RESUMEN
ß-Apocarotenoids are eccentric cleavage products of carotenoids formed by chemical and enzymatic oxidations. They occur in foods containing carotenoids and thus might be directly absorbed from the diet. However, there is limited information about their intestinal absorption. The present research examined the kinetics of uptake and metabolism of ß-apocarotenoids. Caco-2 cells were grown on 6-well plastic plates until a differentiated cell monolayer was achieved. ß-Apocarotenoids were prepared in Tween 40 micelles, delivered to differentiated cells in serum-free medium, and incubated at 37°C for up to 8 h. There was rapid uptake of ß-apo-8'-carotenal into cells, and ß-apo-8'-carotenal was largely converted to ß-apo-8'-carotenoic acid and a minor metabolite that we identified as 5,6-epoxy-ß-apo-8'-carotenol. There was also rapid uptake of ß-apo-10'-carotenal into cells, and ß-apo-10'-carotenal was converted into a major metabolite identified as 5,6-epoxy-ß-apo-10'-carotenol and a minor metabolite that is likely a dihydro-ß-apo-10'-carotenol. Finally, there was rapid cellular uptake of ß-apo-13-carotenone, and this compound was extensively degraded. These results suggest that dietary ß-apocarotenals are extensively metabolized in intestinal cells via pathways similar to the metabolism of retinal. Thus, they are likely not absorbed directly from the diet.
Asunto(s)
Carotenoides/metabolismo , Células CACO-2 , Cromatografía Líquida de Alta Presión , Humanos , Cinética , Espectrometría de Masas , Vitamina A/metabolismo , beta Caroteno/metabolismoRESUMEN
Consumption of the tomato carotenoid, lycopene, has been associated with favorable health benefits. Some of lycopene's biological activity may be due to metabolites resulting from cleavage of the lycopene molecule. Because of their structural similarity to the retinoic acid receptor (RAR) antagonist, ß-apo-13-carotenone, the "first half" putative oxidative cleavage products of the symmetrical lycopene have been synthesized. All transformations proceed in moderate to good yield and some with high stereochemical integrity allowing ready access to these otherwise difficult to obtain terpenoids. In particular, the methods described allow ready access to the trans isomers of citral (geranial) and pseudoionone, important flavor and fragrance compounds that are not readily available isomerically pure and are building blocks for many of the longer apolycopenoids. In addition, all of the apo-11, apo-13, and apo-15 lycopenals/lycopenones/lycopenoic acids have been prepared. These compounds have been evaluated for their effect on RAR-induced genes in cultured hepatoma cells and, much like ß-apo-13-carotenone, the comparable apo-13-lycopenone and the apo-15-lycopenal behave as RAR antagonists. Furthermore, molecular modeling studies demonstrate that the apo-13-lycopenone efficiently docked into the ligand binding site of RARα. Finally, isothermal titration calorimetry studies reveal that apo-13-lycopenone acts as an antagonist of RAR by inhibiting coactivator recruitment to the receptor.
Asunto(s)
Carotenoides/síntesis química , Carotenoides/farmacología , Receptores de Ácido Retinoico/antagonistas & inhibidores , Carotenoides/química , Carotenoides/metabolismo , Técnicas de Química Sintética , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Licopeno , Simulación del Acoplamiento Molecular , Conformación Proteica , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismoRESUMEN
Provitamin A carotenoids are oxidatively cleaved by ß-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, ß-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids ß-carotene, α-carotene, and ß-cryptoxanthin. Its catalytic activity with ß-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-ß-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and ß-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-ß-apo-8'-carotenal were also consistently detected in BCO2-ß-cryptoxanthin reaction mixtures. With the exception of this activity with ß-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this.
Asunto(s)
Pollos/metabolismo , Dioxigenasas/metabolismo , beta Caroteno/metabolismo , Secuencia de Aminoácidos , Animales , Carotenoides/química , Carotenoides/metabolismo , Pollos/genética , Criptoxantinas/química , Criptoxantinas/metabolismo , Dioxigenasas/química , Dioxigenasas/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , beta Caroteno/químicaRESUMEN
ß-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, ß-carotene 15,15'-oxygenase (BCO1) converts ß-carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitamin A that acts as a transcription factor ligand to regulate gene expression. ß-Carotene can also be cleaved by ß-carotene 9',10'-oxygenase (BCO2) to form ß-apo-10'-carotenal, a precursor of retinoic acid and a transcriptional regulator per se The mammalian embryo obtains ß-carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that ß-carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show that ß-carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that ß-apo-10'-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4α and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of ß-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that ß-carotene induces a feed-forward mechanism in the placenta to enhance the assimilation of ß-carotene for proper embryogenesis.
Asunto(s)
Proteínas Portadoras/biosíntesis , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Gestacionales/biosíntesis , Embarazo/metabolismo , beta Caroteno/metabolismo , Animales , Transporte Biológico Activo/fisiología , Proteínas Portadoras/genética , Dioxigenasas/genética , Dioxigenasas/metabolismo , Femenino , Ratones , Ratones Noqueados , Embarazo/genética , Proteínas Gestacionales/genética , beta-Caroteno 15,15'-Monooxigenasa/genética , beta-Caroteno 15,15'-Monooxigenasa/metabolismoRESUMEN
Carotenoids are polyenes synthesized in plants and certain microorganisms and are pigments used by plants and animals in various physiological processes. Some of the over 600 known carotenoids are capable of metabolic conversion to the essential nutrient vitamin A (retinol) in higher animals. Vitamin A also gives rise to a number of other metabolites which, along with their analogs, are known as retinoids. To facilitate discussion about these important molecules, a nomenclature is required to identify specific substances. The generally accepted rules for naming these important molecules have been agreed to by various Commissions of the International Union of Pure and Applied Chemistry and International Union of Biochemistry. These naming conventions are explained along with comparisons to more systematic naming rules that apply for these organic chemicals. Identification of the carotenoids and retinoids has been advanced by their chemical syntheses, and here, both classical and modern methods for synthesis of these molecules, as well as their analogs, are described. Because of their importance in biological systems, sensitive methods for the detection and quantification of these compounds from various sources have been essential. Early analyses that relied on liquid adsorption and partition chromatography have given way to high-performance liquid chromatography (HPLC) coupled with various detection methods. The development of HPLC coupled to mass spectrometry, particularly LC/MS-MS with Multiple Reaction Monitoring, has resulted in the greatest sensitivity and specificity in these analyses.
Asunto(s)
Carotenoides , Animales , Carotenoides/análisis , Carotenoides/química , Carotenoides/clasificación , Cromatografía/métodos , Predicción , Humanos , Estructura Molecular , Plantas/química , Retinoides/análisis , Retinoides/química , Retinoides/clasificación , Terminología como Asunto , Vitamina A/química , Vitamina A/metabolismoRESUMEN
Retinoid X receptor (RXRα) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that ß-apo-13-carotenone, an eccentric cleavage product of ß-carotene, antagonizes the activation of RXRα by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of ß-apo-13-carotenone's modulation on the transcriptional activity of RXRα is not understood and is the subject of this report. We performed transactivation assays using full-length RXRα and reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. ß-Apo-13-carotenone was compared with the RXRα antagonist UVI3003. The results showed that both ß-apo-13-carotenone and UVI3003 shifted the dose-dependent RXRα activation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXRαLBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXRαLBD, but ß-apo-13-carotenone did not. However, both ß-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7. To resolve this apparent contradiction, we investigated the effect of ß-apo-13-carotenone on the oligomeric state of purified recombinant RXRαLBD. ß-Apo-13-carotenone induces tetramerization of the RXRαLBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that ß-apo-13-carotenone regulates RXRα transcriptional activity by inducing the formation of the "transcriptionally silent" RXRα tetramer.
Asunto(s)
Carotenoides/farmacología , Multimerización de Proteína/efectos de los fármacos , Receptor alfa X Retinoide/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Células COS , Caspasa 9/biosíntesis , Caspasa 9/genética , Línea Celular Tumoral , Chlorocebus aethiops , Ácidos Cumáricos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Ratones , Multimerización de Proteína/fisiología , Receptor alfa X Retinoide/antagonistas & inhibidores , Receptor alfa X Retinoide/genética , Tetrahidronaftalenos/farmacología , Transcripción Genética/fisiologíaRESUMEN
ß-Carotene 15-15'-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and ß-carotene in either (16)O2-H2(18)O or (18)O2-H2(16)O medium for 15 min at 37 °C, and the relative amounts of (18)O-retinal and (16)O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from (18)O-retinal-H2(16)O and (16)O-retinal-H2(18)O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase.
Asunto(s)
Dioxigenasas/química , Oxígeno/química , Retinaldehído/química , Vitamina A/biosíntesis , Dioxigenasas/genética , Dioxigenasas/metabolismo , Humanos , Oxígeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinaldehído/genética , Retinaldehído/metabolismo , Vitamina A/química , Vitamina A/genéticaRESUMEN
ß-Apo-carotenoids, including ß-apo-13-carotenone and ß-apo-14'-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of ß-apo-13-carotenone and ß-apo-14'-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these ß-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of ß-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two ß-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either ß-apo-13-carotenone or ß-apo-10'-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some ß-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation.
Asunto(s)
Adipocitos/citología , Adipocitos/efectos de los fármacos , Carotenoides/farmacología , Diferenciación Celular/efectos de los fármacos , Células 3T3-L1 , Animales , Ratones , Receptores de Ácido Retinoico/antagonistas & inhibidores , Tretinoina/farmacologíaRESUMEN
Humans cannot synthesize vitamin A and thus must obtain it from their diet. ß-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of ß-carotene with a Vmax = 197.2 nmol retinal/mg BCO1 × h, Km = 17.2 µM and catalytic efficiency kcat/Km = 6098 M(-1) min(-1). The enzyme also catalyzed the oxidative cleavage of α-carotene, ß-cryptoxanthin, and ß-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of ß-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of ß-carotene. The shorter ß-apocarotenals (ß-apo-10'-carotenal, ß-apo-12'-carotenal, ß-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-ß-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.
Asunto(s)
Carotenoides/química , beta-Caroteno 15,15'-Monooxigenasa/química , Carotenoides/metabolismo , Catálisis , Humanos , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , beta-Caroteno 15,15'-Monooxigenasa/genética , beta-Caroteno 15,15'-Monooxigenasa/aislamiento & purificación , beta-Caroteno 15,15'-Monooxigenasa/metabolismoRESUMEN
Dietary carotenoids like ß-carotene are converted within the body either to retinoid, via ß-carotene-15,15'-dioxygenase (BCO1), or to ß-apo-carotenoids, via ß-carotene-9',10'-oxygenase 2. Some ß-apo-carotenoids are potent antagonists of retinoic acid receptor (RAR)-mediated transcriptional regulation, which is required to ensure normal heart development and functions. We established liquid chromatography tandem mass spectrometery methods for measuring concentrations of 10 ß-apo-carotenoids in mouse plasma, liver, and heart and assessed how these are influenced by Bco1 deficiency and ß-carotene intake. Surprisingly, Bco1(-/-) mice had an increase in heart levels of retinol, nonesterified fatty acids, and ceramides and a decrease in heart triglycerides. These lipid changes were accompanied by elevations in levels of genes important to retinoid metabolism, specifically retinol dehydrogenase 10 and retinol-binding protein 4, as well as genes involved in lipid metabolism, including peroxisome proliferator-activated receptor-γ, lipoprotein lipase, Cd36, stearoyl-CoA desaturase 1, and fatty acid synthase. We also obtained evidence of compromised heart function, as assessed by two-dimensional echocardiography, in Bco1(-/-) mice. However, the total absence of Bco1 did not substantially affect ß-apo-carotenoid concentrations in the heart. ß-Carotene administration to matched Bco1(-/-) and wild-type mice elevated total ß-apo-carotenal levels in the heart, liver, and plasma and total ß-apo-carotenoic acid levels in the liver. Thus, BCO1 modulates heart metabolism and function, possibly by altering levels of cofactors required for the actions of nuclear hormone receptors.
Asunto(s)
Cardiopatías/genética , Metabolismo de los Lípidos/genética , Retinoides/metabolismo , beta-Caroteno 15,15'-Monooxigenasa/deficiencia , beta-Caroteno 15,15'-Monooxigenasa/genética , Animales , Carotenoides/metabolismo , Cardiopatías/enzimología , Cardiopatías/metabolismo , Homeostasis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismoRESUMEN
ß-Carotene is the major dietary source of provitamin A. Central cleavage of ß-carotene catalyzed by ß-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA), which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of ß-carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are ß-apocarotenals and ß-apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the ß-apocarotenoids significantly activated RARs. Importantly, however, ß-apo-14'-carotenal, ß-apo-14'-carotenoic acid, and ß-apo-13-carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that ß-apo-13-carotenone can interact directly with the ligand binding site of the retinoid receptors. ß-Apo-13-carotenone and the ß-apo-14'-carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 cells. Finally, we developed an LC/MS method and found 3-5 nm ß-apo-13-carotenone was present in human plasma. These findings suggest that ß-apocarotenoids function as naturally occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary ß-carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer.
Asunto(s)
Carotenoides/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , beta Caroteno/metabolismo , Animales , Unión Competitiva , Células COS , Carotenoides/química , Carotenoides/farmacología , Chlorocebus aethiops , Sistema Enzimático del Citocromo P-450 , Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Modelos Moleculares , Estructura Molecular , Ensayo de Unión Radioligante , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/genética , Ácido Retinoico 4-Hidroxilasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Transcripcional/efectos de los fármacos , Tretinoina/farmacología , Tritio , beta Caroteno/químicaRESUMEN
In this review a discussion of the usual procedures used to synthesize retinoids is followed by an overview of the structure-activity relationships of these molecules. The discussion is then focused on the role and impact of retinoids on metabolic disorders with a particular emphasis on obesity, diabetes, and the metabolic syndrome. In these areas, both natural and synthetic retinoids that are being studied are reviewed and areas where likely future research will occur are suggested. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.
Asunto(s)
Enfermedades Metabólicas/metabolismo , Retinoides/química , Retinoides/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Síndrome Metabólico/metabolismo , Obesidad/metabolismo , Retinoides/síntesis química , Relación Estructura-ActividadRESUMEN
Chirally deuterated benzyl chlorides were prepared using novel, general hexachloroacetone/polymer-supported triphenylphosphine treatment of chirally deuterated benzyl alcohols. Doubly labeled protected tyrosine was obtained in 62% yield with 86% de at the α-carbon and 82% de at the ß-carbon. Key in the synthesis was the alkylation of (15)N-labeled (-)-8-phenylmenthylhippurate with R-(-)-4-triisopropylsilyloxybenzyl-α-d chloride.
Asunto(s)
Compuestos de Bencilo/síntesis química , Deuterio/química , Tirosina/síntesis química , Acetona/análogos & derivados , Acetona/síntesis química , Acetona/química , Alcoholes Bencílicos/síntesis química , Alcoholes Bencílicos/química , Compuestos de Bencilo/química , Isótopos de Nitrógeno/química , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/química , Polímeros/química , Tirosina/químicaRESUMEN
The dihydroceramide desaturase (DES) enzyme is responsible for inserting the 4,5-trans-double bond to the sphingolipid backbone of dihydroceramide. We previously demonstrated that fenretinide (4-HPR) inhibited DES activity in SMS-KCNR neuroblastoma cells. In this study, we investigated whether 4-HPR acted directly on the enzyme in vitro. N-C8:0-d-erythro-dihydroceramide (C(8)-dhCer) was used as a substrate to study the conversion of dihydroceramide into ceramide in vitro using rat liver microsomes, and the formation of tritiated water after the addition of the tritiated substrate was detected and used to measure DES activity. NADH served as a cofactor. The apparent K(m) for C(8)-dhCer and NADH were 1.92 ± 0.36 µm and 43.4 ± 6.47 µm, respectively; and the V(max) was 3.16 ± 0.24 and 4.11 ± 0.18 nmol/min/g protein. Next, the effects of 4-HPR and its metabolites on DES activity were investigated. 4-HPR was found to inhibit DES in a dose-dependent manner. At 20 min, the inhibition was competitive; however, longer incubation times demonstrated the inhibition to be irreversible. Among the major metabolites of 4-HPR, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) showed the highest inhibitory effect with substrate concentration of 0.5 µm, with an IC(50) of 1.68 µm as compared with an IC(50) of 2.32 µm for 4-HPR. N-(4-Methoxyphenyl)retinamide (4-MPR) and 4-Oxo-N-(4-methoxyphenyl)retinamide (4-oxo-4-MPR) had minimal effects on DES activity. A known competitive inhibitor of DES, C(8)-cyclopropenylceramide was used as a positive control. These studies define for the first time a direct in vitro target for 4-HPR and suggest that inhibitors of DES may be used as therapeutic interventions to regulate ceramide desaturation and consequent function.
Asunto(s)
Antineoplásicos/farmacocinética , Fenretinida/farmacocinética , Microsomas Hepáticos/enzimología , Oxidorreductasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Fenretinida/farmacología , Oxidorreductasas/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Retinoids are a class of compounds with structural similarity to vitamin A. These compounds inhibit the proliferation of many cancer cell lines but have had limited medical application as they are often toxic at therapeutic levels. Efforts to synthesize retinoids with a greater therapeutic index have met with limited success. 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic acid (TTNPB) is one of the most biologically active all-trans-retinoic acid (atRA) analogues and is highly teratogenic. In this study, we show that modification of the TTNPB carboxyl group with an N-(4-hydroxyphenyl)amido (4HPTTNPB) or a 4-hydroxybenzyl (4HBTTNPB) group changes the activity of the compound in cell culture and in vivo. Unlike TTNPB, both compounds induce apoptosis in cancer cells and bind poorly to the retinoic acid receptors (RARs). Like the similarly modified all-trans-retinoic acid (atRA) analogues N-(4-hydroxyphenyl)retinamide (4-HPR/fenretinide) and 4-hydroxybenzylretinone (4-HBR), 4HBTTNPB is a potent activator of components of the ER stress pathway. The amide-linked analogue, 4HPTTNPB, is less toxic to developing embryos than the parent TTNPB, and most significantly, the 4-hydroxybenzyl-modified compound (4HBTTNPB) that cannot be hydrolyzed in vivo to the parent TTNPB compound is nearly devoid of teratogenic liability.
Asunto(s)
Antineoplásicos/síntesis química , Benzoatos/síntesis química , Neoplasias de la Mama/tratamiento farmacológico , Retículo Endoplásmico/efectos de los fármacos , Fenretinida/uso terapéutico , Receptores de Ácido Retinoico/metabolismo , Retinoides/síntesis química , Vitamina A/análogos & derivados , Administración Oral , Amidas/química , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Benzoatos/efectos adversos , Benzoatos/uso terapéutico , Unión Competitiva , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Femenino , Fenretinida/síntesis química , Humanos , Fenol/química , Embarazo , Ratas , Ratas Sprague-Dawley , Retinoides/efectos adversos , Retinoides/uso terapéutico , Teratógenos , Factor de Transcripción CHOP/biosíntesis , Vitamina A/síntesis química , Vitamina A/uso terapéuticoRESUMEN
In this study, we investigated the effects of eccentric cleavage products of ß-carotene, i.e. ß-apocarotenoids (BACs), on retinoid X receptor alpha (RXRα) signaling. Transactivation assays were performed to test whether BACs activate or antagonize RXRα. Reporter gene constructs (RXRE-Luc, pRL-tk) and RXRα were transfected into Cos-1 cells and used to perform these assays. None of the BACs tested activated RXRα. Among the compounds tested, ß-apo-13-carotenone was found to antagonize the activation of RXRα by 9-cis-retinoic acid and was effective at concentrations as low as 1 nM. Molecular modeling studies revealed that ß-apo-13-carotenone makes molecular interactions like an antagonist of RXRα. The results suggest a possible function of BACs on RXRα signaling.
Asunto(s)
Carotenoides/metabolismo , Carotenoides/farmacología , Receptor alfa X Retinoide/antagonistas & inhibidores , beta Caroteno/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Ligandos , Ratones , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Ratas , Receptor alfa X Retinoide/agonistas , Receptor alfa X Retinoide/química , Receptor alfa X Retinoide/metabolismo , Activación Transcripcional/efectos de los fármacos , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Hepatic stellate cells (HSCs) are responsible for storing 90-95% of the retinoid present in the liver. These cells have been reported in the literature also to accumulate dietary ß-carotene, but the ability of HSCs to metabolize ß-carotene in situ has not been explored. To gain understanding of this, we investigated whether ß-carotene-15,15'-monooxygenase (Bcmo1) and ß-carotene-9',10'-monooxygenase (Bcmo2) are expressed in HSCs. Using primary HSCs and hepatocytes purified from wild type and Bcmo1-deficient mice, we establish that Bcmo1 is highly expressed in HSCs; whereas Bcmo2 is expressed primarily in hepatocytes. We also confirmed that HSCs are an important cellular site within the liver for accumulation of dietary ß-carotene. Bcmo2 expression was found to be significantly elevated for livers and hepatocytes isolated from Bcmo1-deficient compared to wild type mice. This elevation in Bcmo2 expression was accompanied by a statistically significant increase in hepatic apo-12'-carotenal levels of Bcmo1-deficient mice. Although apo-10'-carotenal, like apo-12'-carotenal, was readily detectable in livers and serum from both wild type and Bcmo1-deficient mice, we were unable to detect either apo-8'- or apo-14'-carotenals in livers or serum from the two strains. We further observed that hepatic triglyceride levels were significantly elevated in livers of Bcmo1-deficient mice fed a ß-carotene-containing diet compared to mice receiving no ß-carotene. Collectively, our data establish that HSCs are an important cellular site for ß-carotene accumulation and metabolism within the liver.
Asunto(s)
Células Estrelladas Hepáticas/metabolismo , Retinoides/metabolismo , beta Caroteno/metabolismo , Animales , Femenino , Regulación Enzimológica de la Expresión Génica , Hepatocitos/metabolismo , Masculino , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , beta-Caroteno 15,15'-Monooxigenasa/deficiencia , beta-Caroteno 15,15'-Monooxigenasa/genética , beta-Caroteno 15,15'-Monooxigenasa/metabolismoRESUMEN
The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) induces apoptosis in a variety of cell lines and has shown promise as an anticancer agent both in vitro and in vivo. The clinical dose of 4-HPR, however, is limited by residual-associated toxicities, indicating a need for a less toxic drug. In this study, we show that 4-hydroxybenzylretinone (4-HBR), the unhydrolyzable analogue of 4-HPR, is effective in producing apoptosis in a variety of 4-HPR-sensitive cell lines, including breast cancer, neuroblastoma, and leukemia cells. We also show through the use of a pan-caspase inhibitor that this 4-HBR-induced apoptosis is dependent, at least in part, on caspase activity. 4-HBR is shown to exhibit binding to the retinoic acid receptors (RAR) at concentrations necessary to induce cell death and induces expression of all-trans-retinoic acid-responsive genes that can be blocked by a RAR pan-antagonist. However, through the use of this RAR pan-antagonist, 4-HBR-induced apoptosis and cell death is shown to be independent of the RAR signaling pathway. To further characterize the mechanism of action of 4-HBR, expression of the endoplasmic reticulum stress-induced genes GADD153 and Bcl-2-binding component 3 was examined. These mRNAs are shown to be rapidly induced in 4-HBR-treated and 4-HPR-treated breast cancer cells, and this up-regulation is also shown to be independent of the RARs. These results suggest that a stress-mediated apoptotic cascade is involved in the mechanism of action of these retinoids.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Apoptosis , Fenretinida/análogos & derivados , Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Ácido Retinoico/metabolismo , Factor de Transcripción CHOP/biosíntesis , Vitamina A/análogos & derivados , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Células HL-60 , Humanos , Hidrólisis , Leucemia/metabolismo , ARN Mensajero/metabolismo , Retinoides/metabolismo , Vitamina A/farmacologíaRESUMEN
Recent studies exhibit that 4-hydroxyphenylretinamide (4HPR) decreases aromatase activity in breast and placental cells. The effect of synthetic 4HPR analogs on aromatase and expression was examined in three breast cancer cell lines. Most derivatives did not decrease cellular aromatase activity. Two of the analogs even stimulated aromatase activity at the transcriptional level. Only one derivative significantly decreased aromatase in all three breast cancer cell lines and also suppressed CYP19 gene expression in one of the cell line. Placental microsomal aromatase assay rule out the possibility that this compound directly inhibits the aromatase enzyme. A non-genomic mechanism in suppression of cellular aromatase activity of this compound is proposed.
Asunto(s)
Inhibidores de la Aromatasa/farmacología , Aromatasa/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Fenretinida/análogos & derivados , Antineoplásicos/química , Antineoplásicos/farmacología , Aromatasa/genética , Inhibidores de la Aromatasa/química , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cartilla de ADN/genética , Femenino , Fenretinida/química , Fenretinida/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Microsomas/efectos de los fármacos , Microsomas/enzimología , Placenta/enzimología , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismoRESUMEN
4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a recently identified metabolite of fenretinide (4-HPR). We explored the effectiveness of 4-oxo-4-HPR in inducing cell growth inhibition in ovarian, breast, and neuroblastoma tumor cell lines; moreover, we investigated the molecular events mediating this effect in two ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/HPR) to 4-HPR. 4-oxo-4-HPR was two to four times more effective than 4-HPR in most cell lines, was effective in both 4-HPR-sensitive and 4-HPR-resistant cells, and, in combination with 4-HPR, caused a synergistic effect. The tumor growth-inhibitory effects of 4-oxo-4-HPR seem to be independent of nuclear retinoid receptors (RAR), as indicated by the failure of RAR antagonists to inhibit its effects and by its poor ability to bind and transactivate RARs. Unlike 4-HPR, which only slightly affected the G(1) phase of the cell cycle, 4-oxo-4-HPR caused a marked accumulation of cells in G(2)-M. This effect was associated with a reduction in the expression of regulatory proteins of G(2)-M (cyclin-dependent kinase 1 and cdc25c) and S (cyclin A) phases, and with an increase in the expression of apoptosis-related proteins, such as p53 and p21. Apoptosis was induced by 4-oxo-4-HPR in both 4-HPR-sensitive and 4-HPR-resistant cells and involved activation of caspase-3 and caspase-9 but not caspase-8. We also showed that 4-oxo-4-HPR, similarly to 4-HPR, increased reactive oxygen species generation and ceramide levels by de novo synthesis. In conclusion, 4-oxo-4-HPR is an effective 4-HPR metabolite that might act as therapeutic agent per se and, when combined with 4-HPR, might improve 4-HPR activity or overcome 4-HPR resistance.