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1.
Nature ; 569(7755): 270-274, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31043744

RESUMEN

Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8, there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc-, impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc- was negatively associated, in cancer patients, with CD8+ T cell signature, IFNγ expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ferroptosis , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Antígeno B7-H1/antagonistas & inhibidores , Línea Celular Tumoral , Cisteína/metabolismo , Femenino , Ferroptosis/efectos de los fármacos , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Humanos , Interferón gamma/inmunología , Peroxidación de Lípido , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/terapia , Ratones , Neoplasias/metabolismo , Nivolumab/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Resultado del Tratamiento
2.
J Lipid Res ; 59(3): 542-549, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29353239

RESUMEN

The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B2, PGE2, and PGD2, and the 5-lipoxygenase pathway, leukotriene (LT)B4, LTC4, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB3, all trans-LTB3, LTC3, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.


Asunto(s)
Técnicas de Cultivo de Célula , Eicosanoides/biosíntesis , Fosfolípidos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Eicosanoides/análisis , Ratones , Fosfolípidos/análisis , Células RAW 264.7 , Espectrometría de Masas en Tándem , Factor de Necrosis Tumoral alfa/biosíntesis
3.
J Immunol ; 196(2): 891-901, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26663781

RESUMEN

Eicosanoids, including PGs, produced by cyclooxygenases (COX), and leukotrienes, produced by 5-lipoxygenase (5-LO) have been implicated in cancer progression. These molecules are produced by both cancer cells and the tumor microenvironment (TME). We previously reported that both COX and 5-LO metabolites increase during progression in an orthotopic immunocompetent model of lung cancer. Although PGs in the TME have been well studied, less is known regarding 5-LO products produced by the TME. We examined the role of 5-LO in the TME using a model in which Lewis lung carcinoma cells are directly implanted into the lungs of syngeneic WT mice or mice globally deficient in 5-LO (5-LO-KO). Unexpectedly, primary tumor volume and liver metastases were increased in 5-LO-KO mice. This was associated with an ablation of leukotriene (LT) production, consistent with production mainly mediated by the microenvironment. Increased tumor progression was partially reproduced in global LTC4 synthase KO or mice transplanted with LTA4 hydrolase-deficient bone marrow. Tumor-bearing lungs of 5-LO-KO had decreased numbers of CD4 and CD8 T cells compared with WT controls, as well as fewer dendritic cells. This was associated with lower levels of CCL20 and CXL9, which have been implicated in dendritic and T cell recruitment. Depletion of CD8 cells increased tumor growth and eliminated the differences between WT and 5-LO mice. These data reveal an antitumorigenic role for 5-LO products in the microenvironment during lung cancer progression through regulation of T cells and suggest that caution should be used in targeting this pathway in lung cancer.


Asunto(s)
Araquidonato 5-Lipooxigenasa/deficiencia , Carcinoma Pulmonar de Lewis/patología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Carcinoma Pulmonar de Lewis/enzimología , Carcinoma Pulmonar de Lewis/inmunología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Citometría de Flujo , Inmunohistoquímica , Neoplasias Pulmonares/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica/inmunología , Invasividad Neoplásica/patología , Trasplante de Neoplasias , Reacción en Cadena en Tiempo Real de la Polimerasa
4.
J Lipid Res ; 58(6): 1174-1185, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28416579

RESUMEN

Acyl-CoA thioesterase 7 (ACOT7) is an intracellular enzyme that converts acyl-CoAs to FFAs. ACOT7 is induced by lipopolysaccharide (LPS); thus, we investigated downstream effects of LPS-induced induction of ACOT7 and its role in inflammatory settings in myeloid cells. Enzymatic thioesterase activity assays in WT and ACOT7-deficient macrophage lysates indicated that endogenous ACOT7 contributes a significant fraction of total acyl-CoA thioesterase activity toward C20:4-, C20:5-, and C22:6-CoA, but contributes little activity toward shorter acyl-CoA species. Lipidomic analyses revealed that LPS causes a dramatic increase, primarily in bis(monoacylglycero)phosphate species containing long (≥C20) polyunsaturated acyl-chains in macrophages, and that the limited effect observed by ACOT7 deficiency is restricted to glycerophospholipids containing 20-carbon unsaturated acyl-chains. Furthermore, ACOT7 deficiency did not detectably alter the ability of LPS to induce cytokines or prostaglandin E2 production in macrophages. Consistently, although ACOT7 was induced in macrophages from diabetic mice, hematopoietic ACOT7 deficiency did not alter the stimulatory effect of diabetes on systemic inflammation or atherosclerosis in LDL receptor-deficient mice. Thus, inflammatory stimuli induce ACOT7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages, and, although ACOT7 has preferential thioesterase activity toward these lipid species, loss of ACOT7 has no major detrimental effect on macrophage inflammatory phenotypes.≥.


Asunto(s)
Macrófagos/metabolismo , Palmitoil-CoA Hidrolasa/biosíntesis , Fosfolípidos/metabolismo , Animales , Citocinas/biosíntesis , Dinoprostona/metabolismo , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glicerofosfolípidos/metabolismo , Inflamación/enzimología , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Palmitoil-CoA Hidrolasa/deficiencia , Palmitoil-CoA Hidrolasa/genética , Palmitoil-CoA Hidrolasa/metabolismo
5.
Int J Mass Spectrom ; 378: 255-263, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25883529

RESUMEN

The tandem mass spectrometry, ion mobility, and normal phase HPLC of isomeric phosphatidylglycerol (PG) and bis(monoacylglycerol)phosphate (BMP) have been investigated in this study with the objective of differentiating these unique classes of lipids. Measurement of ion mobility using the traveling wave method for negative molecular and product ions from isomeric PG and BMP yielded identical results, but different ion mobilities were observed for positive product ions arising from collision-induced dissociation (CID). The fastest moving positive product ions from the ion mobility analysis of BMP(18:1/18:1) were monoglyceride-like, and the slowest moving product ions from this BMP corresponded to [M+H-2H2O]+, which were readily observed for BMP but were only at very low abundance in the CID spectra of PG. The major product ions observed from the sodium adduct of PG(18:1/18:1) were consistent with diglyceride-like ion formation, but for BMP(18:1/18:1) only monoglyceride-like product ions were formed. The usefulness of ion mobility separation was tested with the selection of positive product ions derived from the isomeric PG and BMP molecular species in the lipid extract of RAW 264.7 cells. The ion mobility spectra of monoglyceride-like ions derived from BMP species with various esterified fatty acyl groups displayed some separation in ion mobility based on fatty acyl chain length and presence of a double bond in the acyl chain. The mechanism of ion formation of the diglyceride- and monoglyceride-like ions from PG and BMP respectively was examined using deuterium-labeled species including PG(D3116:0/18:1) and PG and BMP labeled by deuterium exchange.

6.
J Lipid Res ; 55(4): 782-91, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24563510

RESUMEN

Lysophospholipid acyltransferases (LPATs) incorporate fatty acyl chains into phospholipids via a CoA-dependent mechanism and are important in remodeling phospholipids to generate the molecular species of phospholipids found in cells. These enzymes use one lysophospholipid and one acyl-CoA ester as substrates. Traditional enzyme activity assays engage a single substrate pair, whereas in vivo multiple molecular species exist. We describe here an alternative biochemical assay that provides a mixture of substrates presented to the microsomal extracts. Microsomal preparations from RAW 264.7 cells were used to compare traditional LPAT assays with data obtained using a dual substrate choice assay using six different lysophospholipids and eight different acyl-CoA esters. The complex mixture of newly synthesized phospholipid products was analyzed using LC-MS/MS. Both types of assays provided similar results, but the dual choice assay provided information about multiple fatty acyl chain incorporation into various phospholipid classes in a single reaction. Engineered suppression of LPCAT3 activity in RAW 264.7 cells was easily detected by the dual choice method. These findings demonstrate that this assay is both specific and sensitive and that it provides much richer biochemical detail than traditional assays.


Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/química , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Animales , Unión Competitiva , Pruebas de Enzimas , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Lisofosfolípidos/química , Ratones , Microsomas/enzimología
7.
Prostaglandins Other Lipid Mediat ; 113-115: 52-61, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25175316

RESUMEN

Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation-reacylation cycle from a well-established, versatile vertebrate model species.


Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Eicosanoides/biosíntesis , Células Mieloides/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Animales , Calcimicina/administración & dosificación , Ionóforos de Calcio/administración & dosificación , Lisofosfolípidos/metabolismo , Modelos Animales , Células Mieloides/enzimología , Espectrometría de Masa por Ionización de Electrospray , Pez Cebra
8.
JCI Insight ; 2024 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-39287988

RESUMEN

End stage liver disease is marked by portal hypertension, systemic elevations in ammonia, and development of hepatocellular carcinoma (HCC). While these clinical consequences of cirrhosis are well described, it remains poorly understood whether hepatic insufficiency and the accompanying elevations in ammonia contribute to HCC carcinogenesis. Using preclinical models, we discovered that ammonia entered the cell through the transporter SLC4A11 and served as a nitrogen source for amino acid and nucleotide biosynthesis. Elevated ammonia promoted cancer stem cell properties in vitro and tumor initiation in vivo. Enhancing ammonia clearance reduced HCC stemness and tumor growth. In patients, elevations in serum ammonia were associated with an increased incidence of HCC. Taken together, this study forms the foundation for clinical investigations using ammonia lowering agents as potential therapies to mitigate HCC incidence and aggressiveness.

9.
Proc Natl Acad Sci U S A ; 106(20): 8296-301, 2009 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-19416808

RESUMEN

Leukotrienes (LTs) are lipid mediators of inflammation formed by enzymatic oxidation of arachidonic acid. One intriguing aspect of LT production is transcellular biosynthesis: cells expressing 5-lipoxygenase (5LO) form LTA(4) and transfer it to cells expressing LTA(4) hydrolase (LTA(4)H) or LTC(4) synthase (LTC(4)S) to produce LTB(4) or LTC(4). This process has been demonstrated in vivo for LTB(4), but not for cysteinyl LTs (cysLTs). We examined transcellular cysLT synthesis during zymosan-induced peritonitis, using bone marrow transplants with transgenic mice deficient in key enzymes of LT synthesis and analyzing all eicosanoids by liquid chromatography/tandem mass spectrometry. WT mice time-dependently produced LTB(4) and cysLTs (LTC(4), LTD(4), and LTE(4)). 5LO(-/-) mice were incapable of producing LTs. WT bone marrow cells restored this biosynthetic ability, but 5LO(-/-) bone marrow did not rescue LT synthesis in irradiated WT mice, demonstrating that bone marrow-derived cells are the ultimate source of all LTs in this model. Total levels of 5LO-derived products were comparable in LTA(4)H(-/-) and WT mice, but were reduced in LTC(4)S(-/-) animals. No differences in prostaglandin production were observed between these transgenic or chimeric mice. Bone marrow cells from LTA(4)H(-/-) or LTC(4)S(-/-) mice injected into 5LO(-/-) mice restored the ability to synthesize LTB(4) and cysLTs, providing unequivocal evidence of efficient transcellular biosynthesis of cysLTs. These results highlight the potential relevance of transcellular exchange of LTA(4) for the synthesis of LTs mediating biological activities during inflammatory events in vivo.


Asunto(s)
Médula Ósea/metabolismo , Comunicación Celular , Cisteína/biosíntesis , Leucotrienos/biosíntesis , Peritonitis/metabolismo , Animales , Araquidonato 5-Lipooxigenasa , Trasplante de Médula Ósea , Enzimas/deficiencia , Inflamación/metabolismo , Mediadores de Inflamación , Redes y Vías Metabólicas , Ratones , Ratones Transgénicos , Peritonitis/inducido químicamente , Peritonitis/patología , Zimosan
10.
Cancer Cell ; 40(4): 365-378.e6, 2022 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-35216678

RESUMEN

Tumor cell intrinsic ferroptosis-initiating mechanisms are unknown. Here, we discover that T cell-derived interferon (IFN)γ in combination with arachidonic acid (AA) induces immunogenic tumor ferroptosis, serving as a mode of action for CD8+ T cell (CTL)-mediated tumor killing. Mechanistically, IFNγ stimulates ACSL4 and alters tumor cell lipid pattern, thereby increasing incorporations of AA into C16 and C18 acyl chain-containing phospholipids. Palmitoleic acid and oleic acid, two common C16 and C18 fatty acids in blood, promote ACSL4-dependent tumor ferroptosis induced by IFNγ plus AA. Moreover, tumor ACSL4 deficiency accelerates tumor progression. Low-dose AA enhances tumor ferroptosis and elevates spontaneous and immune checkpoint blockade (ICB)-induced anti-tumor immunity. Clinically, tumor ACSL4 correlates with T cell signatures and improved survival in ICB-treated cancer patients. Thus, IFNγ signaling paired with selective fatty acids is a natural tumor ferroptosis-promoting mechanism and a mode of action of CTLs. Targeting the ACSL4 pathway is a potential anti-cancer approach.


Asunto(s)
Ferroptosis , Neoplasias , Linfocitos T CD8-positivos/metabolismo , Coenzima A Ligasas/metabolismo , Ácidos Grasos , Humanos
11.
J Lipid Res ; 52(8): 1551-60, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21508254

RESUMEN

Lipid mediators are important in lung biochemistry and are derived from the enzymatic oxidation of arachidonic and docosahexaenoic acids, which are PUFAs that are present in phospholipids in cell membranes. In this study, MALDI imaging MS was used to determine the localization of arachidonate- and docosahexaenoate-containing phospholipids in mouse lung. These PUFA-containing phospholipids were determined to be uniquely abundant at the lining of small and large airways, which were unequivocally identified by immunohistochemistry. In addition, it was found that the blood vessels present in the lung were characterized by sphingomyelin molecular species, and lung surfactant phospholipids appeared evenly distributed throughout the lung parenchyma, indicating alveolar localization. This technique revealed unexpected high concentrations of arachidonate- and docosahexaenoate-containing phospholipids lining the airways in pulmonary tissue, which could serve as precursors of lipid mediators affecting airways biology.


Asunto(s)
Vasos Sanguíneos/química , Metabolismo de los Lípidos , Pulmón/química , Fosfolípidos/análisis , Esfingomielinas/análisis , Animales , Ácido Araquidónico/análisis , Ácido Araquidónico/metabolismo , Vasos Sanguíneos/metabolismo , Ácidos Docosahexaenoicos/análisis , Ácidos Docosahexaenoicos/metabolismo , Técnica del Anticuerpo Fluorescente , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfolípidos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Esfingomielinas/metabolismo
12.
J Leukoc Biol ; 84(3): 798-806, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18550790

RESUMEN

In resident mouse peritoneal macrophages, group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) mediates arachidonic acid (AA) release and eicosanoid production in response to diverse agonists such as A23187, phorbol myristate acetate, zymosan, and the enterotoxin, okadaic acid (OA). cPLA(2)alpha is regulated by phosphorylation and by calcium that binds to the C2 domain and induces translocation from the cytosol to membranes. In contrast, OA activates cPLA(2)alpha-induced AA release and translocation to the Golgi in macrophages without an apparent increase in calcium. Inhibitors of heat shock protein 90 (hsp90), geldanamycin, and herbimycin blocked AA release in response to OA but not to A23187, PMA, or zymosan. OA, but not the other agonists, induced activation of a cytosolic serine/threonine 54-kDa kinase (p54), which phosphorylated cPLA(2)alpha in in-gel kinase assays and was associated with cPLA(2)alpha in immunoprecipitates. Activation of the p54 kinase was inhibited by geldanamycin. The kinase coimmunoprecipitated with hsp90 in unstimulated macrophages, and OA induced its loss from hsp90, concomitant with its association with cPLA(2)alpha. The results demonstrate a role for hsp90 in regulating cPLA(2)alpha-mediated AA release that involves association of a p54 kinase with cPLA(2)alpha upon OA stimulation.


Asunto(s)
Fosfolipasas A2 Grupo IV/metabolismo , Proteínas HSP90 de Choque Térmico/fisiología , Macrófagos Peritoneales/efectos de los fármacos , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Ácido Ocadaico/farmacología , Animales , Ácido Araquidónico/metabolismo , Benzoquinonas/farmacología , Calcimicina/farmacología , Calcio/metabolismo , Carcinógenos/farmacología , Citosol/enzimología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Immunoblotting , Inmunoprecipitación , Ionóforos/farmacología , Lactamas Macrocíclicas/farmacología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos ICR , Células 3T3 NIH , Ácidos Fosfoaminos/metabolismo , Fosforilación/efectos de los fármacos , Transporte de Proteínas , Rifabutina/análogos & derivados , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Zimosan/farmacología
13.
Mol Biol Cell ; 17(1): 155-62, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16221889

RESUMEN

The eicosanoids are centrally involved in the onset and resolution of inflammatory processes. A key enzyme in eicosanoid biosynthesis during inflammation is group IVA phospholipase A2 (also known as cytosolic phospholipase A2alpha, cPLA2alpha). This enzyme is responsible for generating free arachidonic acid from membrane phospholipids. cPLA2alpha translocates to perinuclear membranes shortly after cell activation, in a process that is governed by the increased availability of intracellular Ca2+. However, cPLA2alpha also catalyzes membrane phospholipid hydrolysis in response to agonists that do not mobilize intracellular Ca2+. How cPLA2alpha interacts with membranes under these conditions is a major, still unresolved issue. Here, we report that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] promotes translocation of cPLA2alpha to perinuclear membranes of intact cells in a manner that is independent of rises in the intracellular Ca2+ concentration. PtdIns(4,5)P2 anchors the enzyme to perinuclear membranes and allows for a proper interaction with its phospholipid substrate to release arachidonic acid.


Asunto(s)
Calcio/metabolismo , Citosol/enzimología , Hígado/enzimología , Membrana Nuclear/enzimología , Fosfatidilinositol 4,5-Difosfato/farmacología , Fosfolipasas A/metabolismo , Animales , Ácido Araquidónico/metabolismo , Calcio/farmacología , Línea Celular , Supervivencia Celular , Fosfolipasas A2 Grupo IV , Humanos , Hígado/efectos de los fármacos , Ratones , Mutación/genética , Membrana Nuclear/efectos de los fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipasas A/genética , Fosfolipasas A2 , Transporte de Proteínas
14.
Methods Mol Biol ; 1978: 81-105, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31119658

RESUMEN

Mass spectrometry has played a critical role in the identification and quantitation of lipids present in biological extracts. Various strategies have emerged in order to carry out lipidomic studies. These include both shotgun approaches as well as those engaging liquid chromatographic separation of lipid species prior to mass spectrometric analysis. Nonetheless challenges remain at every level of the lipidomic experiment, including extraction of lipids, identification of specific species, and quantitation of the vast array of lipids present in the sample extract. New strategies have emerged to address some of these issues; however, precise quantitation remains a significant challenge. The use of the ratio of the abundance of the molecular ion species to that of an internal standard enables quite accurate assessment of fold changes within complex lipid species without the need for exact quantitation. Challenges continue to remain in terms of availability of reference standard material as well as relevant internal standards.


Asunto(s)
Metabolismo de los Lípidos , Espectrometría de Masas/métodos , Metabolómica/métodos , Cromatografía Liquida , Humanos , Lípidos/química , Espectrometría de Masa por Ionización de Electrospray
15.
Biochem J ; 405(3): 379-95, 2007 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-17623009

RESUMEN

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.


Asunto(s)
Ácido Araquidónico/metabolismo , Leucotrienos/biosíntesis , Lipooxigenasa/química , Lipooxigenasa/metabolismo , Estructura Molecular , Conformación Proteica
16.
Sci Rep ; 7(1): 10981, 2017 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-28887514

RESUMEN

The differentiation of resident tissue macrophages from embryonic precursors and that of inflammatory macrophages from bone marrow cells leads to macrophage heterogeneity. Further plasticity is displayed through their ability to be polarized as subtypes M1 and M2 in a cell culture microenvironment. However, the detailed regulation of eicosanoid production and its involvement in macrophage biology remains unclear. Using a lipidomics approach, we demonstrated that eicosanoid production profiles between bone marrow-derived (BMDM) and peritoneal macrophages differed drastically. In polarized BMDMs, M1 and M2 phenotypes were distinguished by thromboxane B2, prostaglandin (PG) E2, and PGD2 production, in addition to lysophospholipid acyltransferase activity. Although Alox5 expression and the presence of 5-lipoxygenase (5-LO) protein in BMDMs was observed, the absence of leukotrienes production reflected an impairment in 5-LO activity, which could be triggered by addition of exogenous arachidonic acid (AA). The BMDM 5-LO regulatory mechanism was not responsive to PGE2/cAMP pathway modulation; however, treatment to reduce glutathione peroxidase activity increased 5-LO metabolite production after AA stimulation. Understanding the relationship between the eicosanoids pathway and macrophage biology may offer novel strategies for macrophage-associated disease therapy.


Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Ácido Araquidónico/metabolismo , Macrófagos/metabolismo , Animales , Ácido Araquidónico/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , AMP Cíclico , Eicosanoides/metabolismo , Eicosanoides/farmacología , Femenino , Regulación de la Expresión Génica , Lipopolisacáridos/inmunología , Lipooxigenasa/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Fosfolípidos/metabolismo , Transducción de Señal , Espectrometría de Masas en Tándem
17.
Oncotarget ; 8(34): 56051-56065, 2017 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-28915573

RESUMEN

Prostate cancer (PCa) is the most common malignancy among Western men and the second leading-cause of cancer related deaths. For men who develop metastatic castration resistant PCa (mCRPC), survival is limited, making the identification of novel therapies for mCRPC critical. We have found that deficient lipid oxidation via carnitine palmitoyltransferase (CPT1) results in decreased growth and invasion, underscoring the role of lipid oxidation to fuel PCa growth. Using immunohistochemistry we have found that the CPT1A isoform is abundant in PCa compared to benign tissue (n=39, p<0.001) especially in those with high-grade tumors. Since lipid oxidation is stimulated by androgens, we have evaluated the synergistic effects of combining CPT1A inhibition and anti-androgen therapy. Mechanistically, we have found that decreased CPT1A expression is associated with decreased AKT content and activation, likely driven by a breakdown of membrane phospholipids and activation of the INPP5K phosphatase. This results in increased androgen receptor (AR) action and increased sensitivity to the anti-androgen enzalutamide. To better understand the clinical implications of these findings, we have evaluated fat oxidation inhibitors (etomoxir, ranolazine and perhexiline) in combination with enzalutamide in PCa cell models. We have observed a robust growth inhibitory effect of the combinations, including in enzalutamide-resistant cells and mouse TRAMPC1 cells, a more neuroendocrine PCa model. Lastly, using a xenograft mouse model, we have observed decreased tumor growth with a systemic combination treatment of enzalutamide and ranolazine. In conclusion, our results show that improved anti-cancer efficacy can be achieved by co-targeting the AR axis and fat oxidation via CPT1A, which may have clinical implications, especially in the mCRPC setting.

18.
Front Cell Neurosci ; 8: 374, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25426024

RESUMEN

Management of lipids, particularly signaling lipids that control neuroinflammation, is crucial for the regeneration capability of a damaged nervous system. Knowledge of pro- and anti-inflammatory signals after nervous system injury is extensive, most of them being proteins acting through well-known receptors and intracellular cascades. However, the role of lipid binding extracellular proteins able to modify the fate of lipids released after injury is not well understood. Apolipoprotein D (ApoD) is an extracellular lipid binding protein of the Lipocalin family induced upon nervous system injury. Our previous study shows that axon regeneration is delayed without ApoD, and suggests its participation in early events during Wallerian degeneration. Here we demonstrate that ApoD is expressed by myelinating and non-myelinating Schwann cells and is induced early upon nerve injury. We show that ApoD, known to bind arachidonic acid (AA), also interacts with lysophosphatidylcholine (LPC) in vitro. We use an in vivo model of nerve crush injury, a nerve explant injury model, and cultured macrophages exposed to purified myelin, to uncover that: (i) ApoD regulates denervated Schwann cell-macrophage signaling, dampening MCP1- and Tnf-dependent macrophage recruitment and activation upon injury; (ii) ApoD controls the over-expression of the phagocytosis activator Galectin-3 by infiltrated macrophages; (iii) ApoD controls the basal and injury-triggered levels of LPC and AA; (iv) ApoD modifies the dynamics of myelin-macrophage interaction, favoring the initiation of phagocytosis and promoting myelin degradation. Regulation of macrophage behavior by Schwann-derived ApoD is therefore a key mechanism conditioning nerve injury resolution. These results place ApoD as a lipid binding protein controlling the signals exchanged between glia, neurons and blood-borne cells during nerve recovery after injury, and open the possibility for a therapeutic use of ApoD as a regeneration-promoting agent.

19.
Mol Cancer Ther ; 13(10): 2361-71, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25122071

RESUMEN

Prostate cancer is the most commonly diagnosed malignancy among Western men and accounts for the second leading cause of cancer-related deaths. Prostate cancer tends to grow slowly and recent studies suggest that it relies on lipid fuel more than on aerobic glycolysis. However, the biochemical mechanisms governing the relationships between lipid synthesis, lipid utilization, and cancer growth remain unknown. To address the role of lipid metabolism in prostate cancer, we have used etomoxir and orlistat, clinically safe drugs that block lipid oxidation and lipid synthesis/lipolysis, respectively. Etomoxir is an irreversible inhibitor of the carnitine palmitoyltransferase (CPT1) enzyme that decreases ß oxidation in the mitochondria. Combinatorial treatments using etomoxir and orlistat resulted in synergistic decreased viability in LNCaP, VCaP, and patient-derived benign and prostate cancer cells. These effects were associated with decreased androgen receptor expression, decreased mTOR signaling, and increased caspase-3 activation. Knockdown of CPT1A enzyme in LNCaP cells resulted in decreased palmitate oxidation but increased sensitivity to etomoxir, with inactivation of AKT kinase and activation of caspase-3. Systemic treatment with etomoxir in nude mice resulted in decreased xenograft growth over 21 days, underscoring the therapeutic potential of blocking lipid catabolism to decrease prostate cancer tumor growth.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Compuestos Epoxi/farmacología , Lactonas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/administración & dosificación , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacología , Lactonas/administración & dosificación , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Metabolismo , Ratones , Ratones Desnudos , Orlistat , Oxidación-Reducción/efectos de los fármacos , Neoplasias de la Próstata/patología , Distribución Aleatoria , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto
20.
PLoS One ; 8(11): e79633, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24244531

RESUMEN

Eicosanoids are bioactive lipid mediators derived from arachidonic acid(1) (AA), which is released by cytosolic phospholipase A2 (cPLA2). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA2 knockout vs wild-type mice, we demonstrated that prostaglandins (PGE2, PGD2 and PGF2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB4, LTC4, LTD4, LTE4) production required cPLA2 expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.


Asunto(s)
Eicosanoides/metabolismo , Leucotrienos/metabolismo , Neoplasias Pulmonares/metabolismo , Microambiente Tumoral , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Eliminación de Gen , Fosfolipasas A2 Grupo IV/genética , Humanos , Inflamación/metabolismo , Inflamación/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Redes y Vías Metabólicas , Metabolómica/métodos , Ratones , Ratones Noqueados , Microambiente Tumoral/genética
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