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1.
Prostate ; 79(2): 206-214, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30345525

RESUMEN

INTRODUCTION AND OBJECTIVES: Multiple androgen receptor (AR)-dependent and -independent resistance mechanisms limit the efficacy of current castration-resistant prostate cancer (CRPC) treatment. Novel N-terminal domain (NTD) binding AR-targeting compounds, including EPI-001 (EPI), have the promising ability to block constitutively active splice variants, which represent a major resistance mechanism in CRPC. Autophagy is a conserved lysosomal degradation pathway that acts as survival mechanism in cells exposed to anticancer treatments. We hypothesized, that promising NTD-AR treatment may upregulate autophagy and that a combination of NTD-AR and autophagy inhibition might therefore increase antitumor effects. METHODS: AR-expressing prostate cancer cell lines (LNCaP, LNCaP-EnzR) were treated with different concentrations of EPI (10, 25, 50 µM) and in combination with the autophagy inhibitors chloroquine (CHQ, 20 µM) or 3-methyladenine (3-MA, 5 mM). Cell proliferation was assessed by WST-1-assays after 1 and 7 days. Ethidium bromide and Annexin V were used to measure viability and apoptosis on day 7 after treatment. Autophagosome formation was detected by AUTOdot staining. In addition, autophagic activity was monitored by immunocytochemistry and Western blot (WES) for the expression of ATG5, Beclin1, LC3-I/II and p62. RESULTS: Treatment with EPI resulted in a dose-dependent reduction of cell growth and increased apoptosis in both cancer cell lines on day 7. In addition, EPI treatment demonstrated an upregulated autophagosome formation in LNCaP and LNCaP-EnzR cells. Assessment of autophagic activity by immunocytochemistry and WES revealed an increase of ATG5 and LC3-II expression and a decreased p62 expression in all EPI-treated cells. A combined treatment of EPI with autophagy inhibitors led to a further significant reduction of cell viability in both cell lines. CONCLUSIONS: Our results demonstrate that NTD targeting AR inhibition using EPI leads to an increased autophagic activity in LNCaP and LNCaP-EnzR prostate cancer cells. A combination of NTD AR blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI in prostate cancer cells. Double treatment may offer a promising strategy to overcome resistance mechanisms in advanced prostate cancer.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Compuestos de Bencidrilo/farmacología , Clorhidrinas/farmacología , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia/biosíntesis , Proteína 5 Relacionada con la Autofagia/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Células PC-3 , Feniltiohidantoína/farmacología , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología
2.
Exp Cell Res ; 359(1): 215-225, 2017 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-28736082

RESUMEN

Prostate cancer (PC) is one of the leading causes of cancer death in men. It commonly develops in older males, but the number of younger men diagnosed with the disease has increased in recent years. Hormone therapies, such as chemical and surgical methods that inhibit androgen synthesis or androgen receptor (AR) activation, have been used for advanced disease. However, castration-resistant PC (CRPC), which exhibits androgen-independent mechanisms for activating AR, develops after a few years of such treatment and no therapy is available. In this study, we examined differences in the proteomes associated with the androgen-dependent (DHT treatment) and -independent (FSK, forskolin treatment) AR signaling conditions in LNCaP prostate cancer cells. Moreover, we used EPI-001, which inhibits AR-mediated transcriptional activity, to examine whether the observed differences in protein expression were directly affected by AR-mediated mechanisms. A total of 213 protein spots were matched in our 2-dimensional gel electrophoresis (2DE) analysis and 8 proteins with significant expression changes in our 5 different treatment groups were identified by mass spectrometry. Among these proteins, expression levels of PEPCK-M, catalase, tubulin alpha chain, alpha-enolase, and endoplasmic reticulum resident protein 29 were significantly altered by DHT and the levels of HSP 90 and EF-Tu were changed by FSK. These changes were blocked by EPI-001 in DHT-regulated proteins, PEPCK-M, catalase, and tubulin alpha chain and FSK-regulated EF-Tu protein. The results from our immunohistochemical analysis using in vivo xenograft models were consistent with the 2DE data. We therefore report the first identification of differences in proteins that are significantly regulated under androgen-dependent and -independent AR signaling conditions. Our findings could suggest a possible molecular mechanism through which AR is activated in the absence and/or presence of androgen, and might help explain the inhibitory action of EPI-001 on CRPC.


Asunto(s)
Andrógenos/farmacología , Neoplasias de la Próstata/metabolismo , Proteoma/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Compuestos de Bencidrilo/farmacología , Línea Celular Tumoral , Clorhidrinas/farmacología , Colforsina/farmacología , Dihidrotestosterona/farmacología , Electroforesis en Gel Bidimensional , Humanos , Inmunohistoquímica , Masculino , Ratones , Factor Tu de Elongación Peptídica/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Neoplasias de la Próstata/patología , Proteómica , Espectrometría de Masas en Tándem , Tubulina (Proteína)/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
3.
ACS Chem Biol ; 11(9): 2499-505, 2016 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-27356095

RESUMEN

Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.


Asunto(s)
Compuestos de Bencidrilo/farmacología , Clorhidrinas/farmacología , Orquiectomía , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Compuestos de Bencidrilo/uso terapéutico , Clorhidrinas/uso terapéutico , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Activación Transcripcional
4.
Clin Cancer Res ; 22(17): 4466-77, 2016 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-27140928

RESUMEN

PURPOSE: Persistent androgen receptor (AR) transcriptional activity is clinically evident in castration-resistant prostate cancer (CRPC). Therefore, AR remains as a viable therapeutic target for CRPC. All current hormonal therapies target the C-terminus ligand-binding domain (LBD) of AR. By using EPI to target AR activation function-1 (AF-1), in the N-terminal domain that is essential for AR transactivation, we evaluate the ability of EPI to overcome several clinically relevant AR-related mechanisms of resistance. EXPERIMENTAL DESIGN: To study the effect of EPI on AR transcriptional activity against overexpressed coactivators, such as SRC1-3 and p300, luciferase reporter assays were performed using LNCaP cells. AR-negative COS-1 cells were employed for reporter assays to examine whether the length of polyglutamine tract affects inhibition by EPI. The effect of EPI on constitutively active AR splice variants was studied in LNCaP95 cells, which express AR-V7 variant. To evaluate the effect of EPI on the proliferation of LNCaP95 cells, we performed in vitro BrdUrd incorporation assay and in vivo studies using xenografts in mice. RESULTS: EPI effectively overcame several molecular alterations underlying aberrant AR activity, including overexpressed coactivators, AR gain-of-function mutations, and constitutively active AR-V7. EPI inhibited AR transcriptional activity regardless of the length of polyglutamine tract. Importantly, EPI significantly inhibited the in vitro and in vivo proliferation of LNCaP95 prostate cancer cells, which are androgen independent and enzalutamide resistant. CONCLUSIONS: These findings support EPI as a promising therapeutic agent to treat CRPC, particularly against tumors driven by constitutively active AR splice variants that are resistant to LBD-targeting drugs. Clin Cancer Res; 22(17); 4466-77. ©2016 AACRSee related commentary by Sharp et al., p. 4280.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Compuestos de Bencidrilo/farmacología , Clorhidrinas/farmacología , Resistencia a Antineoplásicos , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Mutación , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Unión Proteica , Empalme del ARN , Receptores Androgénicos/genética , Transducción de Señal/efectos de los fármacos , Activación Transcripcional , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Oncotarget ; 6(6): 3811-24, 2015 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-25669987

RESUMEN

The androgen receptor (AR) is a driver of prostate cancer (PCa) cell growth and disease progression. Therapies for advanced PCa exploit AR dependence by blocking the production or action of androgens, but these interventions inevitably fail via multiple mechanisms including mutation or deletion of the AR ligand binding domain (LBD). Thus, the development of new inhibitors which act through non-LBD interfaces is an unmet clinical need. EPI-001 is a bisphenol A-derived compound shown to bind covalently and inhibit the AR NH2-terminal domain (NTD). Here, we demonstrate that EPI-001 has general thiol alkylating activity, resulting in multilevel inhibitory effects on AR in PCa cell lines and tissues. At least one secondary mechanism of action associated with AR inhibition was found to be selective modulation of peroxisome proliferator activated receptor-gamma (PPARγ). These multi-level effects of EPI-001 resulted in inhibition of transcriptional activation units (TAUs) 1 and 5 of the AR NTD, and reduced AR expression. EPI-001 inhibited growth of AR-positive and AR-negative PCa cell lines, with the highest sensitivity observed in LNCaP cells. Overall, this study provides new mechanistic insights to the chemical biology of EPI-001, and raises key issues regarding the use of covalent inhibitors of the intrinsically unstructured AR NTD.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Compuestos de Bencidrilo/farmacología , Clorhidrinas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/biosíntesis , Receptores Androgénicos/genética , Transfección
6.
J Appl Toxicol ; 35(7): 831-41, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25424538

RESUMEN

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.


Asunto(s)
Marcadores Genéticos/efectos de los fármacos , Inmunotoxinas/farmacología , Células Jurkat/efectos de los fármacos , Aldicarb/farmacología , Aldicarb/toxicidad , Compuestos Azo/farmacología , Compuestos Azo/toxicidad , Benzopirenos/farmacología , Benzopirenos/toxicidad , Biomarcadores Farmacológicos , Clorhidrinas/farmacología , Clorhidrinas/toxicidad , Cloropirifos/farmacología , Cloropirifos/toxicidad , Humanos , Imidazoles/farmacología , Imidazoles/toxicidad , Técnicas In Vitro , Neonicotinoides , Nitrocompuestos/farmacología , Nitrocompuestos/toxicidad , Piretrinas/farmacología , Piretrinas/toxicidad , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Pruebas de Toxicidad , ortoaminobenzoatos/farmacología , ortoaminobenzoatos/toxicidad
7.
Biochim Biophys Acta ; 1842(9): 1879-86, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24959971

RESUMEN

Lipids play a role in acute pancreatitis (AP) progression. We investigate the ability of pancreatic acinar cells to trigger inflammatory response in the presence of lipid compounds generated in necrotic areas of peripancreatic adipose tissue (AT) during AP induced in rats by 5% sodium taurocholate. Lipid composition of AT was analyzed by HPLC-mass spectrometry. Acinar inflammatory response to total lipids as well as to either the free fatty acid (FFA) fraction or their chlorinated products (Cl-FFAs) was evaluated. For this, mRNA expression of chemokine (C-C motif) ligand 2 (CCL2) and P-selectin as well as the activation of MAPKs, NF-κB and STAT-3 were analyzed in pancreatic acini. Myeloperoxidase (MPO) activity, as an inducer of Cl-FFA generation, was also analyzed in AT. MPO activity significantly increased in necrotic (AT-N) induced changes in lipid composition of necrotic fat, such as increase in FFA and phospholipid (PL) content, generation of Cl-FFAs and increases in saturated FFAs and in the poly-:mono-unsaturated FFA ratio. Total lipids from AT-N induced overexpression of CCL2 and P-selectin in pancreatic acini as well as MAPKs phosphorylation and activation of NF-κB and STAT3. FFAs, but not Cl-FFAs, up-regulated CCL2 and P-selectin in acinar cells. We conclude that FFAs are capable of up-regulating inflammatory mediators in pancreatic acini and given that they are highly produced during AP, mainly may contribute to the inflammatory response triggered in acinar cells by fat necrosis. No role is played by Cl-FFAs generated as a result of neutrophil infiltration.


Asunto(s)
Células Acinares/inmunología , Tejido Adiposo/patología , Inflamación/etiología , Lípidos/efectos adversos , Páncreas/inmunología , Pancreatitis Aguda Necrotizante/fisiopatología , Células Acinares/metabolismo , Células Acinares/patología , Animales , Biomarcadores/análisis , Western Blotting , Proliferación Celular/efectos de los fármacos , Clorhidrinas/farmacología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lípidos/análisis , Masculino , Páncreas/metabolismo , Páncreas/patología , Peroxidasa/metabolismo , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
8.
J Clin Invest ; 123(7): 2948-60, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23722902

RESUMEN

Hormone therapies for advanced prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD), but these ultimately fail and the disease progresses to lethal castration-resistant prostate cancer (CRPC). The mechanisms that drive CRPC are incompletely understood, but may involve constitutively active AR splice variants that lack the LBD. The AR N-terminal domain (NTD) is essential for AR activity, but targeting this domain with small-molecule inhibitors is complicated by its intrinsic disorder. Here we investigated EPI-001, a small-molecule antagonist of AR NTD that inhibits protein-protein interactions necessary for AR transcriptional activity. We found that EPI analogs covalently bound the NTD to block transcriptional activity of AR and its splice variants and reduced the growth of CRPC xenografts. These findings suggest that the development of small-molecule inhibitors that bind covalently to intrinsically disordered proteins is a promising strategy for development of specific and effective anticancer agents.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Antineoplásicos Hormonales/farmacología , Compuestos de Bencidrilo/farmacología , Clorhidrinas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Antagonistas de Receptores Androgénicos/química , Animales , Antineoplásicos Hormonales/química , Compuestos de Bencidrilo/química , Células COS , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Clorhidrinas/química , Química Clic , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Orquiectomía , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Estructura Terciaria de Proteína , Receptores Androgénicos/química , Receptores Androgénicos/genética , Estereoisomerismo , Activación Transcripcional/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Chem Phys Lipids ; 163(7): 639-47, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20513376

RESUMEN

Hypochlorite generated in vivo under pathological conditions is a known oxidant and chlorinating agent, able to react with proteins and lipids, which affects the stability of biological membranes. Reaction with unsaturated fatty acyl chains in glycerophospholipids such as phosphatidylcholine results in the formation of chlorohydrins. The aim of this study was to determine the effects of chlorohydrins formed by the reaction of hypochlorite with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonylphosphatidylcholine on biophysical properties of bilayers and their effects on human erythrocytes. Using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in a decrease in the rotational correlation time and an increase in the order parameter of liposomes. Unilamellar chlorohydrin liposomes had a lower permeation coefficient for calcein than liposomes made of parent lipids. Flow cytometry demonstrated fast incorporation of uni and multilamellar chlorohydrin liposomes labeled with NBD-phosphatidylethanolamine into erythrocytes. This effect was accompanied by changes in erythrocyte shape (echinocyte formation) and aggregation. Similar but less pronounced effects were noticed for parent lipids only after longer incubation. Chlorohydrins showed also a stronger hemolytic action, proportional to the lipid:erythrocyte ratio. These results are important for understanding the effects of HOCl on mammalian cells, such as might occur in inflammatory pathology.


Asunto(s)
Clorhidrinas/farmacología , Eritrocitos/efectos de los fármacos , Fosfatidilcolinas/farmacología , Agregación Eritrocitaria/efectos de los fármacos , Membrana Eritrocítica/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Ácido Hipocloroso/farmacología , Liposomas/sangre , Fluidez de la Membrana/efectos de los fármacos
10.
Cancer Cell ; 17(6): 535-46, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20541699

RESUMEN

Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.


Asunto(s)
Antagonistas de Receptores Androgénicos , Antineoplásicos Hormonales/uso terapéutico , Compuestos de Bencidrilo/uso terapéutico , Castración , Clorhidrinas/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Andrógenos/farmacología , Animales , Antineoplásicos Hormonales/efectos adversos , Antineoplásicos Hormonales/farmacología , Apoptosis/efectos de los fármacos , Compuestos de Bencidrilo/efectos adversos , Compuestos de Bencidrilo/farmacología , Proteína de Unión a CREB/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Clorhidrinas/efectos adversos , Clorhidrinas/farmacología , ADN/genética , ADN/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Estructura Molecular , Recurrencia Local de Neoplasia/patología , Próstata/anatomía & histología , Próstata/efectos de los fármacos , Próstata/patología , Antígeno Prostático Específico/sangre , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Conformación Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Receptores Androgénicos/metabolismo , Receptores de Esteroides/efectos de los fármacos , Elementos de Respuesta/genética , Serina Endopeptidasas/genética , Activación Transcripcional/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
11.
Lipids ; 43(3): 243-9, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18256867

RESUMEN

Lysophosphatidylcholine (LysoPtdCho) levels are elevated in sera in patients with atherosclerosis and in atherosclerotic tissue. Previous studies have shown that reactive chlorinating species attack plasmalogens in human coronary artery endothelial cells (HCAEC), forming lysoPtdCho and lysoPtdCho-chlorohydrin (lysoPtdCho-ClOH). The results herein demonstrate for the first time that lysoPtdCho-ClOH is elevated over 60-fold in human atherosclerotic lesions. In cultured HCAEC, 18:0 lysoPtdCho-ClOH led to a statistically significant increase in P-selectin cell-surface expression, but unlike 18:1 lysoPtdCho did not lead to cyclooxygenase-2 protein expression. These data show that 18:0 lysoPtdCho-ClOH is elevated in atherosclerotic tissue and may have unique pro-atherogenic properties compared to lysoPtdCho.


Asunto(s)
Aterosclerosis/metabolismo , Clorhidrinas/análisis , Vasos Coronarios/metabolismo , Endotelio Vascular/metabolismo , Lisofosfatidilcolinas/análisis , Selectina-P/metabolismo , Aorta/química , Células Cultivadas , Clorhidrinas/sangre , Clorhidrinas/farmacología , Ciclooxigenasa 2/metabolismo , Células Endoteliales/metabolismo , Humanos , Lisofosfatidilcolinas/sangre , Lisofosfatidilcolinas/farmacología , Espectrometría de Masa por Ionización de Electrospray
12.
Free Radic Biol Med ; 44(3): 452-63, 2008 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-18005671

RESUMEN

HOCl-modified low-density lipoprotein (LDL) has proinflammatory effects, including induction of inflammatory cytokine production, leukocyte adhesion, and ROS generation, but the components responsible for these effects are not completely understood. HOCl and the myeloperoxidase-H(2)O(2)-halide system can modify both protein and lipid moieties of LDL and react with unsaturated phospholipids to form chlorohydrins. We investigated the proinflammatory effects of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine (SOPC) chlorohydrin on artery segments and spleen-derived leukocytes from ApoE(-/-) and C57 Bl/6 mice. Treatment of ApoE(-/-) artery segments with SOPC chlorohydrin, but not unmodified SOPC, caused increased leukocyte-arterial adhesion in a time- and concentration-dependent manner. This could be prevented by pretreatment of the artery with P-selectin or ICAM-1-blocking antibodies, but not anti-VCAM-1 antibody, and immunohistochemistry showed that P-selectin expression was upregulated. However, chlorohydrin treatment of leukocytes did not increase expression of adhesion molecules LFA-1 or PSGL-1, but caused increased release of ROS from PMA-stimulated leukocytes by a CD36-dependent mechanism. The SOPC chlorohydrin-induced adhesion and ROS generation could be abrogated by pretreatment of the ApoE(-/-) mice with pravastatin or a nitrated derivative, NCX 6550. These findings suggest that phospholipid chlorohydrins formed in HOCl-treated LDL could contribute to the proinflammatory effects observed for this modified lipoprotein in vitro.


Asunto(s)
Aterosclerosis/inmunología , Clorhidrinas/metabolismo , Leucocitos/inmunología , Lipoproteínas LDL/metabolismo , Selectina-P/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Animales , Anticolesterolemiantes/farmacología , Apolipoproteínas E/genética , Arterias/efectos de los fármacos , Arterias/inmunología , Adhesión Celular/efectos de los fármacos , Clorhidrinas/farmacología , Ácido Hipocloroso/química , Inflamación/inmunología , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/efectos de los fármacos , Lipoproteínas LDL/química , Ratones , Ratones Noqueados , Nitratos/farmacología , Selectina-P/análisis , Fosfatidilcolinas/farmacología , Fosfolípidos/farmacología , Pravastatina/análogos & derivados , Pravastatina/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba
13.
Acta Biochim Pol ; 53(4): 761-8, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17128291

RESUMEN

The oxidation of low-density lipoprotein (LDL) is thought to contribute to atherogenesis, which is an inflammatory disease involving activation of phagocytic cells. Myeloperoxidase, an enzyme which is able to produce hypochlorous acid (HOCl), is released from these phagocytic cells, and has been found in an active form in atherosclerotic plaques. HOCl can oxidize both the lipid and protein moiety of LDL, and HOCl-modified LDL has been found to be pro-inflammatory, although it is not known which component is responsible for this effect. As HOCl can oxidize lipids to give chlorohydrins, we hypothesized that phospholipid chlorohydrins might have toxic and pro-inflammatory effects. We have formed chlorohydrins from fatty acids (oleic, linoleic and arachidonic acids) and from phospholipids (stearoyl-oleoyl phosphatidylcholine, stearoyl-linoleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine), and investigated various biological effects of these oxidation products. Fatty acid and phospholipid chlorohydrins were found to deplete ATP levels in U937 cells in a concentration-dependent manner, with significant effects observed at concentrations of 25 microM and above. Low concentrations (25 microM) of stearoyl-oleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine chlorohydrins were also found to increase caspase-3 activity. Finally, stearoyl-oleoyl phosphatidylcholine chlorohydrin increased leukocyte adhesion to artery segments isolated from C57Bl/6 mice. These results demonstrate potentially harmful effects of lipid chlorohydrins, and suggest that they may contribute to some of the pro-inflammatory effects that HOCl-modified low density lipoprotein has been found to induce.


Asunto(s)
Clorhidrinas/farmacología , Endotelio Vascular/citología , Estrés Oxidativo , Adenosina Trifosfato/análisis , Animales , Caspasa 3/metabolismo , Adhesión Celular , Línea Celular , Clorhidrinas/química , Ácidos Grasos , Humanos , Inflamación/etiología , Leucocitos/citología , Ratones , Ratones Endogámicos C57BL , Fosfolípidos
14.
Biofactors ; 24(1-4): 17-31, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16403960

RESUMEN

The oxidation of lipids is important in many pathological conditions and lipid peroxidation products such as 4-hydroxynonenal (HNE) and other aldehydes are commonly measured as biomarkers of oxidative stress. However, it is often useful to complement this with analysis of the original oxidized phospholipid. Electrospray mass spectrometry (ESMS) provides an informative method for detecting oxidative alterations to phospholipids, and has been used to investigate oxidative damage to cells, and low-density lipoprotein, as well as for the analysis of oxidized phosphatidylcholines present in atherosclerotic plaque material. There is increasing evidence that intact oxidized phospholipids have biological effects; in particular, oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholine (PAPC) have been found to cause inflammatory responses, which could be potentially important in the progression of atherosclerosis. The effects of chlorohydrin derivatives of lipids have been much less studied, but it is clear that free fatty acid chlorohydrins and phosphatidylcholine chlorohydrins are toxic to cells at concentrations above 10 micromolar, a range comparable to that of HNE and oxidized PAPC. There is some evidence that chlorohydrins have biological effects that may be relevant to atherosclerosis, but further work is needed to elucidate their pro-inflammatory properties, and to understand the mechanisms and balance of biological effects that could result from oxidation of complex mixtures of lipids in a pathophysiological situation.


Asunto(s)
Peroxidación de Lípido , Fosfolípidos/química , Espectrometría de Masa por Ionización de Electrospray , Animales , Aterosclerosis/metabolismo , Clorhidrinas/química , Clorhidrinas/farmacología , Cromatografía Liquida , Eritrocitos/química , Ácidos Grasos/química , Humanos , Ácido Hipocloroso/química , Ácido Hipocloroso/metabolismo , Ácido Hipocloroso/farmacología , Lipoproteínas LDL/química , Espectrometría de Masas , Oxidación-Reducción , Estrés Oxidativo , Fosfatidilcolinas/análisis , Fosfatidilcolinas/química , Conejos
15.
FEBS Lett ; 540(1-3): 245-50, 2003 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-12681516

RESUMEN

Chlorohydrins of stearoyl-oleoyl phosphatidylcholine (SOPC), stearoyl-linoleoyl phosphatidylcholine, and stearoyl-arachidonyl phosphatidylcholine were incubated with cultured myeloid cells (HL60) for 24 h, and the cellular ATP level was measured using a bioluminescent assay. The chlorohydrins caused significant depletion of cellular ATP in the range 10-100 microM. The ATP depletion by the phospholipid chlorohydrins was slightly less than that of 4-hydroxy-2-nonenal, but greater than that of hexanal, trans-2-nonenal, and autoxidised palmitoyl-arachidonoyl phosphatidylcholine. SOPC chlorohydrin was also found to cause loss of viability in U937 cells, and thus phospholipid chlorohydrins could contribute to the formation of a necrotic core in advanced atherosclerotic lesions.


Asunto(s)
Adenosina Trifosfato/metabolismo , Clorhidrinas/farmacología , Fosfolípidos/farmacología , Células HL-60 , Humanos , Luminiscencia , Espectrometría de Masa por Ionización de Electrospray , Células U937
16.
Eur J Pharmacol ; 354(1): 25-31, 1998 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-9726627

RESUMEN

The ability of 2,2,2-trichloroethanol (TCE) and related alcohols to modify the 5-hydroxytryptamine3 (5-HT3) receptor-mediated depolarisation of the rat isolated cervical vagus nerve were investigated by extracellular electrophysiological recording using the 'grease gap' technique. TCE at millimolar concentrations increased the magnitude of the 5-HT3 receptor-mediated depolarisations of the rat vagus nerve by a number of agonists (5-HT, phenylbiguanide (PBG), quipazine). Concentration response curves generated for the 5-HT3 receptor agonists. 5-HT and PBG, in the absence and presence of TCE (5 mM) indicated that the potentiation in agonist-induced depolarisation was due to an increase in both agonist potency and apparent efficacy. Following apparent complete 5-HT3 receptor desensitisation (induced by either 5-HT or PBG; 100 microM for 90 min), application of TCE (5 mM) in the continued presence of either agonist induced a depolarisation of the vagus nerve. In addition to TCE, a number of related alcohols (tribromoethanol, isopentanol and 5-chloropentanol but not ethanol) at millimolar concentrations also potentiated depolarisation of the vagus nerve induced by 5-HT. Combined application of both TCE (0.1-20 mM) and isopentanol (20 mM) indicated that the potentiation of the 5-HT3 receptor-mediated depolarisation by these alcohols was not additive. The present studies indicate that the 5-HT3 receptor expressed on the cervical vagus nerve is susceptible to allosteric modulation by a number of alcohols including the anaesthetic agent TCE. Such an interaction may have relevance to the nausea and vomiting experienced by some patients following recovery from general anaesthesia.


Asunto(s)
Etilenclorhidrina/análogos & derivados , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/fisiología , Nervio Vago/efectos de los fármacos , Nervio Vago/ultraestructura , Animales , Clorhidrinas/farmacología , Interacciones Farmacológicas , Etanol/análogos & derivados , Etanol/farmacología , Etilenclorhidrina/farmacología , Femenino , Técnicas In Vitro , Indoles/farmacología , Pentanoles/farmacología , Ratas , Ratas Wistar , Receptores de Serotonina 5-HT3 , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Sulfonamidas/farmacología , Nervio Vago/fisiología
17.
Biochem Biophys Res Commun ; 231(2): 295-8, 1997 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-9070266

RESUMEN

The hydrogen peroxide (H2O2)-myeloperoxidase system can convert linoleic acid (LA) to monochlorohydrins. LA monochlorohydrins can also be produced when LA monoepoxides are subjected to low pH solutions. We chemically synthesized and purified LA monochlorohydrins and examined their effect on the developed tension of guinea-pig papillary muscles in vitro in terms of both concentration and the duration of exposure. A decrease in muscular tension was observed immediately after addition of LA monochlorohydrins at concentrations of 30, 100, and 300 microM. We also compared the effect of LA monochlorohydrins on muscle tension with those of LA monoepoxides.


Asunto(s)
Clorhidrinas/farmacología , Corazón/efectos de los fármacos , Ácidos Linoleicos/química , Contracción Miocárdica/efectos de los fármacos , Animales , Clorhidrinas/análisis , Femenino , Cromatografía de Gases y Espectrometría de Masas , Cobayas , Corazón/fisiología , Técnicas In Vitro , Ácido Linoleico , Miocardio/química , Ratas
18.
Redox Rep ; 3(5-6): 263-71, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9754324

RESUMEN

Hypochlorous acid (HOCl), a strong oxidant generated by the myeloperoxidase system of neutrophils and monocytes, has been implicated in inflammatory tissue damage by these cells. Reaction of HOCl with the double bonds of unsaturated lipids produces alpha, beta-chlorohydrin isomers. We have exposed red cell membranes to HOCl and used thin layer chromatography (TLC) of the extracted lipids and enzyme-linked immunosorbent assay (ELISA), using an antichlorohydrin monoclonal antibody, to show that fatty acyl chlorohydrins are formed. The ELISA was approximately 25 fold more sensitive than TLC, and chlorohydrins were detected when membranes from 10(6) cells were treated with > or = 0.16 nmoles HOCl. Lipid chlorohydrins are more polar and bulky than their parent lipids and as such could affect membrane stability and function. To determine the effect of incorporation of lipid chlorohydrins into cell membranes, preformed fatty acid and cholesterol chlorohydrins were incubated with red cells. Lysis was measured as release of haemoglobin and incorporation of lipids was determined by 14C scintillation counting. Addition of HOCl-treated oleic acid to red cells resulted in rapid lysis of a fraction of the cells in a concentration dependent manner. HOCl-treated cholesterol also caused a small amount of cell lysis that was predominantly due to chlorohydrin 3, one of the three major cholesterol chlorohydrin products. Chlorohydrin 3, which has a decreased planarity and polarity, was also primarily responsible for altering the critical micelle concentration of HOCl-treated cholesterol-containing liposomes.


Asunto(s)
Clorhidrinas/farmacología , Membrana Eritrocítica/fisiología , Hemólisis/fisiología , Ácido Hipocloroso/metabolismo , Lípidos de la Membrana/sangre , Anticuerpos Monoclonales , Clorhidrinas/sangre , Cromatografía en Capa Delgada , Ensayo de Inmunoadsorción Enzimática , Membrana Eritrocítica/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Cinética , Lípidos de la Membrana/aislamiento & purificación , Micelas , Ácido Oléico/sangre , Ácido Oléico/farmacología , Sensibilidad y Especificidad
20.
Contraception ; 41(2): 207-12, 1990 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-2311406

RESUMEN

The effect of the male contraceptive, alpha-chlorohydrin, on in situ pH in seminiferous tubules and epididymal duct of the rat has been studied employing in vivo microelectrode techniques. After eight days of low-dose alpha-chlorohydrin administration (15mg/kg/day), a significant increase in acidity of luminal fluid in seminiferous tubules, proximal caput, middle caput, and proximal cauda epididymidis was observed. Increased acidity in the testis and epididymis may play an important role in the antifertility effect of alpha-chlorohydrin.


Asunto(s)
Clorhidrinas/farmacología , Anticonceptivos Masculinos/farmacología , Epidídimo/efectos de los fármacos , Testículo/efectos de los fármacos , alfa-Clorhidrina/farmacología , Animales , Epidídimo/fisiología , Concentración de Iones de Hidrógeno , Masculino , Ratas , Ratas Endogámicas , Túbulos Seminíferos/efectos de los fármacos , Testículo/fisiología
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