RESUMEN
Glioblastoma is the most common malignant brain tumor, which, despite combined radio- and chemotherapy, recurs and is invariably fatal for affected patients. Members of the sphingolipid (SL) family are potent effectors of glioma cell proliferation. In particular sphingosine-1-phosphate (S1P) and the corresponding G protein-coupled S1P receptors transmit proliferative signals to glioma cells. To investigate the contribution to glioma cell proliferation we inhibited the first step of de novo SL synthesis in p53(wt) and p53(mut) glioma cells, and interfered with S1P signaling specifically in p53(wt) U87MG cells. Subunit silencing (RNAi) or pharmacological antagonism (using myriocin) of serine palmitoyltransferase (SPT; catalyzing the first committed step of SL biosynthesis) reduced proliferation of p53(wt) but not p53(mut) GBM cells. In U87MG cells these observations were accompanied by decreased ceramide, sphingomyelin, and S1P content. Inhibition of SPT upregulated p53 and p21 expression and induced an increase in early and late apoptotic U87MG cells. Exogenously added S1P (complexed to physiological carriers) increased U87MG proliferation. In line, silencing of individual members of the S1P receptor family decreased U87MG proliferation. Silencing and pharmacological inhibition of the ATP-dependent cassette transporter A1 (ABCA1) that facilitates S1P efflux in astrocytes attenuated U87MG growth. Glyburide-mediated inhibition of ABCA1 resulted in intracellular accumulation of S1P raising the possibility that ABCA1 promotes S1P efflux in U87MG glioma cells thereby contributing to inside-out signaling. Our findings indicate that de novo SL synthesis, S1P receptor-mediated signaling, and ABCA1-mediated S1P efflux could provide pharmacological targets to interfere with glioma cell proliferation.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Esfingolípidos/biosíntesis , Transportador 1 de Casete de Unión a ATP/metabolismo , Apoptosis , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular , Silenciador del Gen , Glioma/patología , Humanos , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Transducción de SeñalRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo. METHODS: The outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling. RESULTS: RNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53(wt) (U87MG, A172, and primary GBM2), and p53(mut) (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53(wt) and p53(mut) cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53(wt) and p53(mut) GBM cells. PRKD2 knockdown in p53(wt) cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53(mut) GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation. CONCLUSIONS: PRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Senescencia Celular/fisiología , Glioma/metabolismo , Proteínas Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Silenciador del Gen , Xenoinjertos , Humanos , Inmunoprecipitación , Ratones , Microscopía Fluorescente , Proteína Quinasa D2 , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , TransfecciónRESUMEN
Neuronal sphingolipids (SL) play important roles during axonal extension, neurotrophic receptor signaling and neurotransmitter release. Many of these signaling pathways depend on the presence of specialized membrane microdomains termed lipid rafts. Sphingomyelin (SM), one of the main raft constituents, can be formed de novo or supplied from exogenous sources. The present study aimed to characterize fluorescently-labeled SL turnover in a murine neuronal cell line (CATH.a). Our results demonstrate that at 4°C exogenously added BODIPY-SM accumulates exclusively at the plasma membrane. Treatment of cells with bacterial sphingomyelinase (SMase) and back-exchange experiments revealed that 55-67% of BODIPY-SM resides in the outer leaflet of the plasma membrane. Endocytosis of BODIPY-SM occurs via caveolae with part of internalized BODIPY-fluorescence ending up in the Golgi and the ER. Following endocytosis BODIPY-SM undergoes hydrolysis, a reaction substantially faster than BODIPY-SM synthesis from BODIPY-ceramide. RNAi demonstrated that both, acid (a)SMase and neutral (n)SMases contribute to BODIPY-SM hydrolysis. Finally, high-density lipoprotein (HDL)-associated BODIPY-SM was efficiently taken up by CATH.a cells. Our findings indicate that endocytosis of exogenous SM occurs almost exclusively via caveolin-dependent pathways, that both, a- and nSMases equally contribute to neuronal SM turnover and that HDL-like particles might represent physiological SM carriers/donors in the brain.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Microdominios de Membrana/metabolismo , Neuronas/enzimología , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Animales , Compuestos de Boro , Caveolinas/genética , Caveolinas/metabolismo , Línea Celular , Endocitosis , Retículo Endoplásmico/efectos de los fármacos , Colorantes Fluorescentes , Regulación de la Expresión Génica , Aparato de Golgi/efectos de los fármacos , Hidrólisis , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lipoproteínas HDL/metabolismo , Microdominios de Membrana/efectos de los fármacos , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/genética , Esfingomielinas/farmacología , TemperaturaRESUMEN
Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun(S73) phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Proteínas Quinasas/fisiología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Evaluación Preclínica de Medicamentos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Lisofosfolípidos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Invasividad Neoplásica , Proteína Quinasa D2 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Pirimidinas/farmacología , Interferencia de ARN/fisiología , ARN Interferente Pequeño/farmacología , Esfingosina/análogos & derivados , Esfingosina/farmacologíaAsunto(s)
Transición Epitelial-Mesenquimal/fisiología , Factor de Crecimiento de Hepatocito/metabolismo , Melanoma/fisiopatología , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Cadherinas , Línea Celular Tumoral , Inhibidores Enzimáticos/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/patología , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Proteína 1 Relacionada con Twist/genéticaRESUMEN
The regulation of CD44v6, a variant of the CD44 family of glycosylated adhesion molecules, through hepatocyte growth factor (HGF) has implications for motility in primary human melanocytes. We show that exposure of primary human melanocytes to HGF results in an increase of CD44v6 expression. Immunostaining of melanocytic lesions revealed low cytoplasmic positivity of CD44v6 in some nevi but high membranous expression in primary cutaneous melanomas, and cutaneous and lymph node metastases. HGF-dependent CD44v6 regulation in melanocytes is NF-kappaB dependent because BAY 11-7082, an inhibitor of NF-kappaB activation, but not interference with the mitogen-activated protein kinase or phosphatidylinositol 3-kinase cascade, antagonized HGF-induced CD44v6 expression. NF-kappaB-mediated transcriptional regulation of CD44v6 involves the transcription factors Egr-1 and CCAAT enhancer-binding protein-beta (C/EBP-beta). In gel shift assays, the initial binding of p100/p52 NF-kappaB, C/EBP-beta, and Egr-1 to the CD44 promoter experienced reshuffling toward increased affinity of C/EBP-beta after HGF stimulation. A blocking antibody to CD44v6 decreased HGF-induced c-Met phosphorylation as well as enhanced random- and site-directed migration. Our data show that HGF-induced motility in primary human melanocytes depends on c-Met-CD44v6 interaction, and that HGF-enhanced CD44v6 expression is required for motility and transcriptional upregulation of CD44v6, presumably mediated through a complex comprising NF-kappaB/C/EBP-beta and Egr-1.
Asunto(s)
Movimiento Celular/fisiología , Receptores de Hialuranos/metabolismo , Melanocitos/patología , Melanoma/secundario , Neoplasias Cutáneas/patología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Receptores de Hialuranos/genética , Inmunohistoquímica , Metástasis Linfática , Melanocitos/metabolismo , Melanoma/metabolismo , Melanoma/fisiopatología , FN-kappa B/metabolismo , Nevo/metabolismo , Nevo/patología , Transducción de Señal/fisiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/fisiopatología , Transcripción Genética/fisiologíaRESUMEN
We present images of a rare case where a primary lung tumour was visualised by transthoracic echocardiography. The patient was a 78-year-old male where Chest X-ray had revealed a tumour-suspected structure in the left lung. Both transthoracic echocardiography and combined PET/CT images showed a large tumour located close to the heart. Fine-needle biopsy showed non-small cell lung cancer.