RESUMEN
Oxysterols play significant roles in many physiological and pathological processes including cancer. They modulate some of the cancer hallmarks pathways, influence the efficacy of anti-cancer drugs, and associate with patient survival. In this study, we aimed to analyze the role of 7-ketocholesterol (7-KC) in breast carcinoma cells and its potential modulation of the tamoxifen effect. 7-KC effects were studied in two estrogen receptor (ER)-positive (MCF-7 and T47D) and one ER-negative (BT-20) breast cancer cell lines. First, we tested the viability of cells in the presence of 7-KC. Next, we co-incubated cells with tamoxifen and sublethal concentrations of 7-KC. We also tested changes in caspase 3/7 activity, deregulation of the cell cycle, and changes in expression of selected genes/proteins in the presence of tamoxifen, 7-KC, or their combination. Finally, we analyzed the effect of 7-KC on cellular migration and invasion. We found that the presence of 7-KC slightly decreases the efficacy of tamoxifen in MCF-7 cells, while an increased effect of tamoxifen and higher caspase 3/7 activity was observed in the BT-20 cell line. In the T47D cell line, we did not find any modulation of tamoxifen efficacy by the presence of 7-KC. Expression analysis showed the deregulation in CYP1A1 and CYP1B1 with the opposite trend in MCF-7 and BT-20 cells. Moreover, 7-KC increased cellular migration and invasion potential regardless of the ER status. This study shows that 7-KC can modulate tamoxifen efficacy as well as cellular migration and invasion, making 7-KC a promising candidate for future studies.
Asunto(s)
Neoplasias de la Mama , Tamoxifeno , Humanos , Femenino , Tamoxifeno/farmacología , Células MCF-7 , Caspasa 3/genética , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Antineoplásicos Hormonales/farmacologíaRESUMEN
Oxygenated metabolites of cholesterol (oxysterols) have been previously demonstrated to contribute to progression of various cancers and to modulate resistance to breast cancer endocrine therapy in vitro. We measured prognostic roles of circulating levels of seven major oxysterols in the progression of luminal subtype breast carcinoma. Liquid chromatography coupled with tandem mass spectrometry was used for determination of levels of non-esterified 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α,6α-epoxide, cholesterol-5ß,6ß-epoxide, and cholestane-3ß,5α,6ß-triol in plasma samples collected from patients (nâ¯=â¯58) before surgical removal of tumors. Oxysterol levels were then associated with clinical data of patients. All oxysterols except cholesterol-5α,6α-epoxide were detected in patient plasma samples. Circulating levels of 7α-hydroxycholesterol and 27-hydroxycholesterol were significantly lower in patients with small tumors (pT1) and cholesterol-5ß,6ß-epoxide and cholestane-3ß,5α,6ß-triol were lower in patients with stage IA disease compared to larger tumors or more advanced stages. Patients with higher than median cholestane-3ß,5α,6ß-triol levels had significantly worse disease-free survival than patients with lower levels (pâ¯=â¯0.037 for all patients and pâ¯=â¯0.015 for subgroup treated only with tamoxifen). In conclusion, this study shows, for the first time, that circulating levels of oxysterols, especially cholestane-3ß,5α,6ß-triol, may have prognostic roles in patients with luminal subtype breast cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Oxiesteroles/sangre , Neoplasias de la Mama/sangre , Carcinoma Ductal de Mama/sangre , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Tasa de SupervivenciaRESUMEN
Twenty to thirty percent of patients undergoing cardiac surgery develop acute kidney injury (AKI). In mice, inhibition of soluble epoxide hydrolase (sEH) attenuates renal injury following ischemia-reperfusion. We tested the hypothesis that functional variants of EPHX2, encoding sEH, are associated with AKI after cardiac surgery. We genotyped patients in two independent cardiac surgery cohorts for functional EPHX2 polymorphisms, Lys55Arg and Arg287Gln, and determined AKI using Acute Kidney Injury Network criteria. The 287Gln variant was not associated with AKI. In the discovery cohort, the gain-of-function 55Arg variant was associated with an increased incidence of AKI in univariate (p = 0.03) and multivariable (p = 0.04) analyses. In white patients without chronic kidney disease (CKD), the 55Arg variant was independently associated with AKI with an OR of 2.04 (95% CI 0.95-4.42) for 55Arg heterozygotes and 31.53 (1.57-633.19) for homozygotes (p = 0.02), after controlling for age, sex, body mass index, baseline estimated glomerular filtration rate, and use of cardiopulmonary bypass. These findings were replicated in the second cardiac surgery cohort. 12,13- and total- dihydroxyoctadecanoic acids (DiHOME): epoxyoctadecanoic acids (EpOME) ratios were increased in EPHX2 55Arg variant carriers, consistent with increased hydrolase activity. The EPHX2 Lys55Arg polymorphism is associated with AKI following cardiac surgery in patients without preexisting CKD. Pharmacological strategies to decrease sEH activity might decrease postoperative AKI.
Asunto(s)
Lesión Renal Aguda/etiología , Puente de Arteria Coronaria/efectos adversos , Epóxido Hidrolasas/genética , Lisina/genética , Polimorfismo Genético , Lesión Renal Aguda/genética , Lesión Renal Aguda/fisiopatología , Anciano , Estudios de Cohortes , Ácido Eicosapentaenoico/sangre , Ácido Eicosapentaenoico/metabolismo , Epóxido Hidrolasas/química , Femenino , Genotipo , Tasa de Filtración Glomerular , Humanos , Incidencia , Masculino , Persona de Mediana EdadRESUMEN
Male and female homozygous 129/Sv mice carrying four copies of the human cytochrome P450 4A11 gene (CYP4A11) under control of its native promoter (B-129/Sv-4A11(+/+)) develop hypertension (142 ± 8 versus 113 ± 7 mm Hg systolic blood pressure (BP)), and exhibit increased 20-hydroxyeicosatetraenoic acid (20-HETE) in kidney and urine. The hypertension is reversible by a low-sodium diet and by the CYP4A inhibitor HET0016. B-129/Sv-4A11(+/+) mice display an 18% increase of plasma potassium (p < 0.02), but plasma aldosterone, angiotensin II (ANGII), and renin activities are unchanged. This phenotype resembles human genetic disorders with elevated activity of the sodium chloride co-transporter (NCC) and, accordingly, NCC abundance is increased by 50% in transgenic mice, and NCC levels are normalized by HET0016. ANGII is known to increase NCC abundance, and renal mRNA levels of its precursor angiotensinogen are increased 2-fold in B-129/Sv-4A11(+/+), and blockade of the ANGII receptor type 1 with losartan normalizes BP. A pro-hypertensive role for 20-HETE was implicated by normalization of BP and reversal of renal angiotensin mRNA increases by administration of the 20-HETE antagonists 2-((6Z,15Z)-20-hydroxyicosa-6,15-dienamido)acetate or (S)-2-((6Z,15Z)-20-hydroxyicosa-6,15-dienamido)succinate. SGK1 expression is also increased in B-129/Sv-4A11(+/+) mice and paralleled increases seen for NCC. Losartan, HET0016, and 20-HETE antagonists each normalized SGK1 mRNA expression. These results point to a potential 20-HETE dependence of intrarenal angiotensinogen production and ANGII receptor type 1 activation that are associated with increases in NCC and SGK1 and identify elevated P450 4A11 activity and 20-HETE as potential risk factors for salt-sensitive human hypertension by perturbation of the renal renin-angiotensin axis.
Asunto(s)
Presión Sanguínea , Citocromo P-450 CYP4A/biosíntesis , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensión/enzimología , Sistema Renina-Angiotensina , Angiotensinas/genética , Angiotensinas/metabolismo , Animales , Citocromo P-450 CYP4A/genética , Femenino , Humanos , Ácidos Hidroxieicosatetraenoicos/genética , Hipertensión/genética , Losartán/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Cloruro de Sodio Dietético/farmacología , Miembro 3 de la Familia de Transportadores de Soluto 12/biosíntesis , Miembro 3 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (â¼ 44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.
Asunto(s)
Empalme Alternativo/genética , Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP2C8/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Ácido Araquidónico/metabolismo , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Biocatálisis , Células COS , Respiración de la Célula , Chlorocebus aethiops , Simulación por Computador , Citocromo P-450 CYP2C8/química , Citocromo P-450 CYP2C8/metabolismo , Hemo/metabolismo , Células Hep G2 , Humanos , Isoenzimas/metabolismo , Microsomas Hepáticos/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Estrés Oxidativo , Unión Proteica , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Alineación de SecuenciaRESUMEN
Cytochrome P450 (P450) 2U1 has been shown to be expressed, at the mRNA level, in human thymus, brain, and several other tissues. Recombinant P450 2U1 was purified and used as a reagent in a metabolomic search for substrates in bovine brain. In addition to fatty acid oxidation reactions, an oxidation of endogenous N-arachidonoylserotonin was characterized. Subsequent NMR and mass spectrometry and chemical synthesis showed that the main product was the result of C-2 oxidation of the indole ring, in contrast to other human P450s that generated different products. N-Arachidonoylserotonin, first synthesized chemically and described as an inhibitor of fatty acid amide hydrolase, had previously been found in porcine and mouse intestine; we demonstrated its presence in bovine and human brain samples. The product (2-oxo) was 4-fold less active than N-arachidonoylserotonin in inhibiting fatty acid amide hydrolase. The rate of oxidation of N-arachidonoylserotonin was similar to that of arachidonic acid, one of the previously identified fatty acid substrates of P450 2U1. The demonstration of the oxidation of N-arachidonoylserotonin by P450 2U1 suggests a possible role in human brain and possibly other sites.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Oxígeno/metabolismo , Serotonina/análogos & derivados , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Catálisis , Bovinos , Cromatografía Liquida , Familia 2 del Citocromo P450 , Eritrocitos/enzimología , Escherichia coli/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Indoles/metabolismo , Hígado/enzimología , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteómica , Homología de Secuencia de Aminoácido , Serotonina/metabolismoRESUMEN
Epoxyeicosatrienoic acids (EETs) contribute importantly to the regulation of vascular tone and blood pressure control. The purpose of this study was to develop stable EET analogs and test their in vivo blood pressure lowering effects in hypertensive rats. Using the pharmacophoric moiety of EETs, ether EET analogs were designed with improved solubility and resistance to auto-oxidation and metabolism by soluble epoxide hydrolase. Ether EET analogs were chosen based on their ability to dilate afferent arterioles and subsequently tested for blood pressure lowering effects in rodent models of hypertension. Initially, 11,12-ether-EET-8-ZE failed to lower blood pressure in angiotensin hypertension or spontaneously hypertensive rats (SHR). Esterification of the carboxylic group of 11,12-ether-EET-8-ZE prevented blood pressure increase in SHR when injected at 2 mg/day for 12 days (MAP Δ change at day 8 of injection was -0.3 ± 2 for treated and 12 ± 1 mmHg for control SHR). Amidation of the carboxylic group with aspartic acid produced another EET analog (NUDSA) with a blood pressure lowering effect when injected at 3 mg/day in SHR for 5 days. Amidation of the carboxylic group with lysine amino acid produced another analog with minimal blood pressure lowering effect. These data suggest that esterification of the carboxylic group of 11,12-ether-EET-8-ZE produced the most effective ether-EET analog in lowering blood pressure in SHR and provide the first evidence to support the use of EET analogs in treatment of cardiovascular diseases.
RESUMEN
Cultured lung endothelial cells (LEC) respond to VEGF or arachidonic acid with increases in cell proliferation, the formation of tube-like structures, and the activation of Akt and ERK1/2 mediated growth pathways. LECs express a VEGF inducible Cyp2c44 epoxygenase and its 11,12- and 14,15-EET metabolites increase cell proliferation, tubulogenic activity, and the phosphorylation states of the ERK1/2 and Akt kinases. Ketoconazole, an epoxygenase inhibitor, blocks the cellular responses to VEGF. LECs expressing a Cyp2c44 epoxygenase small interference RNA show reductions in Cyp2c44 mRNA levels, and in their VEGF-stimulated proliferative and tubulogenic capacities; effects that are associated with decreases in VEGF-induced phosphorylation of the ERK1/2 and Akt kinases. We conclude that the Cyp2c44 arachidonic acid epoxygenase is a component of the signaling pathways associated with VEGF-stimulated angiogenesis, and suggest a role for EETs in the growth factor-induced changes in the activation states of the ERK1/2 and Akt kinase pathways.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Células Endoteliales/enzimología , Pulmón/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Antifúngicos/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocromo P-450 CYP2J2 , Inhibidores Enzimáticos del Citocromo P-450 , Familia 2 del Citocromo P450 , Células Endoteliales/citología , Activación Enzimática/efectos de los fármacos , Cetoconazol/farmacología , Pulmón/citología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Transgénicos , 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 , Neovascularización Fisiológica/fisiología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Functional and biochemical data have suggested a role for the cytochrome P450 arachidonate monooxygenases in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. We show here that disruption of the murine cytochrome P450, family 4, subfamily a, polypeptide 10 (Cyp4a10) gene causes a type of hypertension that is, like most human hypertension, dietary salt sensitive. Cyp4a10-/- mice fed low-salt diets were normotensive but became hypertensive when fed normal or high-salt diets. Hypertensive Cyp4a10-/- mice had a dysfunctional kidney epithelial sodium channel and became normotensive when administered amiloride, a selective inhibitor of this sodium channel. These studies (a) establish a physiological role for the arachidonate monooxygenases in renal sodium reabsorption and blood pressure regulation, (b) demonstrate that a dysfunctional Cyp4a10 gene causes alterations in the gating activity of the kidney epithelial sodium channel, and (c) identify a conceptually novel approach for studies of the molecular basis of human hypertension. It is expected that these results could lead to new strategies for the early diagnosis and clinical management of this devastating disease.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Hipertensión , Riñón/metabolismo , Canales de Sodio/metabolismo , Sodio en la Dieta , Amilorida/metabolismo , Animales , Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Presión Sanguínea/fisiología , Familia 2 del Citocromo P450 , Electrofisiología , Canales Epiteliales de Sodio , Femenino , Humanos , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Ratones , Ratones Noqueados , Pirimidinas/metabolismo , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/metabolismo , Canales de Sodio/genéticaRESUMEN
An HPLC method for the chiral analysis of the four regioisomeric epoxyeicosatrienoic acids (EETs) is described. The cytochrome P450 arachidonic acid epoxygenase metabolites are resolved, without the need for derivatization, by chiral-phase HPLC on a Chiralcel OJ column. Application of this methodology to the analysis of the liver endogenous EETs demonstrates stereospecific biosynthesis and corroborates the role of cytochrome P450 as the endogenous arachidonic acid epoxygenase.