RESUMEN
Pancreatic adenocarcinoma is one of the most deadly malignancies, and endometrial cancer represents the most common gynecologic cancer in the USA. Better understanding on the pathologic mechanisms and pathways is required for effective treatment of these malignancies. Recently, human epididymis protein 4 (HE4 or WFDC2), a secretory glycoprotein, was found to be overexpressed in pancreatic and endometrial cancers. In addition, studies have shown that HE4 overexpression in endometrial cancer cell lines led to faster cancer progression in a mouse subcutaneous model. These findings raise a question on the role(s) of secretory, extracellular HE4 in cancer development. In the present study, we found that treatment of pancreatic and endometrial cancer cell lines with purified, extracellular HE4 protein led to a significant increase in cell viability and proliferation. Moreover, extracellular HE4 protein was able to increase DNA synthesis, and modulate the mRNA and protein levels of cell cycle marker PCNA and cell cycle inhibitor p21. These effects appeared to be robust and sustainable and required a relatively low concentration of HE4 protein. The findings indicated the secreted, extracellular HE4 may carry some physiopathological functions. Via paracrine/endocrine actions, circulatory HE4 produced by malignant cells may contribute to pancreatic and endometrial cancer progression and/or metastasis.
Asunto(s)
Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias Endometriales/genética , Femenino , Humanos , Neoplasias Pancreáticas/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
Syncytin-1 is a member of human endogenous retroviral W gene family (HERVW1). Known to be expressed in human placental trophoblast, syncytin-1 protein mediates the fusion of cytotrophoblasts for the formation of syncytiotrophoblasts, the terminally differentiated form of trophoblast lineage. In addition, in vitro studies indicate that syncytin-1 possessed nonfusogenic functions such as those for immune suppression, cell cycle regulation and anti-apoptotic activities. Overexpression of syncytin-1 has been observed in various malignant tissues including breast, endometrial and ovarian cancers. It was reported that syncytin-1 gene expression is associated with dynamic changes of DNA hypomethylation in the 5' LTR. In this study, applying the real-time PCR, Western blot analysis and immunohistochemistry methods, we demonstrate a constitutive expression of syncytin-1 in normal pancreas tissues as well as normal tissues adjacent to cancer lesions. Moreover, a reduced expression is found in the pancreatic adenocarcinoma tissues. The expression levels of syncytin-1 are not correlated with the stage, historical grade and gender, but inversely correlated with patients' age. Furthermore, COBRA and bisulfite sequencing results indicated that the lower expression of syncytin-1 is correlated with the hypermethylation of two CpG dinucleotides in the 5' LTR of syncytin-1 gene. The nonfusogenic function of syncytin-1 in normal pancreas as well as its role(s) in the pathogenesis and progression of pancreatic cancers remains to be investigated. Identification of the two CpG dinucleotides around transcription start site as key epigenetic elements has provided valuable information for further studies on the epigenetic regulation of syncytin-1 in pancreatic cancer cells.
Asunto(s)
Adenocarcinoma/genética , Metilación de ADN , Productos del Gen env/genética , Neoplasias Pancreáticas/genética , Proteínas Gestacionales/genética , Regiones Promotoras Genéticas , Estudios de Casos y Controles , Humanos , ARN Mensajero/genética , Secuencias Repetidas Terminales , Análisis de Matrices TisularesRESUMEN
Human epididymis protein 4 (HE4) is a recognized biomarker in ovarian and endometrial cancer and over-expressed in pancreatic adenocarcinoma. The diagnostic value of HE4 in pancreatic adenocarcinoma remains unknown. Here we elucidate mRNA, protein and serum level of HE4 in pancreatic adenocarcinoma. HE4 mRNA level in tumor adjacent tissues and pancreatic adenocarcinoma tissues were tested by real time-PCR. Tissue microarray containing normal, adenocarcinoma, and adjacent pancreatic tissue was tested by immunohistochemistry (IHC). Serum level of HE4, carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 15-3 (CA15-3) and carbohydrate antigen 125 (CA125) were detected by ELISA assay in control and tumor patients. Further we compared the sensitivity and specificity of determining HE4, CA19-9, CA15-3, and CA125 for diagnosis of pancreatic adenocarcinoma and assessed the complementary diagnostic value of HE4, CA19-9, CA15-3 and CA125. Real time PCR showed significantly increased HE4 mRNA level in pancreatic adenocarcinoma compared with control. Result of IHC showed that HE4 significantly higher expressed in the human pancreatic carcinoma tissues than in both normal and adjacent non-tumorous pancreatic tissues, and the staining intensity is inversely correlated with the clinical stage. HE4 was highly expressed in early stage of pancreatic adenocarcinoma. Serum HE4 level is higher in cases with pancreatic adenocarcinoma than in the controls. Serum HE4 levels could research to a sensitivity of 45.83% and specificity of 93.75% when the Cutoff was set at 4.59 ng/mL. The Combined HE4 and CA19-9 increased the sensitivity to 83.33%; and interestingly, the combination of HE4 with CA15-3 led to the most powerful sensitivity of 87.5%. Combined with CA19-9 and CA15-3, HE4 could be a potential biomarker to improve the diagnostic power for pancreatic adenocarcinoma.
Asunto(s)
Adenocarcinoma/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Proteínas/análisis , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Antígenos de Carbohidratos Asociados a Tumores/sangre , Área Bajo la Curva , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Antígeno Ca-125/sangre , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Estadificación de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
BACKGROUND: The results of prostate specific antigen (PSA) and digital rectal examination (DRE) screenings lead to both under and over treatment of prostate cancer (PCa). As such, there is an urgent need for the identification and evaluation of new markers for early diagnosis and disease prognosis. Studies have shown a link between PCa, lipids and lipid metabolism. Therefore, the aim of this study was to examine the concentrations and distribution of serum lipids in patients with PCa as compared with serum from controls. METHOD: Using Electrospray ionization mass spectrometry (ESI-MS/MS) lipid profiling, we analyzed serum phospholipids from age-matched subjects who were either newly diagnosed with PCa or healthy (normal). RESULTS: We found that cholester (CE), dihydrosphingomyelin (DSM), phosphatidylcholine (PC), egg phosphatidylcholine (ePC) and egg phosphatidylethanolamine (ePE) are the 5 major lipid groups that varied between normal and cancer serums. ePC 38:5, PC 40:3, and PC 42:4 represent the lipids species most prevalent in PCa as compared with normal serum. Further analysis revealed that serum ePC 38:5 ≥0.015 nmoles, PC 40.3 ≤0.001 nmoles and PC 42:4 ≤0.0001 nmoles correlated with the absence of PCa at 94% prediction. Conversely, serum ePC 38:5 ≤0.015 nmoles, PC 40:3 ≥0.001 nmoles, and PC 42:4 ≥0.0001 nmoles correlated with the presence of PCa. CONCLUSION: In summary, we have demonstrated that ePC 38:5, PC 40:3, and PC 42:4 may serve as early predictive serum markers for the presence of PCa.
Asunto(s)
Diagnóstico Precoz , Fosfolípidos/sangre , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Diagnóstico Diferencial , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Fosfatidilcolinas/sangre , Fosfatidiletanolaminas/sangre , Pronóstico , Antígeno Prostático Específico/sangre , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodos , Esfingomielinas/sangreRESUMEN
With an estimated 37,000 deaths per year, pancreatic cancer is the fourth leading cause of cancer deaths in the USA. A total of 95% of pancreatic cancers are exocrine neoplasms, known as pancreatic ductal adenocarcinomas (PDACs). The difficulty of early diagnosis and the high prevalence of metastasis associated with PDAC contribute to its dismal prognosis. The past decade has witnessed intensive study and impressive progress in searching for more sensitive, specific and cost-effective biomarkers. This review focuses on the epigenetic biomarkers potentially useful for the management of PDAC. The authors begin with an overview on the available biomarkers, and subsequently discuss the recent development in epigenetic biomarkers, including DNA methylation, miRNA and histone modifications in diversified specimens of cell lines, xenograft, cancer tissues, pancreatic juice and patient blood. These findings raise the possibility for clinical application of epigenetic biomarkers towards screening, early diagnosis, prognosis, chemosensitivity prediction and recurrence surveillance of PDAC patients.
Asunto(s)
Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Epigénesis Genética/genética , Conductos Pancreáticos/patología , Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Metilación de ADN , Detección Precoz del Cáncer , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , MicroARNs , Metástasis de la Neoplasia/diagnóstico , Metástasis de la Neoplasia/patología , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Conductos Pancreáticos/metabolismo , PronósticoRESUMEN
To understand the role of human 15-lipoxygenase 1 (15-LOX1) in vascular wall remodeling, we have studied the effect of the major 15-LOX1 metabolite of arachidonic acid, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), on vascular smooth muscle cell (VSMC) migration both in vitro and in vivo. Among 5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, 15(S)-HETE potentially stimulated more vascular smooth muscle cell (VSMC) migration. In addition, 15(S)-HETE-induced VSMC migration was dependent on Src-mediated activation of signal transducer and activator of transcription-3 (STAT-3). 15(S)-HETE also induced monocyte chemoattractant protein-1 (MCP-1) expression via Src-STAT-3 signaling, and neutralizing anti-MCP-1 antibodies completely negated 15(S)-HETE-induced VSMC migration. Cloning and characterization of a 2.6-kb MCP-1 promoter revealed the presence of four putative STAT-binding sites, and the site that is proximal to the transcription start site was found to be essential for 15(S)-HETE-induced Src-STAT-3-mediated MCP-1 expression. Rat carotid arteries that were subjected to balloon injury and transduced with Ad-15-LOX1 upon exposure to [(3)H]arachidonic acid ex vivo produced 15-HETE as a major eicosanoid and enhanced balloon injury-induced expression of MCP-1 in smooth muscle cells in Src and STAT-3-dependent manner in vivo. Adenovirus-mediated delivery of 15-LOX1 into rat carotid artery also led to recruitment and homing of macrophages to medial region in response to injury. In addition, transduction of Ad-15-LOX1 into arteries enhanced balloon injury-induced smooth muscle cell migration from media to intima and neointima formation. These results show for the first time that 15-LOX1-15(S)-HETE axis plays a major role in vascular wall remodeling after balloon angioplasty.
Asunto(s)
Movimiento Celular , Quimiocina CCL2/genética , Regulación de la Expresión Génica , Ácidos Hidroxieicosatetraenoicos/metabolismo , Miocitos del Músculo Liso/citología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Línea Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Humanos , Técnicas In Vitro , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Ratas , Factor de Transcripción STAT3/genética , Transducción de SeñalRESUMEN
OBJECTIVE: Migration of vascular smooth muscle cells (VSMCs) from media to intima is a key event in the pathophysiology of atherosclerosis and restenosis. The lipoxygenase products of polyunsaturated fatty acids (PUFA) were shown to play a role in these diseases. cAMP response element binding protein (CREB) has been implicated in the regulation of VSMC growth and motility in response to thrombin and angiotensin II. The aim of the present study was to test the role of CREB in an oxidized lipid molecule, 15(S)-HETE-induced VSMC migration and neointima formation. METHODS AND RESULTS: 15(S)-HETE stimulated VSMC migration in CREB-dependent manner, as measured by the modified Boyden chamber method. Blockade of MEK1, JNK1, or p38MAPK inhibited 15(S)-HETE-induced CREB phosphorylation and VSMC migration. 15(S)-HETE induced expression and secretion of interleukin-6 (IL-6), as analyzed by RT-PCR and ELISA, respectively. Neutralizing anti-IL-6 antibodies blocked 15(S)-HETE-induced VSMC migration. Dominant-negative mutant-mediated blockade of ERK1/2, JNK1, p38MAPK, or CREB suppressed 15(S)-HETE-induced IL-6 expression in VSMCs. Serial 5' deletions and site-directed mutagenesis of IL-6 promoter along with chromatin immunoprecipitation using anti-CREB antibodies showed that cAMP response element is essential for 15(S)-HETE-induced IL-6 expression. Dominant-negative CREB also suppressed balloon injury-induced IL-6 expression, SMC migration from media to intimal region, and neointima formation. Adenovirus-mediated transduction of 15-lipoxygenase 2 (15-LOX2) caused increased production of 15-HETE in VSMCs and enhanced IL-6 expression, SMC migration from media to intimal region, and neointima formation in response to arterial injury. CONCLUSIONS: The above results suggest a role for 15-LOX2-15-HETE in the regulation of VSMC migration and neointima formation involving CREB-mediated IL-6 expression.
Asunto(s)
Traumatismos de las Arterias Carótidas/metabolismo , Movimiento Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Interleucina-6/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Angioplastia de Balón/efectos adversos , Animales , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Traumatismos de las Arterias Carótidas/enzimología , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Modelos Animales de Enfermedad , Interleucina-6/genética , MAP Quinasa Quinasa 1/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Mutación , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Fosforilación , Regiones Promotoras Genéticas , Ratas , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
15-LOX-1 and its metabolites are involved in colorectal cancer. Recently, we reported that 15-LOX-1 overexpression in HCT-116 human colorectal cancer cells inhibited cell growth by induction of p53 phosphorylation (4). To determine whether the 15-LOX-1 protein or its metabolites are responsible for phosphorylation of p53 in HCT-116 cells, we used HCT-116 cells that expressed a mutant 15-LOX-1. The mutant 15-LOX-1 enzyme, with a substitution of Leu at residue His361, was devoid of enzymatic activity. HCT-116 cells transiently transfected with either native or mutant 15-LOX-1 showed an increase in p53 phosphorylation and an increase in the expression of downstream genes. Thus, 15-LOX-1 induces p53 phosphorylation independent of enzymatic activity. Treatment of A549 human lung carcinoma cells with IL-4 increased the expression of 15-LOX-1 and also increased the expression of downstream targets of p53. This confirmed that the activation of p53 was also observed in wild-type cells expressing physiological 15-LOX-1. Immunoprecipitation experiments revealed that 15-LOX-1 interacts with, and binds to, DNA-dependent protein kinase (DNA-PK). The binding of 15-LOX-1 to DNA-PK caused an approximate 3.0-fold enhancement in kinase activity, resulting in increased p53 phosphorylation at Ser15. Knockdown of DNA-PK by small interfering RNA (siRNA) significantly reduced p53 phosphorylation. Furthermore, confocal microscopy demonstrated a colocalization of 15-LOX and DNA-PK in the cells. We propose that the 15-LOX-1 protein binds to DNA-PK, increasing its kinase activity and results in downstream activation of the tumor suppressor p53, thus revealing a new mechanism by which lipoxygenases (LOX) may influence the phenotype of tumor cells.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Neoplasias Colorrectales/enzimología , Neoplasias Pulmonares/enzimología , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , Neoplasias Colorrectales/metabolismo , ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Inmunoprecipitación , Ácido Linoleico/metabolismo , Neoplasias Pulmonares/metabolismo , Fenotipo , Fosforilación , Proteínas Quinasas/genética , ARN Interferente Pequeño , TransfecciónRESUMEN
PURPOSE: To examine for the expression of 15-lipoxygenase 1 (15-LOX1) and 15-LOX2 in human retinal microvascular endothelial cells (HRMVECs) and study the role of arachidonic acid metabolites of these enzymes in angiogenesis. METHODS: Quantitative RT-PCR and reverse-phase HPLC analyses were used to determine 15-LOX1/2 expression and their arachidonic acid metabolites in HRMVECs. The role of MEK1 in 15(S)-HETE-induced angiogenesis was studied using HRMVEC migration, tube formation, and basement membrane matrix plug angiogenesis. RESULTS: HRMVECs expressed both 15-LOX1 and 15-LOX2. Hypoxia induced the expression of 15-LOX1 and the production of its arachidonic acid metabolites 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 15(S)-HETE stimulated HRMVEC migration and tube formation as potently as 20 ng/mL fibroblast growth factor-2 (FGF-2). In addition, 15(S)-HETE stimulated the phosphorylation of ERK1/2, JNK1, p38 MAPK, and MEK1 in a time-dependent manner in these cells. Inhibition of MEK1 by pharmacologic and dominant-negative mutant approaches attenuated 15(S)-HETE-induced phosphorylation of ERK1/2 and JNK1 but not p38 MAPK. Blockade of ERK1/2 and JNK1 activation suppressed 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. Inhibition of p38 MAPK attenuated 15(S)-HETE-induced HRMVEC migration only. Inhibition of MEK1 also blocked 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. CONCLUSIONS: These results suggest that hypoxia, through the induction of 15-LOX1 expression, leads to the production of 15(S)-HETE in HRMVECs. In addition, 15(S)-HETE, through MEK1-dependent activation of ERK1/2 and JNK1, stimulates the angiogenic differentiation of HRMVECs and basement membrane matrix plug angiogenesis.
Asunto(s)
Endotelio Vascular/metabolismo , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Hipoxia/metabolismo , MAP Quinasa Quinasa 1/fisiología , Neovascularización Retiniana/metabolismo , Vasos Retinianos/citología , Adenoviridae/genética , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Membrana Basal , Movimiento Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/farmacología , Vectores Genéticos , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Satellite cells are stem cells that are critical for the formation and growth of skeletal muscle during myogenesis. To differentiate and fuse, proliferating satellite cells or myoblasts must migrate and establish stable cell-cell contacts. However, the factors that regulate myoblast migration and fusion are not understood completely. We have identified PGI2 as a novel regulator of myogenesis in vitro. PGI2 is a member of the family of prostaglandins (PG), autocrine/paracrine signaling molecules synthesized via the cyclooxygenase-1 and -2 pathways. Primary mouse muscle cells both secrete PGI2 and express the PGI2 receptor, IP, at various stages of myogenesis. Using genetic and pharmacological approaches, we show that PGI2 is a negative regulator of myoblast migration that also enhances cell fusion. Thus, PGI2 may act as a "brake" on migrating cells to facilitate cell-cell contact and fusion. Together, our results highlight the importance of the balance between positive and negative regulators in cell migration and myogenesis. This work may have implications for migration of other populations of adult stem cells and/or cells that undergo fusion.
Asunto(s)
Fusión Celular , Movimiento Celular/fisiología , Epoprostenol/metabolismo , Desarrollo de Músculos/fisiología , Mioblastos/fisiología , Animales , Antihipertensivos/metabolismo , Diferenciación Celular , Células Cultivadas , Sistema Enzimático del Citocromo P-450/metabolismo , Epoprostenol/agonistas , Oxidorreductasas Intramoleculares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mioblastos/citología , Receptores de Epoprostenol/genética , Receptores de Epoprostenol/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: The haplotypes in the gene vitamin K epoxide reductase complex subunit 1 (VKORC1) have been found to affect warfarin dose response through effects on the formation of reduced-form vitamin K, a cofactor for gamma-carboxylation of vitamin K-dependent proteins, which is involved in the coagulation cascade and has a potential impact on atherosclerosis. We hypothesized that VKORC1-dependent effects on the coagulation cascade and atherosclerosis would contribute to susceptibility for vascular diseases. METHODS AND RESULTS: To test the hypothesis, we studied the association of polymorphisms of VKORC1 with stroke (1811 patients), coronary heart disease (740 patients), and aortic dissection (253 patients) compared with matched controls (n=1811, 740, and 416, respectively). Five common noncoding single-nucleotide polymorphisms of VKORC1 were identified in a natural haplotype block with strong linkage disequilibrium (D'>0.9, r2>0.9), then single-nucleotide polymorphism (SNP) +2255 in the block was selected for the association study. We found that the presence of the C allele of the +2255 locus conferred almost twice the risk of vascular disease (odds ratio [OR] 1.95, 95% confidence interval [CI] .58 to 2.41, P<0.001 for stroke; OR 1.72, 95% CI 1.24 to 2.38, P<0.01 for coronary heart disease; and OR 1.90, 95% CI 1.04 to 3.48, P<0.05 for aortic dissection). We also observed that subjects with the CC and CT genotypes had lower levels of undercarboxylated osteocalcin (a regulator for the bone), probably vascular calcification, and lower levels of protein induced in vitamin K absence or antagonism II (PIVKA-II, a des-gamma-carboxy prothrombin) than those with TT genotypes. CONCLUSIONS: The haplotype of VKORC1 may serve as a novel genetic marker for the risk of stroke, coronary heart disease, and aortic dissection.
Asunto(s)
Haplotipos , Oxigenasas de Función Mixta/genética , Enfermedades Vasculares/genética , Anciano , Rotura de la Aorta/diagnóstico , Rotura de la Aorta/genética , Arterias , Estudios de Casos y Controles , Enfermedad Coronaria/diagnóstico , Enfermedad Coronaria/genética , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Oxigenasas de Función Mixta/fisiología , Polimorfismo Genético , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/genética , Enfermedades Vasculares/diagnóstico , Vitamina K Epóxido ReductasasRESUMEN
Curcumin (diferuloylmethane) is one of the phytophenolic compounds found in the turmeric plant with anti-inflammatory and anticarcinogenic activities. One possible mechanism for these activities is the inhibition of prostaglandin (PG) E(2) formation. In this study and other reports, curcumin suppresses interleukin-1beta-induced formation of prostaglandin E(2) in a concentration-dependent manner. Interleukin-1beta-induced microsomal prostaglandin E synthase 1 (mPGES-1) and cyclooxygenase-2 were attenuated by curcumin at the protein and mRNA levels, but a more dramatic inhibition of mPGES-1 expression was observed at lower concentrations of curcumin in A549 human lung epithelial cells. The inhibition of mPGES-1 expression by curcumin shifted the arachidonic acid profile from PGE(2) to PGF(2alpha) and 6-keto-PGF(1alpha) as major metabolites. The expression of early growth response gene 1 (EGR-1), a key transcription factor of cytokine-induced mPGES-1, was inhibited by curcumin. Incubation with siRNA for EGR-1 inhibited interleukin (IL)-1beta-induced mPGES-1, and the controlled expression of EGR-1 increased the mPGES-1 expression. Several proinflammatory signaling molecules, such as nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinases, are also known to affect curcumin-regulated gene expression. Curcumin inhibited IkappaBalpha phosphorylation and degradation and thus reduced the expression of mPGES-1. Curcumin suppressed cytokine-induced mPGES-1 by inhibiting phosphorylation of Jun N-terminal kinase (JNK)1/2. However, EGR-1 expression was suppressed by lower concentrations of curcumin, as compared with JNK1/2 and IkappaBalpha. These results indicate that curcumin inhibits IL-1beta-induced PGE(2) formation by inhibiting the expression of mPGES-1 that is mediated by suppression of EGR-1 expression as well as NF-kappaB and JNK1/2.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Anticarcinógenos/farmacología , Curcumina/farmacología , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Interleucina-1/fisiología , Oxidorreductasas Intramoleculares/metabolismo , Microsomas Hepáticos/enzimología , Transducción de Señal/efectos de los fármacos , Ácido Araquidónico/metabolismo , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Microsomas Hepáticos/efectos de los fármacos , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Plásmidos/genética , Prostaglandina-E Sintasas , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.
Asunto(s)
Araquidonato 15-Lipooxigenasa/fisiología , Ácido Araquidónico/fisiología , Neoplasias de la Mama/enzimología , MAP Quinasa Quinasa 6/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Ácido Araquidónico/metabolismo , Carcinoma/enzimología , Adhesión Celular/fisiología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Activación Enzimática/fisiología , Femenino , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inhibidores de la Lipooxigenasa , MAP Quinasa Quinasa Quinasa 5/metabolismo , Masoprocol/farmacología , FosforilaciónRESUMEN
Changes in the expression and activity of lipid-metabolizing enzymes, including the linoleic acid (LA)-metabolizing enzyme 15-lipoxygenase-1 (15-LO-1), may play a role in the development and progression of human prostate carcinoma (PCa). We reported that human 15-LO-1 (designated as leukocyte type 12-LO or 12/15-LO in mouse) is expressed in human prostate and increased in PCa, particularly high-grade PCa. Genetically engineered mouse (GEM) models of PCa could facilitate the study of this gene and its regulation and function in PCa progression. In this study, we examine the protein expression and enzyme activity levels of 12/15-LO associated with PCa progression in the TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) model of PCa. This GEM model develops prostatic intraepithelial neoplasia (PIN), followed by invasive gland-forming PCa and invasive and metastatic less differentiated PCa, with neuroendocrine (NE) differentiation (NE Ca). In the wild-type and TRAMP prostates, the most prominent LA metabolite was 13-hydroxyoctadecadienoic acid (13-HODE). Lesser amounts of 12-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid (HETE) were made from arachidonic acid (AA). In TRAMP prostates, 12/15-LO activity was increased compared to wild type at 20, 29, 39, and 49 weeks, as assessed by LA conversion to 13-HODE, and by AA conversion to 12/15-HETE, respectively. Immunostaining demonstrated that the increased capacity to generate 13-HODE was paralleled by an increase in neoplastic epithelial expression of 12/15-LO in PIN and invasive carcinomas. In conclusion, although there is a basal 12/15-LO activity in the wild-type mouse prostate, there is a marked increase in the expression of 12/15-LO with TRAMP PCa progression, paralleling our previously reported increased expression of the ortholog 15-LO-1 in high-grade human PCa. Thus, 12/15-LO and LA metabolism in the TRAMP model shares similarities to human PCa, and may allow to confirm a role for LA metabolism and other biologic functions of 15-LO-1 in human PCa. In addition, the TRAMP model will serve as a tool for testing the suitability of 12/15-LO-and ultimately human 15-LO--as a therapeutic target during PCa progression.
Asunto(s)
Adenocarcinoma/enzimología , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Neoplasias de la Próstata/enzimología , Adenocarcinoma/patología , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Ácido Araquidónico/metabolismo , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Activación Enzimática/fisiología , Humanos , Inmunohistoquímica , Ácido Linoleico/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neoplasias de la Próstata/patología , Regulación hacia ArribaAsunto(s)
Enfermedades de los Trabajadores Agrícolas/inducido químicamente , Enfermedades de los Trabajadores Agrícolas/diagnóstico , Ansiedad/inducido químicamente , Depresión/inducido químicamente , Fatiga/inducido químicamente , Hidrocarburos Clorados , Insecticidas/envenenamiento , Adulto , Enfermedad Crónica , Diagnóstico Diferencial , Femenino , Humanos , Tamizaje Masivo , Anamnesis , Residuos de Plaguicidas/análisis , Residuos de Plaguicidas/metabolismoRESUMEN
Lipoxygenases oxidatively metabolize polyunsaturated fatty acids to a rich spectrum of biologically active products. One enzyme of the lipoxygenase family, Arachidonate 15-lipoxygenase-1 (arachidonate:oxygen 15-oxidoreductase, EC 1.13.11.33), is implicated in several forms of epithelial cell cancers, and provides a rationale for exploring its role in the progression and metastasis of cancers. Thus, 15-LO-1 could be a marker of the growth and spread of cancers. The importance of 15-LO-1 expression is imparted by its metabolic product of linoleic acid, yielding 13-(S)-hydroxyoctadecadienoic acid (13-(S)-HODE). In order to maximize our understanding of the roles of lipoxygenases and cyclooxygenases in cancer, it is necessary to correlate their metabolic pathways to the respective expression levels, and not exaggerate the presence or absence of an enzyme or its function when it clearly is not there. It is also important to acknowledge that some forms of these enzymes may not even be relevant to human disease. Although it is unlikely that any single gene is sufficient to account for the complex etiology of cancer, 15-LO-1 overexpression is clearly a key component in the cell proliferation process. This article is divided into three sections emphasizing the key role of 15-LO-1 in prostate, colorectal, and breast cancers.
Asunto(s)
Araquidonato 15-Lipooxigenasa/biosíntesis , Neoplasias/enzimología , Neoplasias/patología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Humanos , Masculino , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patologíaRESUMEN
Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2alpha. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.