RESUMEN
BACKGROUND: Osteosarcoma (OS) patients with metastasis have very dismal prognoses, and lack effective target therapies. Overexpression of cytosolic phospholipase A2 (cPLA2) has been shown to promote progression in several types of cancers, but its functions in OS have not been investigated. MATERIALS AND METHODS: In our study, the expression of cPLA2a was detected with immunohistochemistry in 102 cases of OS. The clinical significance of cPLA2a was evaluated by analyzing its correlation with clinicopathological factors. The prognostic significance of cPLA2a was estimated by univariate and multivariate analysis. The oncogenic functions of cPLA2a on cell proliferation and invasion were investigated by MTT assay and tranwell assay respectively. Western blotting was applied to detect the markers of epithelial-mesenchymal transition (EMT) after silencing cPLA2a expression or inhibiting its activity by a specific antagonist. RESULTS: In our study, high expression of cPLA2a was significantly associated with metastasis and advanced Enneking stage. High cPLA2a expression was significantly associated with poor prognosis and it was an independent prognostic biomarker of OS. By silencing cPLA2a or inhibiting its activity by a specific antagonist, we demonstrated that cPLA2a promoted cell invasion of OS cells via inducing the EMT process. CONCLUSIONS: High cPLA2a expression was an independent prognostic biomarker of OS, and cPLA2a could promote OS cell invasion via inducing the EMT process, indicating that cPLA2a was an independent prognostic biomarker and may be an effective drug target for OS.
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Neoplasias Óseas/patología , Transición Epitelial-Mesenquimal/fisiología , Fosfolipasas A2 Grupo IV/biosíntesis , Osteosarcoma/patología , Adulto , Biomarcadores de Tumor/análisis , Neoplasias Óseas/mortalidad , Niño , Femenino , Humanos , Masculino , Invasividad Neoplásica/patología , Osteosarcoma/mortalidad , Pronóstico , Análisis de SupervivenciaRESUMEN
Analysis of gill transcriptome of the Atlantic cod from the Baltic Sea demonstrated that alternatively spliced (AS) variants may be actively involved in the process of adaptation to altered salinity. Some AS variants of different genes, like phospholipase A2 group IVC (PLA2G4C), appeared only in fish exposed to altered salinity, while other isoforms of the same genes were present in all experimental groups. Novel sequence arrangements represent 89% of all AS in the Baltic cod compared to the Atlantic population. Profiles of modified pathways suggest that regulation by AS can afford specific changes of genes expressed in response to the environment. The AS variants appear to be involved in the response to stress by modifications of signalling in apoptosis pathways, an innate immunological response and pro-inflammatory process. Present results support the hypothesis that developing new AS variants could support genome complexity and reinforce the ability to fast adapt to local environments.
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Aclimatación/fisiología , Empalme Alternativo/fisiología , Proteínas de Peces/biosíntesis , Gadus morhua/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Fosfolipasas A2 Grupo IV/biosíntesis , Salinidad , Animales , Proteínas de Peces/genética , Gadus morhua/genética , Fosfolipasas A2 Grupo IV/genéticaRESUMEN
A high incidence of tumor recurrence and metastasis has been reported in breast cancer patients; nevertheless, the underlying molecular mechanisms are largely unknown. Epithelial-mesenchymal transition (EMT), which is induced by transforming growth factor-ß (TGF-ß), has been implicated in tumorigenesis and breast cancer metastasis. EMT events are now directly associated with tumor metastasis, and this progress is dependent on the inflammatory microenvironment. Cytosolic phospholipase A2α (cPLA2α) has been shown to participate in a series of biological processes including inflammation and cancer development. However, the role and molecular mechanism of cPLA2α in breast cancer EMT and metastasis remain enigmatic. In this study, we found that cPLA2α was commonly overexpressed in most human breast cancer tissues and significantly correlated with a poor prognosis for human breast cancer. Functional studies demonstrated that cPLA2α overexpression was significantly associated with elevated migration and invasion in MDA-MB-231 and T47D cells. Conversely, reduced cPLA2α expression strongly attenuated metastasis and the EMT program of MDA-MB-231 cells. Further study found that knockdown of cPLA2α in MDA-MB-231 cells inhibited TGF-ß-induced EMT through the PI3K/Akt signaling pathway. Animal experiments revealed that cPLA2α downregulation in MDA-MB-231 cells markedly restrained tumorigenesis and metastasis in vivo. This study indicates the potential role of cPLA2α in breast cancer metastasis and indicates that this molecule is a promising therapeutic target for breast cancer.
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Neoplasias de la Mama/metabolismo , Transición Epitelial-Mesenquimal , Fosfolipasas A2 Grupo IV/biosíntesis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Fosfolipasas A2 Grupo IV/genética , Humanos , Ratones , Metástasis de la Neoplasia , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal , Factor de Crecimiento Transformador beta/genéticaRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by selective death of motor neurons in the cortex, brainstem, and spinal cord. Cytosolic phospholipase A2 alpha (cPLA2α) upregulation and activation in the spinal cord of patients with sporadic ALS and in the spinal cord of human mutant SOD1G93A (hmSOD1) transgenic mice were recently reported. METHODS: cPLA2α upregulation in the brainstem and spinal cord was reduced by brain infusion of a specific antisense oligonucleotide against cPLA2α (AS), and the effect was evaluated on disease progression and brain cell activation. RESULTS: We found that the elevation of cPLA2α protein expression in the spinal cord was first detected at 6-week-old hmSOD1 mice and remained elevated during their whole life span. Reduction of the elevated expression of cPLA2α in the spinal cord of hmSOD1 mice by brain infusion of an AS at week 15 (shortly before the appearance of the disease symptoms), for a duration of 6 weeks, delayed the loss of motor neuron function in comparison with hmSOD1 mice and with sense brain-infused hmSOD1 mice. To characterize the effect of cPLA2α upregulation on different processes taking place at the appearance of the disease symptoms, mice were brain infused with AS or with sense at week 15 for 3-4 weeks. The AS treatment that reduced cPLA2α upregulation in the spinal cord of AS-treated hmSOD1 mice (as analyzed at week 18-19) prevented the reduction in the number of the neurons (detected by NeuN) and inhibited astrocyte activation (detected by GFAP) and microglia activation (detected by Iba-1 and by CD40). In addition, AS treatment blunted the upregulation of the proinflammatory enzyme-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) detected in hmSOD1 mice. CONCLUSIONS: Since specific reduction of cPLA2α in the brainstem and spinal cord significantly attenuated the development of the disease, cPLA2α may offer an efficient target for treatment of ALS.
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Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/prevención & control , Modelos Animales de Enfermedad , Fosfolipasas A2 Grupo IV/biosíntesis , Superóxido Dismutasa/biosíntesis , Regulación hacia Arriba/fisiología , Esclerosis Amiotrófica Lateral/patología , Animales , Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa/genéticaRESUMEN
Aberrant increase in protein kinase B (AKT) phosphorylation (pAKT), due to a gain-of-function mutation of phosphatidylinositol-3-kinase (PI3K) or loss-of-function mutation or deletion of phosphatase and tensin homolog (PTEN), is a common alteration in prostate cancer and associated with poor prognosis. Cytosolic phospholipase A2α (cPLA2α) is a lipid modifying enzyme by catalyzing the hydrolysis of arachidonic acid from membrane phospholipid. The released arachidonic acid and its metabolites contribute to survival and proliferation of prostate cancer cells. In this mini-review, we summarize the relationship between pAKT and cPLA2α in prostate cancer cells. There was a concordant increase in pAKT and cPLA2α levels in prostate tissue of prostate epithelial-specific PTEN-knockout mice compared to PTEN-wild type mice. Restoration of PTEN expression or inhibition of PI3K action decreased cPLA2α expression in PTEN-mutated or deleted prostate cancer cells. An increase in AKT phosphorylation elevated, whereas inhibition of AKT phosphorylation diminished, cPLA2α protein levels. pAKT had no influence on cPLA2α expression at mRNA levels but stabilized cPLA2α at protein levels by protecting it from degradation. Conversely, an induction of cPLA2α expression led to an increase in pAKT levels in PTEN-mutated or deleted prostate cancer cells, while silencing of cPLA2α expression or pharmacological blocking cPLA2α action decreased pAKT levels. The diminishment of pAKT by either genetic silencing or pharmacological blocking of cPLA2α was mitigated by the addition of arachidonic acid. The stimulatory effect of arachidonic acid on pAKT levels was lessened by inhibiting the production of arachidonic acid metabolites. These studies have revealed a link between oncogenic pathway and lipid metabolism and provided potential molecular targets for treating prostate cancer.
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Fosfolipasas A2 Grupo IV/biosíntesis , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Transducción de Señal/fisiología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Silenciador del Gen/efectos de los fármacos , Silenciador del Gen/fisiología , Fosfolipasas A2 Grupo IV/genética , Humanos , Masculino , Fosfohidrolasa PTEN/biosíntesis , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Angiotensin II activates cytosolic phospholipase A(2)α (cPLA2α) and releases arachidonic acid from tissue phospholipids, which mediate or modulate ≥1 cardiovascular effects of angiotensin II and has been implicated in hypertension. Because arachidonic acid release is the rate limiting step in eicosanoid production, cPLA2α might play a central role in the development of angiotensin II-induced hypertension. To test this hypothesis, we investigated the effect of angiotensin II infusion for 13 days by micro-osmotic pumps on systolic blood pressure and associated pathogenesis in wild type (cPLA2α(+/+)) and cPLA2α(-/-) mice. Angiotensin II-induced increase in systolic blood pressure in cPLA2α(+/+) mice was abolished in cPLA2α(-/-) mice; increased systolic blood pressure was also abolished by the arachidonic acid metabolism inhibitor, 5,8,11,14-eicosatetraynoic acid in cPLA2α(+/+) mice. Angiotensin II in cPLA2α(+/+) mice increased cardiac cPLA2 activity and urinary eicosanoid excretion, decreased cardiac output, caused cardiovascular remodeling with endothelial dysfunction, and increased vascular reactivity in cPLA2α(+/+) mice; these changes were diminished in cPLA2α(-/-) mice. Angiotensin II also increased cardiac infiltration of F4/80(+) macrophages and CD3(+) T lymphocytes, cardiovascular oxidative stress, expression of endoplasmic reticulum stress markers p58(IPK), and CHOP in cPLA2α(+/+) but not cPLA2α(-/-) mice. Angiotensin II increased cardiac activity of ERK1/2 and cSrc in cPLA2α(+/+) but not cPLA2α(-/-) mice. These data suggest that angiotensin II-induced hypertension and associated cardiovascular pathophysiological changes are mediated by cPLA2α activation, most likely through the release of arachidonic acid and generation of eicosanoids with predominant prohypertensive effects and activation of ≥1 signaling molecules, including ERK1/2 and cSrc.
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Presión Sanguínea/fisiología , Citosol/enzimología , Regulación de la Expresión Génica , Fosfolipasas A2 Grupo IV/genética , Hipertensión/genética , Estrés Oxidativo , ARN/genética , Angiotensina II/toxicidad , Animales , Modelos Animales de Enfermedad , Fosfolipasas A2 Grupo IV/biosíntesis , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos BALB C , Miocardio/enzimología , Miocardio/patología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Formation of the powerful osteogenic prostaglandin E2 by osteoblasts, a key modulatory event in the paracrine and autocrine regulation of bone cell activity, is preceded by release of the precursor arachidonic acid from phospholipid stores. The main routes of arachidonate liberation may involve phospholipase enzymes such as group IVA phospholipase A2 which is believed to be the main effector in many cell system due to its preference for arachidonate-containing lipids. MC3T3-E1 cells are non-transformed osteoblasts and are widely used as an in vitro model of osteoblast function. In these cells there is still no clarity about the main release pathway of arachidonic acid. Besides cytosolic phospholipase A2, phospholipase C and D pathways may play a key role in arachidonate release. Despite the crucial role of osteoblastic prostgalandin synthesis information on the occurrence of involved enzymes at the molecular level is scarse in MC3T3-E1 cells. We have characterised group IVA phospholipase A2 at the mRNA in these cells as a constitutively expressed enzyme which is cytosolic and translocates to the membrane upon endothelin-1 stimulation. Using immunopurification combined with Western blotting and high-resolution mass spectrometry, the enzyme was also identified at the protein level. Using specific gene silencing we were able to show that osteoblastic cytosolic phospholipase A2 is crucially involved in ET-1-induced prostaglandin formation.
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Endotelina-1/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Fosfolipasas A2 Grupo IV/biosíntesis , Osteoblastos/enzimología , Animales , Ácido Araquidónico/genética , Ácido Araquidónico/metabolismo , Línea Celular , Endotelina-1/genética , Fosfolipasas A2 Grupo IV/genética , Ratones , Osteoblastos/citología , Prostaglandinas/biosíntesis , Prostaglandinas/genética , Transporte de Proteínas/fisiologíaRESUMEN
The role and diagnostic efficacy of gene and protein products RB1, CDK4, CHMP2B, HSP90, and cPLA2G4A, all previously shown to be involved in tumor genesis and cell proliferation, were examined by immunohistochemical techniques in 32 cases of myxofibrosarcomas and 29 myxoid liposarcomas (all diagnosis had been confirmed by fluorescence in situ hybridization). HSP90 demonstrated strong nuclear and cytoplasmic positivity in all myxoid liposarcoma cases, while only 4 myxofibrosarcomas showed scattered HSP90 positivity. All but 4 cases of myxofibrosarcoma displayed strong positivity for cPLA2G4A, while only 2 myxoid liposarcoma cases were cPLA2G4A positive and both were CHMP2B negative. Overexpression of both cPLA2G4A and CHMP2B also suggested higher tumor grade. In conclusion, HSP90 and cPLA2G4A immunohistochemical stains are useful markers to distinguish myxofibrosarcoma from myxoid liposarcoma.
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Biomarcadores de Tumor/análisis , Fibrosarcoma/diagnóstico , Liposarcoma Mixoide/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Quinasa 4 Dependiente de la Ciclina/análisis , Quinasa 4 Dependiente de la Ciclina/biosíntesis , Diagnóstico Diferencial , Complejos de Clasificación Endosomal Requeridos para el Transporte/análisis , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Femenino , Fosfolipasas A2 Grupo IV/análisis , Fosfolipasas A2 Grupo IV/biosíntesis , Proteínas HSP90 de Choque Térmico/análisis , Proteínas HSP90 de Choque Térmico/biosíntesis , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteína de Retinoblastoma/análisis , Proteína de Retinoblastoma/biosíntesis , Adulto JovenRESUMEN
Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4(-/-) and Myd88(-/-) mice, but not in Cd44(-/-) mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2(high) and COX1/ALOX15/ALOX5/LTA4H(low) gene and PGE2/PGD2/15-HETE(high) and LXA4(low) eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.
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Eicosanoides/biosíntesis , Fosfolipasas A2 Grupo IV/biosíntesis , Ácido Hialurónico/farmacología , Metabolismo de los Lípidos/fisiología , Macrófagos/metabolismo , Monocitos/metabolismo , Animales , Línea Celular , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Eicosanoides/genética , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Fosfolipasas A2 Grupo IV/genética , Humanos , Ácido Hialurónico/genética , Ácido Hialurónico/metabolismo , Inflamación/genética , Inflamación/metabolismo , Macrófagos/citología , Masculino , Ratones , Ratones Noqueados , Monocitos/citología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) is an inflammatory disease of the joint characterized by chronic synovitis causing pain, swelling and loss of function due to destruction of cartilage and bone. The complex series of pathological events occurring in RA is largely regulated via excessive production of pro-inflammatory cytokines, the most prominent being tumor necrosis factor (TNF). The objective of this work was to elucidate possible involvement of group IVA cytosolic phospholipase A2 (cPLA2α) in TNF-induced regulation of synovitis and joint destructive effectors in RA, to evaluate the potential of cPLA2α as a future therapeutic target. METHODS: The involvement of cPLA2α in tumor necrosis factor (TNF)-induced intracellular signaling cascades in synoviocytes (synovial fibroblast-like cells) was analyzed by arachidonic acid (AA) release assay, synoviocyte enzyme activity assay, gene expression analysis by real-time PCR and ELISA immunoassay for the detection of prostaglandin E2 (PGE2), interleukin 8 (IL8) and stromelysin-1 (MMP3), respectively. RESULTS: Inhibitors of cPLA2α enzyme activity (AVX002, ATK) significantly reduced TNF-induced cellular release of AA, PGE2, IL8 and MMP3. This reduction was evident both at transcriptional, protein or metabolite levels. Interestingly, cPLA2α inhibition affected several key points of the arachidonyl cascade; AA-release, cyclooxygenase-2 (COX2) expression and PGE2 production. Furthermore, the results suggest that cPLA2α is subject to transcriptional auto-regulation as inhibition of cPLA2α resulted in reduced PLA2G4A gene expression in TNF-stimulated synoviocytes. CONCLUSIONS: cPLA2α appears to be an important regulator of central effectors of inflammation and joint destruction, namely MMP3, IL8, COX2, and PGE2. Decreased transcription of the PLA2G4A and COX2 genes in response to cPLA2α enzyme inhibition further suggest a self-reinforcing effect of cPLA2α inhibition in response to TNF. Collectively, these results support that cPLA2α is an attractive therapeutic target candidate as its inhibition reduces the production of multiple key pro-inflammatory factors involved in RA pathogenesis.
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Artritis Reumatoide/enzimología , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Fosfolipasas A2 Grupo IV/biosíntesis , Mediadores de Inflamación/metabolismo , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Artritis Reumatoide/patología , Línea Celular , Fibroblastos/patología , HumanosRESUMEN
Specific control of group IVA cytosolic phospholipase A2 (cPLA2α or PLA2G4A) expression modulates arachidonic acid production, thus tightly regulating the downstream effects of pro- and anti-inflammatory eicosanoids. The significance of this pathway in human disease is apparent in a range of pathologies from inflammation to tumorigenesis. While much of the regulation of cPLA2α has focused on posttranslational phosphorylation of the protein, studies on transcriptional regulation of this gene have focused only on proximal promoter regions. We have identified a DNase I hypersensitive site encompassing a 5' distal enhancer element containing a highly conserved consensus AP-1 site involved in transcriptional activation of cPLA2α by interleukin (IL)-1ß. Chromatin immunoprecipitation (ChIP), knockdown, knockout, and overexpression analyses have shown that c-Jun acts both in a negative and positive regulatory role. Transcriptional activation of cPLA2α occurs through the phosphorylation of c-Jun in conjunction with increased association of C/EBPß with the distal novel enhancer. The association of C/EBPß with the transcriptional activation complex does not require an obvious DNA binding site. These data provide new and important contributions to the understanding of cPLA2α regulation at the transcriptional level, with implications for eicosanoid metabolism, cellular signaling, and disease pathogenesis.
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Citocinas/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Fosfolipasas A2 Grupo IV/genética , Células Cultivadas , Fosfolipasas A2 Grupo IV/biosíntesis , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
ß-cell apoptosis is a significant contributor to ß-cell dysfunction in diabetes and ER stress is among the factors that contributes to ß-cell death. We previously identified that the Ca²âº-independent phospholipase A2ß (iPLA2ß), which in islets is localized in ß-cells, participates in ER stress-induced ß-cell apoptosis. Here, direct assessment of iPLA2ß role was made using ß-cell-specific iPLA2ß overexpressing (RIP-iPLA2ß-Tg) and globally iPLA2ß-deficient (iPLA2ß-KO) mice. Islets from Tg, but not KO, express higher islet iPLA2ß and neutral sphingomyelinase, decrease in sphingomyelins, and increase in ceramides, relative to WT group. ER stress induces iPLA2ß, ER stress factors, loss of mitochondrial membrane potential (∆Ψ), caspase-3 activation, and ß-cell apoptosis in the WT and these are all amplified in the Tg group. Surprisingly, ß-cells apoptosis while reduced in the KO is higher than in the WT group. This, however, was not accompanied by greater caspase-3 activation but with larger loss of ∆Ψ, suggesting that iPLA2ß deficiency impacts mitochondrial membrane integrity and causes apoptosis by a caspase-independent manner. Further, autophagy, as reflected by LC3-II accumulation, is increased in Tg and decreased in KO, relative to WT. Our findings suggest that (1) iPLA2ß impacts upstream (UPR) and downstream (ceramide generation and mitochondrial) pathways in ß-cells and (2) both over- or under-expression of iPLA2ß is deleterious to the ß-cells. Further, we present for the first time evidence for potential regulation of autophagy by iPLA2ß in islet ß-cells. These findings support the hypothesis that iPLA2ß induction under stress, as in diabetes, is a key component to amplifying ß-cell death processes.
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Apoptosis , Autofagia , Estrés del Retículo Endoplásmico , Regulación Enzimológica de la Expresión Génica , Fosfolipasas A2 Grupo IV/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Ceramidas/metabolismo , Diabetes Mellitus/enzimología , Diabetes Mellitus/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Fosfolipasas A2 Grupo IV/biosíntesis , Fosfolipasas A2 Grupo IV/genética , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Noqueados , Ratones Transgénicos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/biosíntesis , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Técnicas de Cultivo de Tejidos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Arachidonic acid metabolic pathway has been implicated in the inflammation-associated tumorigenesis of gastrointestinal cancers. As the rate-limiting enzyme of arachidonic acid production, group IVA phospholipase A2 (PLA2G4A) is hypothesized to play a fundamental role in gastric tumorigenesis as well as cyclooxygenase-2 (COX-2). However, little is known about the expression and role of PLA2G4A in gastric cancer, and the association of PLA2G4A with COX-2 remains to be elucidated. In this study, the mRNA expression of PLA2G4A and COX-2 in 60 pairs of fresh gastric tumors and corresponding adjacent non-cancerous mucosa was detected by using real-time quantitative PCR and the immunostaining of the both proteins in paired samples from 866 gastric cancer patients were assessed by using immunohistochemistry method. The clinicopathological and the prognostic relevance of PLA2G4A and COX-2 expression were determined. The results revealed a significantly reduced expression of PLA2G4A in gastric tumors compared to in non-cancerous tissues, as opposite to the increased expression of COX-2. PLA2G4A was significantly associated with tumor size (P = 0.003), tumor grade (P < 0.001), intestinal type (P = 0.003), T classification (P < 0.001), N classification (P < 0.001), and thereby TNM stage (P < 0.001). PLA2G4A and COX-2 expression were both identified as independent prognostic factors in multivariate Cox model analysis (P = 0.024 for PLA2G4A and P < 0.001 for COX-2). Moreover, the reduced PLA2G4A and increased COX-2 expression was both associated with unfavorable survival for patients with gastric cancer. PLA2G4A might serve as a promising target for future therapeutic approaches to gastric cancer combined with COX-2 inhibitors.
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Biomarcadores de Tumor/análisis , Fosfolipasas A2 Grupo IV/biosíntesis , Neoplasias Gástricas/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Ciclooxigenasa 2/análisis , Ciclooxigenasa 2/biosíntesis , Femenino , Fosfolipasas A2 Grupo IV/análisis , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Análisis de Matrices Tisulares , Adulto JovenRESUMEN
AIM OF THE STUDY: Botulinum toxin A (BoNT/A) has been recently used in the treatment of benign prostatic hyperplasia due to its apoptotic activity on prostatic epithelium but few data exist on this issue in prostate cancer. Also no information exist on the eventual modulation exerted by the neurotoxin on Phospholipase A2 (PLA2) expression in prostate cancer. The aim of this study was to evaluate the activity of BoNT/A on cell growth and expression of PLA2 in prostate cancer lines. MATERIALS AND METHODS: PC-3 and LNCaP cell lines were exposed to BoNT/A (Xeomin®), different doses and time of exposure. Presence of SV2 receptors (SV2-A and SV2-B) for the neurotoxin was also investigated. The expression of P-Ser505-cPLA2-α (phosphorylated enzyme) was performed immunofluorescence. RESULTS: After 96 hours of BoNT/A administration a 20% reduction of cell growth in LNCaP and 25% in PC-3 were observed. SV-2 receptors were expressed in both cell lines. No cPLA2-α total expression was found in LnCaP. In PC-3 there was a high expression of cPLA2-α total which was not modified after BoNT/A treatment. In both LNCap and PC-3 the expression of P-Ser505-cPLA2-α (phosphorylated enzyme) increases significantly after treatment with [10 U/ml] of BoNT/A. CONCLUSIONS: LNCaP and PC-3 cell lines are sensitive to treatment with BoNT/A which probably enters the cells by SV2 receptors. The increase in the phosphorylated form of cPLA2-a, induced by BoNT/A may represent one mechanism by which the toxin reduces cell growth and proliferation.
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Adenocarcinoma/patología , Toxinas Botulínicas Tipo A/farmacología , Neoplasias de la Próstata/patología , Adenocarcinoma/enzimología , Andrógenos , División Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Ensayos de Selección de Medicamentos Antitumorales , Inducción Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Fosfolipasas A2 Grupo IV/análisis , Fosfolipasas A2 Grupo IV/biosíntesis , Fosfolipasas A2 Grupo IV/genética , Humanos , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/patología , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/enzimología , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA2 (phospholipase A2) [also known as cPLA2γ (cytosolic PLA2γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA2γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA2γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)-c-Jun, p65-p65 and USF (upstream stimulating factor) 1-USF2 complexes are recruited to the cPLA2γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA2γ gene expression with allergic asthma and outline a complex regulatory mechanism.
Asunto(s)
Aspergillus fumigatus/inmunología , Asma/genética , Fosfolipasas A2 Grupo IV/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Factor de Transcripción Activador 2/metabolismo , Animales , Asma/inmunología , Línea Celular , Inducción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción/biosíntesis , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
The activation of cytosolic phospholipase A(2)α (cPLA(2)α) plays an important role in initiating the inflammatory response. The regulation of cPLA(2)α mRNA turnover has been proposed to control cPLA(2)α gene expression under cytokine and growth factor stimulation. However, the detailed mechanism is still unknown. In this report, we have demonstrated that the cPLA(2)α mRNA stability was increased under IL-1ß treatment in A549 cells. By using EMSAs, HuR was identified as binding with the cPLA(2)α mRNA 3'-UTR, and the binding region was located at nucleotides 2716-2807, a fragment containing AUUUA flanked by U-rich sequences. IL-1ß treatment enhanced the association of cPLA(2)α mRNA with cytosolic HuR. The reduction of HuR expression by RNA interference technology inhibited IL-1ß-induced cPLA(2)α mRNA and protein expression. Furthermore, blocking the p38 MAPK signaling pathway with SB203580 abolished the effect of IL-1ß-induced cPLA(2)α gene expression. Phosphorylation at residue Thr-118 of HuR is crucial in regulating the interaction between HuR and its target mRNAs. Mutation of HuR Thr-118 reduced the association between HuR and cPLA(2)α mRNA under IL-1ß treatment. This inhibitory effect was also observed in binding with COX-2 mRNA. This result indicated that p38 MAPK-mediated Thr-118 phosphorylation may play a key role in regulating the interaction of HuR with its target mRNAs in inflammation.
Asunto(s)
Regiones no Traducidas 3' , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas ELAV/metabolismo , Fosfolipasas A2 Grupo IV/biosíntesis , Interleucina-1beta/farmacología , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Neoplásico/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Proteínas ELAV/genética , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Fosfolipasas A2 Grupo IV/genética , Humanos , Imidazoles/farmacología , Inflamación/genética , Inflamación/mortalidad , Neoplasias Pulmonares/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Mutación , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Piridinas/farmacología , ARN Neoplásico/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Phospholipids play an important role in mediating cell migration. In the present study, we investigated the role of cPLA(2)γ in chemotaxis of human breast cancer cells. Inhibition of cPLA(2)γ expression by small interference RNA severely inhibits EGF-induced chemotaxis in a dose-dependent manner in MDA-MB-231, MCF-7, T47D and ZR-75-30 cells. Furthermore, silencing cPLA(2)γ expression also impaired directional migration, adhesion and invasion in MDA-MB-231 cells. In addition, we investigated the molecular mechanism by which cPLA(2)γ regulated migration. Knockdown of cPLA(2)γ suppressed the phosphorylation of Akt at both Thr308 and Ser473. Phosphorylation of PKCζ, downstream of Akt, was also dampened. Knockdown of cPLA(2)γ also impaired the phosphorylation of integrin ß1 and cofilin, key regulators of cell adhesion and actin polymerization, respectively. Taken together, our results suggest that cPLA(2)γ plays an important role in cancer cell chemotaxis.
Asunto(s)
Neoplasias de la Mama/patología , Quimiotaxis , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quimiotaxis/genética , Regulación hacia Abajo , Factor de Crecimiento Epidérmico/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Fosfolipasas A2 Grupo IV/biosíntesis , Fosfolipasas A2 Grupo IV/genética , Humanos , Fosforilación/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
The eicosanoid pathway is activated in many types of cancers including prostate. Eicosanoids are synthesized from intracellular arachidonic acid (AA), which is released from membrane glycerophospholipids mainly by the action of cytosolic phospholipase A(2)alpha (cPLA(2)alpha). Thus, targeting cPLA(2)alpha has been proposed as a treatment option. The aim of this study was to determine the effect of cPLA(2)alpha inhibition on cyclooxygenase (COX) expression and PGE(2) production. Inhibition of cPLA(2)alpha expression by siRNA or activity by Efipladib in prostate cancer cell lines (PC3 and LNCaP) led to an increase in COX-1 protein and PGE(2) levels in a dose-dependent manner from 24 to 72 h. The COX-2 response was less evident. Efipladib treatment increased COX-1 promoter transcriptional activity without changing the rate of COX-1 protein degradation. Treatment with Efipladib also led to a decrease in most LOX products (HETEs) as measured by LC/MS/MS. Replenishing 5- and 12-HETEs abolished Efipladib-induced COX-1 and PGE(2) levels. Decreasing 5- and 12-HETE production, as a result of treating cells with inhibitors MK886 and Baicalein, respectively, mimicked the effect of Efipladib on COX-1 and PGE(2) levels. Hence, the mechanism underlying the cPLA(2)alpha inhibition-induced COX-1 is likely due to a decrease in LOX products, which may exert a negative feedback on COX-1 gene expression in prostate cancer cells. Considering that PGE(2) is a potent promoter of cancer cell proliferation and survival, understanding the mechanism coupling cPLA(2)alpha with COX-1 is of potential clinical significance.
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Ácido Araquidónico/metabolismo , Ciclooxigenasa 1/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Fosfolipasas A2 Grupo IV/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Próstata/enzimología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biosíntesis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Humanos , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Masculino , Proteínas de Neoplasias/antagonistas & inhibidores , Regiones Promotoras Genéticas , Neoplasias de la Próstata/tratamiento farmacológico , Transcripción Genética/efectos de los fármacosRESUMEN
MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of gene expression during development. This study was performed to determine gestational age-dependent changes in miRNA expression in the chorioamniotic membranes and to assess the significance of miRNAs in human pregnancy and parturition. The expression profile of 455 miRNAs was compared between patients at term without labour (TNL: n = 10), in labour (TL: n = 10), and preterm labour (PTL: n = 10) using microarrays. A total of 39 miRNAs were differentially expressed between term and preterm cases, of which 31 (79.5%) were down-regulated at term. Expression of ten miRNAs, including miR-338, differentially expressed between PTL and TL groups was decreased at term. Computational analyses using miRBase Targets have identified PLA2G4B, a phospholipase implicated in parturition, as a putative target of miR-338. Inhibition of endogenous miR-338 with anti-miR-338 increased the mRNA and protein expression of PLA2G4B in decidual cells. Luciferase assay with reporter constructs confirmed that the suppression of PLA2G4B occurs through binding of miR-338 to the 3UTR of PLA2G4B. Interestingly, the expression of Dicer, a key miRNA-processing enzyme, was markedly decreased at term, particularly with labour in the chorioamniotic membranes. Collectively, the novel findings reported herein strongly suggest that post-transcriptional regulation of genes by miRNAs, coupled with the changes of miRNA processing machinery in the chorioamniotic membranes, plays a role in pregnancy and parturition. Furthermore, the expression level of Dicer in the chorioamniotic membranes dichotomizes pathological preterm labour and physiological spontaneous labour at term.
Asunto(s)
Amnios/metabolismo , Corion/metabolismo , MicroARNs/metabolismo , Embarazo/genética , Adolescente , Adulto , Secuencia de Bases , Peso al Nacer , Decidua/metabolismo , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica/métodos , Edad Gestacional , Fosfolipasas A2 Grupo IV/biosíntesis , Fosfolipasas A2 Grupo IV/genética , Humanos , Cariotipificación , MicroARNs/fisiología , Datos de Secuencia Molecular , Trabajo de Parto Prematuro/genética , Trabajo de Parto Prematuro/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Parto/genética , Parto/metabolismo , Embarazo/metabolismo , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Ribonucleasa III/metabolismo , Adulto JovenRESUMEN
Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) plays a critical role in inflammatory disorders including experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Although PAF accumulation in the spinal cord (SC) of EAE mice and cerebrospinal fluid of MS patients has been reported, little is known about the metabolic processing of PAF in these diseases. In this study, we demonstrate that the activities of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase (LysoPAFAT) are elevated in the SC of EAE mice on a C57BL/6 genetic background compared with those of naive mice and correlate with disease severity. Correspondingly, levels of groups IVA, IVB, and IVF cytosolic PLA(2)s, group V secretory PLA(2), and LysoPAFAT transcripts are up-regulated in the SC of EAE mice. PAF acetylhydrolase activity is unchanged during the disease course. In addition, we show that LysoPAFAT mRNA and protein are predominantly expressed in microglia. Considering the substrate specificity and involvement of PAF production, group IVA cytosolic PLA(2) is likely to be responsible for the increased PLA(2) activity. These data suggest that PAF accumulation in the SC of EAE mice is profoundly dependent on the group IVA cytosolic PLA(2)/LysoPAFAT axis present in the infiltrating macrophages and activated microglia.