in healthy controls. CONCLUSIONS: Our study demonstrates for the first time that renal tubular cells express and secrete ADPN, and their concentration increases upon inflammatory stimulus. These results suggest that in renal inflammatory diseases, tubular cells may contribute to the increase in circulating ADPN levels, triggering a feedback response in order to self-mitigate the inflammatory process.
Asunto(s)
Adiponectina/metabolismo , Células Epiteliales/metabolismo , Túbulos Renales Proximales/metabolismo , Adiponectina/análisis , Adiponectina/genética , Células Cultivadas , Medios de Cultivo/análisis , Células Epiteliales/efectos de los fármacos , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Lipopolisacáridos/farmacología , ARN Mensajero/análisis , ARN Mensajero/metabolismoRESUMEN
Well-differentiated papillary and follicular thyroid carcinoma are the most frequent types of thyroid cancer and the prognosis is generally favorable however, a number of patients develops recurrences. Epigallocatechin-3-gallate (EGCG), a major catechin in green tea, was shown to possess remarkable therapeutic potential against various types of human cancers, although data on thyroid cancer cells are still lacking. The aim of this study was to investigate the effect of EGCG on the proliferation and motility of human thyroid papillary (FB-2) and follicular (WRO) carcinoma cell lines. Our results demonstrate that EGCG (10, 40, 60 µM) treatment inhibited the growth of FB-2 and WRO cells in a dose-dependent manner. These changes were associated with reduced cyclin D1, increased p21 and p53 expression. Furthermore, EGCG suppressed phosphorylation of AKT and ERK1/2. In addition EGCG treatment results in reduction of cell motility and migration. Changes in motility and migration in FB-2 were associated with modulation in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. After 24 h EGCG caused an increase of the E-cadherin expression and a concomitant decrease of SNAIL, ZEB and the basic helix-loop-helix transcription factor TWIST. Besides expression of Vimentin, N-cadherin and α5-integrin was down-regulated. These data well correlate with a reduction of MMP9 activity as evidenced by gelatin zymography. Our findings support the inhibitory role of EGCG on thyroid cancer cell proliferation and motility with concomitant loss of epithelial-to-mesenchymal cell transition markers.
Asunto(s)
Catequina/análogos & derivados , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Actinas/genética , Actinas/metabolismo , Apoptosis/efectos de los fármacos , Cadherinas/genética , Cadherinas/metabolismo , Catequina/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , 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 , Regulación hacia Abajo/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Vimentina/genética , Vimentina/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be essential for fibroblast differentiation into fat cells. Because peroxisome proliferator-activated receptor-gamma (PPARγ) is known to be one of the master regulators of adipogenesis, we aimed to study the regulation of CIC by the PPARγ ligand rosiglitazone (BRL) in 3T3-L1 fibroblasts and in adipocytes. We demonstrated that BRL up-regulated CIC mRNA and protein levels in fibroblasts, while it did not elicit any effects in mature adipocytes. The enhancement of CIC levels upon BRL treatment was reversed using the PPARγ antagonist GW9662, addressing how this effect was mediated by PPARγ. Functional experiments using a reporter gene containing rat CIC promoter showed that BRL enhanced CIC promoter activity. Mutagenesis studies, electrophoretic-mobility-shift assay and chromatin-immunoprecipitation analysis revealed that upon BRL treatment, PPARγ and Sp1 are recruited on the Sp1-containing region within the CIC promoter, leading to an increase in CIC expression. In addition, mithramycin, a specific inhibitor for Sp1-DNA binding activity, abolished the PPARγ-mediated up-regulation of CIC in fibroblasts. The stimulatory effects of BRL disappeared in mature adipocytes in which PPARγ/Sp1 complex recruited SMRT corepressor to the Sp1 site of the CIC promoter. Taken together, our results contribute to clarify the molecular mechanisms by which PPARγ regulates CIC expression during the differentiation stages of fibroblasts into mature adipocytes.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis/fisiología , Fibroblastos/metabolismo , Mitocondrias/metabolismo , PPAR gamma/metabolismo , Proteínas Represoras/genética , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Hipoglucemiantes/farmacología , Luciferasas/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Co-Represor 2 de Receptor Nuclear/antagonistas & inhibidores , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , PPAR gamma/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosiglitazona , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Tiazolidinedionas/farmacología , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Obesity condition confers risks to breast cancer development and progression, and several reports indicate that the adipokine leptin, whose synthesis and plasma levels increase with obesity, might play an important role in modulating breast cancer cell phenotype. Functional crosstalk occurring between leptin and different signaling molecules contribute to breast carcinogenesis. In this study, we show, in different human breast cancer cell lines, that leptin enhanced the expression of a chaperone protein Hsp90 resulting in increased HER2 protein levels. Silencing of Hsp90 gene expression by RNA interference abrogated leptin-mediated HER2 up-regulation. Leptin effects were dependent on JAK2/STAT3 activation, since inhibition of this signaling cascade by AG490 or ectopic expression of a STAT3 dominant negative abrogated leptin-induced HER2 and Hsp90 expressions. Functional experiments showed that leptin treatment significantly up-regulated human Hsp90 promoter activity. This occurred through an enhanced STAT3 transcription factor binding to its specific responsive element located in the Hsp90 promoter region as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Analysis of HER2, Akt and MAPK phosphorylation levels revealed that leptin treatment amplified the responsiveness of breast cancer cells to growth factor stimulation. Furthermore, we found that long-term leptin exposure reduced sensitivity of breast cancer cells to the antiestrogen tamoxifen. In the same experimental conditions, the combined treatment of tamoxifen with the Hsp90 inhibitor 17-AAG completely abrogated leptin-induced anchorage-independent breast cancer cell growth. In conclusion, our results highlight, for the first time, the ability of the adipocyte-secreted factor leptin to modulate Hsp90/HER2 expressions in breast cancer cells providing novel insights into the molecular mechanism linking obesity to breast cancer growth and progression.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas HSP90 de Choque Térmico/genética , Leptina/metabolismo , Receptor ErbB-2/genética , Factor de Transcripción STAT3/metabolismo , Regulación hacia Arriba , Antineoplásicos Hormonales/farmacología , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Tamoxifeno/farmacologíaRESUMEN
The omega-3 long chain polyunsaturated fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), elicit anti-proliferative effects in cancer cell lines and in animal models. Dietary DHA and EPA can be converted to their ethanolamide derivatives, docosahexaenoyl ethanolamine (DHEA), and eicosapentaenoyl ethanolamine (EPEA), respectively; however, few studies are reported on their anti-cancer activities. Here, we demonstrated that DHEA and EPEA were able to reduce cell viability in MCF-7 breast cancer cells whereas they did not elicit any effects in MCF-10A non-tumorigenic breast epithelial cells. Since DHA and EPA are ligands of peroxisome proliferator-activated receptor gamma (PPARγ), we sought to determine whether PPARγ may also mediate DHEA and EPEA actions. In MCF-7 cells, both compounds enhanced PPARγ expression, stimulated a PPAR response element-dependent transcription as confirmed by the increased expression of its target gene PTEN, resulting in the inhibition of AKT-mTOR pathways. Besides, DHEA and EPEA treatment induced phosphorylation of Bcl-2 promoting its dissociation from beclin-1 which resulted in autophagy induction. We also observed an increase of beclin-1 and microtubule-associated protein 1 light chain 3 expression along with an enhanced autophagosomes formation as revealed by mono-dansyl-cadaverine staining. Finally, we demonstrated the involvement of PPARγ in DHEA- and EPEA-induced autophagy by using siRNA technology and a selective inhibitor. In summary, our data show that the two omega-3 ethanolamides exert anti-proliferative effects by inducing autophagy in breast cancer cells highlighting their potential use as breast cancer preventive and/or therapeutic agents.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Etanolamina/farmacología , PPAR gamma/agonistas , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Transcripción Genética , Activación Transcripcional , Transfección , Regulación hacia ArribaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Ziziphus extracts have been used in Traditional Chinese Medicine for the treatment of cancer. AIM OF THE STUDY: In the present study we have investigated the effects of Ziziphus jujube extracts (ZEs) on breast cancer. MATERIALS AND METHODS: We evaluated the effects of increasing concentrations of ZEs on ERα positive MCF-7 and ERα negative SKBR3 breast cancer cell proliferation using MTT assays. Apoptosis was analyzed by evaluating the involvement of some pro-apoptotic proteins, including Bax, Bad, Bid and PARP cleavage by immunoblotting analysis. Moreover, the effects of ZEs treatment on apoptosis were tested by both DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. By using chromatographic techniques, we identified the constituents of the effective extracts. RESULTS: ZE1, ZE2, and ZE4 exerted significant antiproliferative effects on estrogen receptor alpha (ERα) positive MCF-7 (IC(50) values of 14.42, 7.64, 1.69µg/mL) and ERα negative SKBR3 (IC(50) values of 14.06, 6.21, 3.70µg/mL) human breast cancer cells. Remarkably, ZEs did not affect cell viability of both normal human fibroblasts BJ1-hTERT and nonmalignant breast epithelial MCF-10A cells. Treatment with ZEs induced cell death by apoptosis in both malignant breast cells. We found that the most effective extracts ZE2 and ZE4 shared a number of triterpenic acids, already known for their anticancer activities. CONCLUSIONS: Our data provide a rational base for the use of Ziziphus extracts in the treatment of breast cancer in Traditional Chinese Medicine.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Medicamentos Herbarios Chinos/uso terapéutico , Fitoterapia , Triterpenos/uso terapéutico , Ziziphus/química , Antineoplásicos Fitogénicos/análisis , Antineoplásicos Fitogénicos/farmacología , Mama/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fragmentación del ADN , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Frutas/química , Humanos , Etiquetado Corte-Fin in Situ , Medicina Tradicional China , Triterpenos/análisis , Triterpenos/farmacologíaRESUMEN
The combined treatment with nanomolar doses of the PPARγ ligand Rosiglitazone (BRL) and the RXR ligand 9-cisretinoic acid (9RA) induces a p53-dependent apoptosis in MCF7, SKBR3 and T47D human breast cancer cells. Since MCF7 cells express a wild-type p53 protein, while SKBR3 and T47D cells harbor endogenous mutant p53, we elucidated the mechanism through which PPARγ and RXR ligands triggered apoptotic processes independently of p53 transcriptional activity. We showed an upregulation of Bid expression enhancing the association between Bid/p53 in both cytosol and mitochondria after the ligand treatment. Particularly in the mitochondria, the complex involves the truncated Bid that plays a key role in the apoptotic process induced by BRL and 9RA, since the disruption of mitochondrial membrane potential, the induction of PARP cleavage and the percentage of TUNEL-positive cells were reversed after knocking down Bid. Moreover, PPARγ and RXR ligands were able to reduce mitochondrial GST activity, which was no longer noticeable silencing Bid expression, suggesting the potential of Bid in the regulation of mitochondrial intracellular reactive oxygen species scavenger activity. Our data, providing new insight into the role of p53/Bid complex at the mitochondria in promoting breast cancer cell apoptosis upon low doses of PPARγ and RXR ligands, address Bid as a potential target in the novel therapeutical strategies for breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Neoplasias de la Mama/metabolismo , Tiazolidinedionas/farmacología , Tretinoina/farmacología , Alitretinoína , Antineoplásicos/uso terapéutico , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/antagonistas & inhibidores , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Ligandos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , PPAR gamma/antagonistas & inhibidores , PPAR gamma/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores X Retinoide/antagonistas & inhibidores , Receptores X Retinoide/metabolismo , Rosiglitazona , Tiazolidinedionas/uso terapéutico , Tretinoina/uso terapéutico , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Obesity is a major risk factor for the development and progression of breast cancer. Leptin, a cytokine mainly produced by adipocytes, plays a crucial role in mammary carcinogenesis and is elevated in hyperinsulinemia and insulin resistance. The antidiabetic thiazolidinediones inhibit leptin gene expression through ligand activation of the peroxisome proliferator-activated receptor-γ (PPARγ) and exert antiproliferative and apoptotic effects on breast carcinoma. In this study, we investigated the ability of PPARγ ligands to counteract leptin stimulatory effects on breast cancer growth in either in vivo or in vitro models. The results show that activation of PPARγ prevented the development of leptin-induced MCF-7 tumor xenografts and inhibited the increased cell-cell aggregation and proliferation observed on leptin exposure. PPARγ ligands abrogated the leptin-induced up-regulation of leptin gene expression and its receptors in breast cancer. PPARγ-mediated repression of leptin gene involved the recruitment of nuclear receptor corepressor protein and silencing mediator of retinoid and thyroid hormone receptors corepressors on the glucocorticoid responsive element site in the leptin gene expression regulatory region in the presence of glucocorticoid receptor and PPARγ. In addition, PPARγ ligands inhibited leptin signaling mediated by MAPK/STAT3/Akt phosphorylation and counteracted leptin stimulatory effect on estrogen signaling. These findings suggest that PPARγ ligands may have potential therapeutic benefits in the treatment of breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Leptina/metabolismo , PPAR gamma/metabolismo , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Femenino , Humanos , Técnicas In Vitro , Ligandos , Neoplasias Mamarias Animales/metabolismo , Ratones , Ratones Desnudos , Obesidad/complicaciones , Interferencia de ARN , Receptores de Glucocorticoides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Factores de Riesgo , Transducción de SeñalRESUMEN
Ligand activation of peroxisome proliferator-activated receptor (PPAR)gamma and retinoid X receptor (RXR) induces antitumor effects in cancer. We evaluated the ability of combined treatment with nanomolar levels of the PPARgamma ligand rosiglitazone (BRL) and the RXR ligand 9-cis-retinoic acid (9RA) to promote antiproliferative effects in breast cancer cells. BRL and 9RA in combination strongly inhibit of cell viability in MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer cells, whereas MCF-10 normal breast epithelial cells are unaffected. In MCF-7 cells, combined treatment with BRL and 9RA up-regulated mRNA and protein levels of both the tumor suppressor p53 and its effector p21(WAF1/Cip1). Functional experiments indicate that the nuclear factor-kappaB site in the p53 promoter is required for the transcriptional response to BRL plus 9RA. We observed that the intrinsic apoptotic pathway in MCF-7 cells displays an ordinated sequence of events, including disruption of mitochondrial membrane potential, release of cytochrome c, strong caspase 9 activation, and, finally, DNA fragmentation. An expression vector for p53 antisense abrogated the biological effect of both ligands, which implicates involvement of p53 in PPARgamma/RXR-dependent activity in all of the human breast malignant cell lines tested. Taken together, our results suggest that multidrug regimens including a combination of PPARgamma and RXR ligands may provide a therapeutic advantage in breast cancer treatment.