RESUMEN
Secondary metastases are the leading cause of mortality in patients with breast cancer. Cytochrome P450 (CYP) 2J2 (CYP2J2) is upregulated in many human tumors and generates epoxyeicosanoids from arachidonic acid that promote tumorigenesis and metastasis, but at present there is little information on the genes that mediate these actions. In this study MDA-MB-468 breast cancer cells were stably transfected with CYP2J2 (MDA-2J2 cells) and Affymetrix microarray profiling was undertaken. We identified 182 genes that were differentially expressed in MDA-2J2 cells relative to control (MDA-CTL) cells (log[fold of control] ≥2). From gene ontology pathway analysis bone morphogenetic protein (BMP) receptor 1B (BMPR1B) emerged as an important upregulated gene in MDA-2J2 cells. Addition of the BMPR1B ligand BMP2 stimulated the migration of MDA-2J2 cells, but not MDA-CTL cells, from 3D-matrigel droplets. Migration of MDA-2J2 cells was prevented by the BMPR antagonist dorsomorphin. These findings indicate that over-expression of CYP2J2 in MDA-MB-468-derived breast cancer cells activates BMPR1B expression that may contribute to increased migration. Targeting BMPR1B may be a novel approach to inhibit the metastatic activity of breast cancers that contain high levels of CYP2J2.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Movimiento Celular/genética , Sistema Enzimático del Citocromo P-450/genética , Activación Transcripcional , Neoplasias de la Mama Triple Negativas/patología , Carcinogénesis/genética , Línea Celular Tumoral , Citocromo P-450 CYP2J2 , Expresión Génica , Ontología de Genes , Humanos , Metástasis de la Neoplasia , Regulación hacia Arriba/genéticaRESUMEN
Tetracyclic triterpenoids, including the dammarane, cucurbitane, cycloartane, lanostane and protostane groups, is a class of triterpenoids widely distributed in various medicinal plants, particularly those commonly used for the treatment of diabetes and its complications, such as Panax ginseng, Panax quinquefolium, Panax notoginseng, Gynostemma pentaphyllum, Astragalus membranaceus, Momordica charantia, and Ganoderma lucidum. This review highlights recent findings on the chemistry and bioactivities of tetracyclic triterpenoids from these plants and other popular herbal medicines.
Asunto(s)
Complicaciones de la Diabetes/tratamiento farmacológico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/aislamiento & purificación , Plantas Medicinales/química , Triterpenos/aislamiento & purificación , Animales , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Hipoglucemiantes/uso terapéutico , Estructura Molecular , Triterpenos/uso terapéuticoRESUMEN
The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2 gene in a cohort of subjects: G550A, G553A, G673A, A775C, and G862A, that encoded the OATP1A2 variants E184K, D185N, V255I, T259P, and D288N, respectively. The function and expression of these variant transporters were assessed in HEK-293 cells. We found that the novel variants, E184K, D185N, T259P, and D288N, were associated with impaired estrone-3-sulfate, imatinib, and methotrexate transport (â¼20-50% of wild-type control); function was retained by OATP1A2-V255I. From biotinylation assays, the decreased function of these variants was due, at least in part, to impaired plasma membrane expression. The four loss-of-function variants were studied further using mutagenesis to produce variants that encode residues with different charges or steric properties. From immunoblotting, the replacement of negatively charged residues at amino acid positions 184 and 185 impaired membrane expression, while either a positive or negative charge at residue 288 supported the correct membrane targeting of OATP1A2. Replacement of T259 with bulky residues disrupted transporter stability. From molecular models, E184, D185, and D288 were located near several charged residues such that intramolecular ionic interactions may stabilize the transporter structure. Individuals who carry these novel SNPs in the SLCO1A2 gene may be at risk from impaired efficacy or enhanced toxicity during treatment with drugs that are substrates for OATP1A2.
Asunto(s)
Transportadores de Anión Orgánico/química , Transportadores de Anión Orgánico/fisiología , Polimorfismo de Nucleótido Simple/genética , Código Genético/fisiología , Variación Genética/fisiología , Células HEK293 , HumanosRESUMEN
The multi-kinase inhibitor sorafenib is used for the treatment of renal and hepatic carcinomas and is undergoing evaluation for treatment of breast cancer in combination with other agents. Cytochrome P450 (CYP) 3A4 converts sorafenib to multiple metabolites that have been detected in patient plasma. However, recent clinical findings suggest that combination therapy may elicit inhibitory pharmacokinetic interactions involving sorafenib that increase toxicity. While sorafenib N-oxide is an active metabolite, information on the anti-tumor actions of other metabolites is unavailable. The present study evaluated the actions of sorafenib and its five major metabolites in human breast cancer cell lines. All agents, with the exception of N'-hydroxymethylsorafenib N-oxide, decreased ATP formation in four breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7 and T-47D). Prolonged treatment of MDA-MB-231 cells with N'-desmethylsorafenib, N'-desmethylsorafenib N-oxide and sorafenib (10 µM, 72 h) produced small increases in caspase-3 activity to 128-139% of control. Sorafenib and its metabolites, again with the exception of N'-hydroxymethylsorafenib N-oxide, impaired MEK/ERK signaling in MDA-MB-231 cells and modulated the expression of cyclin D1 and myeloid cell leukemia sequence-1, which regulate cell viability. When coadministered with doxorubicin (0.5 or 1 µM), sorafenib and N'-desmethylsorafenib (25 µM) produced greater effects on ATP production than either treatment alone. Thus, it emerges that, by targeting the MEK/ERK pathway, multiple sorafenib metabolites may contribute to the actions of sorafenib in breast cancer. Because N'-desmethylsorafenib is not extensively metabolized and does not inhibit major hepatic CYPs, this metabolite may have a lower propensity to precipitate pharmacokinetic drug interactions than sorafenib.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Células MCF-7 , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Niacinamida/uso terapéutico , SorafenibRESUMEN
Cyclooxygenase-2 (COX-2) is overexpressed in many human cancers and converts the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid to prostaglandin E(2) (PGE(2)), which drives tumorigenesis; in contrast, n-3 PUFA inhibit tumorigenesis. We tested the hypothesis that these antitumor actions of n-3 PUFA may involve the n-3 olefinic bond. n-3 Monounsaturated fatty acids (MUFAs) of chain length C16-C22 were synthesized and evaluated in MDA-MB-468 breast cancer cells that stably overexpressed COX-2 (MDA-COX-2 cells). Longer chain (C19-C22) n-3 MUFAs inhibited proliferation, activated apoptosis, decreased PGE(2) formation, and decreased cell invasion; C16-C18 analogues were less active. Molecular modeling showed that interactions of Arg120, Tyr355, and several hydrophobic amino acid residues in the COX-2 active site with C19-C22 MUFA analogues were favored. Thus, longer-chain n-3 MUFAs may be prototypes of novel anticancer agents that decrease the formation of PGE(2) in tumor cells that contain high levels of COX-2.
Asunto(s)
Antineoplásicos/síntesis química , Ciclooxigenasa 2/metabolismo , Ácidos Grasos Omega-3/química , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama , Dominio Catalítico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno , Dinoprostona/biosíntesis , Combinación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Ácidos Grasos Omega-3/farmacología , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Laminina , Modelos Moleculares , Invasividad Neoplásica , Proteoglicanos , Relación Estructura-Actividad , TermodinámicaRESUMEN
The tyrosine kinase inhibitor drug sorafenib is used in the treatment of liver and renal cancers but adverse effects may necessitate dose interruption and under-dosage may lead to therapeutic failure. Sorafenib also undergoes cytochrome P450 (CYP)-dependent biotransformation to the N-oxide and other metabolites. However, although CYPs are major determinants of efficacy and toxicity the roles of these enzymes in the formation of multiple sorafenib metabolites are unclear. In the present study CYP-mediated pathways of sorafenib oxidation in human liver were evaluated. cDNA-expressed CYP3A4 was the major catalyst in the formation of the principal N-oxide and N-hydroxymethyl metabolites of sorafenib, as well as the minor N-desmethyl metabolite. In contrast, CYP3A5 exhibited only ~5% of the activity of CYP3A4 and eleven other CYPs and three flavin-containing monooxygenases were inactive. In human hepatic microsomes metabolite formation was correlated with CYP3A4-mediated midazolam 1'-hydroxylation, but not with other CYP-specific substrate oxidations. In accord with these findings the CYP3A4 inhibitor ketoconazole selectively inhibited microsomal sorafenib oxidation pathways. From computational modeling studies atoms in the structure of sorafenib that undergo biotransformation were within ~5.4 Å of the CYP3A4 heme. Important hydrogen bonding interactions between sorafenib and amino acids Ser-119 and Glu-374 in the active center of CYP3A4 were identified. These findings indicate that sorafenib is oxidized selectively by human CYP3A4. This information could be adapted in individualized approaches to optimize sorafenib safety and efficacy in cancer patients.
Asunto(s)
Bencenosulfonatos/metabolismo , Citocromo P-450 CYP3A/metabolismo , Piridinas/metabolismo , Bencenosulfonatos/farmacocinética , Dominio Catalítico , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/genética , Humanos , Inactivación Metabólica , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Niacinamida/análogos & derivados , Oxidación-Reducción , Compuestos de Fenilurea , Conformación Proteica , Piridinas/farmacocinética , SorafenibRESUMEN
BACKGROUND AND PURPOSE: Dietary intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) like eicosapentaenoic acid (EPA) decreases cancer risk, while arachidonic acid and other ω-6 PUFAs increase risk, but the underlying mechanisms are unclear. Cytochrome P450 (CYP)-derived epoxides contribute to enhanced tumourigenesis due to ω-6 PUFA intake. Thus, ω-6 arachidonic acid epoxides (EETs) inhibit apoptosis and stimulate proliferation by up-regulating cyclin D1 expression in cells. The present study evaluated the corresponding ω-3 PUFA epoxides and assessed their role in the regulation of cell proliferation. EXPERIMENTAL APPROACH: Four chemically stable EPA epoxides (formed at the 8,9-, 11,12-, 14,15- and 17,18-olefinic bonds) were synthesized and tested against growth-related signalling pathways in brain microvascular endothelial bEND.3 cells. Cell cycle distribution was determined by flow cytometry and cyclin gene expression by immunoblotting and real-time PCR. The role of the p38 mitogen-activated protein (MAP) kinase in cyclin D1 dysregulation was assessed using specific inhibitors and dominant-negative expression plasmids. KEY RESULTS: The ω-3 17,18-epoxide of EPA decreased cell proliferation, interrupted the cell cycle in S-phase and down-regulated the cyclin D1/cyclin-dependent kinase (CDK)-4 complex, whereas the 8,9-, 11,12- and 14,15-epoxides were either inactive or enhanced proliferation. Cyclin D1 down-regulation by 17,18-epoxy-EPA was mediated by activation of the growth-suppressing p38 MAP kinase, but the alternate EPA-epoxides were inactive. CONCLUSIONS AND IMPLICATIONS: The present findings suggest that the epoxide formed by CYP enzymes at the ω-3 olefinic bond may contribute to the beneficial effects of ω-3 PUFA by down-regulating cyclin D1 and suppressing cell proliferation.
Asunto(s)
Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Células Endoteliales/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Regulación hacia Abajo/efectos de los fármacos , Ácido Eicosapentaenoico/química , Ácido Eicosapentaenoico/farmacología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Activación Enzimática/efectos de los fármacos , Compuestos Epoxi/química , Compuestos Epoxi/farmacología , Ratones , Neoplasias/etiología , Neoplasias/prevención & control , ARN Mensajero/genética , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/deficiencia , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
BACKGROUND AND PURPOSE: Human cytochrome P450 2J2 (CYP2J2) generates epoxyfatty acids that modulate cellular apoptosis and proliferation. CYP2J2 regulation has not been intensively studied but induction of the activator protein-1 (AP-1) subunit c-fos mediates CYP2J2 down-regulation in hypoxia, a component of ischaemic injury. Decreased CYP2J2 expression may contribute to tissue injury. EXPERIMENTAL APPROACH: HepG2 cells were treated with sodium nitroprusside (SNP) to induce nitrative stress, which has been associated with inflammation and infection in liver and other tissues. CYP2J2 protein and mRNA expression were evaluated by immunoblotting and real-time PCR respectively. The role of mitogen-activated protein (MAP) kinases in CYP2J2 dysregulation was assessed using specific inhibitors and dominant negative MAP kinase expression plasmids. CYP2J2-luciferase reporter constructs and electromobility shift assays (EMSAs) were used to identify SNP-regulated regions in the CYP2J2 gene. KEY RESULTS: Cytochrome P450 2J2 was down-regulated by SNP while the AP-1 proteins c-jun and c-fos were up-regulated; inhibition of p38 and ERK MAP kinases normalized their expression. The gene elements at -105/-95 and -56/-63 were required for the down-regulation of CYP2J2 induced by nitrative stress. CONCLUSIONS AND IMPLICATIONS: p38 and ERK MAP kinases transduce stress stimuli that down-regulate CYP2J2. Targeting these kinases may prevent the loss of CYP2J2 and epoxy-fatty acids that protect cells against deleterious stresses.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Nitratos/metabolismo , Activación Transcripcional , Secuencia de Bases , Línea Celular Tumoral , Citocromo P-450 CYP2J2 , Cartilla de ADN , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitroprusiato/farmacología , Reacción en Cadena de la Polimerasa , Regiones Promotoras GenéticasRESUMEN
Facile syntheses of the monounsaturated omega-3 fatty acids, (Z)-15-octadecenoic acid and (Z)-16-nonadecenoic acid, are presented. Commercially available hydroxy fatty acids were esterified and oxidised, followed by the Wittig reaction to introduce the omega-3 olefinic bond; hydrolysis yielded the omega-3 fatty acids in high purity. An examination of different reaction conditions for the Wittig step found that THF as solvent and coupling temperatures of -78 degrees C gave optimal stereoselectivity, affording the omega-3 olefins in Z:E ratios >or= 97:3. The syntheses have overall yields of approximately 43%, and utilise straightforward, robust chemistry, that may be readily scaled up and reproduced. Also presented is a method for accurately determining the double bond geometry and isomeric purity of the fatty acid products using 1H-13C-HSQC NMR and GC-MS, respectively.
Asunto(s)
Ácidos Grasos Monoinsaturados/síntesis química , Ácidos Grasos Omega-3/síntesis química , Ácidos Oléicos/síntesis química , Resonancia Magnética Nuclear Biomolecular , EstereoisomerismoRESUMEN
Cells undergo phenotypic changes after exposure to a wide range of exogenous stimuli that include growth factors, proinflammatory cytokines and environmental chemicals. Such stimuli may arise as components of disease pathogenesis and cellular injury, or as a result of exposure to environmental chemicals and radiation. These stimuli modulate the proliferation and differentiation of cells by altering the regulation of genes that control homeostasis. A generalized response appears to be a decline in the expression and function of many cytochrome P450 (CYP) genes in liver and other tissues. Thus, individuals who have been exposed to such exogenous stimuli often exhibit a decreased capacity for drug clearance, which has important consequences for concurrent drug therapy. Several signaling pathways transduce exogenous stimuli within cells, with the mitogen-activated protein kinases (MAPKs) being one of the most important. Evidence is increasing that MAPKs may impair the expression of multiple CYP genes by modulating the activity of transcription factors, including nuclear receptors, the aryl hydrocarbon receptor, and the activator protein-1 complex. MAPKs catalyze the phosphorylation of transcription complexes that incorporate these factors, which modulates their capacity to transactivate target genes, including CYPs. An understanding of the mechanisms that account for the regulatory impact of MAPKs on the transcriptional factors that regulate CYP genes will provide critical insight into the consequences from exposure to injurious stresses that impact cellular function.
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Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/enzimología , Hígado/metabolismo , Sistema de Señalización de MAP QuinasasRESUMEN
BACKGROUND AND PURPOSE: The human organic anion transporter (hOAT) family of transmembrane carrier proteins mediate the cellular flux of anionic substances, including certain hormones and anti-cancer drugs. hOAT4 is highly expressed at the apical membrane of the renal tubular cell and facilitates drug re-absorption in the kidney. In the present study, the impact of 10 nonsynonymous single nucleotide polymorphisms (SNPs) of hOAT4 on transport function in COS-7 cells was characterized. EXPERIMENTAL APPROACH: Transport uptake assay was used to assess the function of the variant transporters. Cell surface biotinylation and western blot analysis were used to investigate the expression characteristics of the transporter proteins. Comparative modelling was used to interpret the influence of nonsynonymous changes in terms of hOAT4 structure. KEY RESULTS: Four naturally occurring hOAT4 variants (L29P, R48Y, V155G and T392I) exhibited a significant loss of function. Substitution of leucine-29, which is a conserved residue in OATs, with a proline residue, impaired the synthesis or the apparent stability of the transporter and membrane insertion was disrupted in the R48Y variant. In the case of the V155G and T392I variants, impaired function was due to decreased affinity of the transporter for oestrone sulphate and impaired transporter-substrate turnover respectively. The T392I variant was inhibited more extensively than the wild-type transporter by the cationic substrate tetraethyl ammonium. CONCLUSIONS AND IMPLICATIONS: Several naturally occurring SNPs encode variant hOAT4s that may impair the renal tubular re-absorption of important drug substrates.
Asunto(s)
Transportadores de Anión Orgánico Sodio-Independiente/genética , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Estrona/análogos & derivados , Estrona/metabolismo , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
The activities of cytochrome P450-derived epoxide metabolites of omega-6 polyunsaturated fatty acids (PUFAs) in cellular homeostasis have generated considerable topical interest, but there is less information on the effects of omega-3 PUFA epoxides. Mass spectroscopic data on the epoxides of the omega-3 PUFA eicosapentaenoic acid (EPA) have been reported but the absence of corresponding NMR data currently hinders their biological assessment. In the present study five monoepoxy derivatives of EPA methyl ester were synthesized by treating EPA methyl ester with m-chloroperbenzoic acid. The individual regioisomers were purified by normal-phase chromatography and characterized by LC-MS/MS and a combination of NMR approaches including (1)H-, (13)C-, (1)H-(1)H-COSY, (1)H-(13)C-HSQC, and (1)H-(13)C-HMBC. The chromatographic properties for these monoepoxides were studied in normal-phase and reversephase-HPLC systems and the MS/MS fragmentation patterns using electrospray ionization were established. This paper also focuses on the NMR characterization of epoxide, olefinic and methylenic moieties and the complete assignments of the isomers.
Asunto(s)
Ácido Eicosapentaenoico/análogos & derivados , Benzoatos/química , Ácido Eicosapentaenoico/síntesis química , Ácido Eicosapentaenoico/química , Isomerismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Methyl esters of C18 polyunsaturated fatty acids, including gamma-linolenic acid, alpha-linolenic acid and stearidonic acid, were epoxidised using m-chloroperbenzoic acid. Nine monoepoxides were obtained by normal-phase HPLC, identified using LC-MS and NMR, and characterized by NMR spectroscopy and mass spectrometry. This study is focused on structural characterization using LC-MS and LC/APCI/MS/MS. The elution profiles of these monoepoxides in RP-HPLC are determined as 12,13->9,10->6,7-epoxy, 9,10->15,16->12,13-epoxy and 15,16->12,13->9,10-epoxy derivatives of gamma-linolenic, alpha-linolenic and stearidonic acid methyl esters, respectively. The major diagnostic fragmentations in MS/MS identified are postulated to be induced by cleavages of the epoxide ring and alpha-bond cleavage to the epoxy group from [M+H]+ and/or [M+H-MeOH]+.
Asunto(s)
Bioquímica/métodos , Biofisica/métodos , Compuestos Epoxi/química , Ácidos Grasos/química , Espectrometría de Masas/métodos , Clorobenzoatos/química , Cromatografía Liquida/métodos , Iones , Modelos Químicos , Conformación Molecular , Peso Molecular , Oxígeno/química , Factores de TiempoRESUMEN
Methyl esters of gamma-linolenic acid, alpha-linolenic acid and stearidonic acid were epoxidised using m-chloroperbenzoic acid to achieve nine cis-monoepoxy-C18 fatty acid methyl esters (FAMEs), including novel methyl cis-monoepoxy derivatives of stearidonic acid and a cis-6,7-epoxy derivative of gamma-linolenic acid. These nine monoepoxy FAMEs were purified by normal-phase HPLC, identified by LC-MS, 1H and 13C NMR, and characterized by mass spectrometry and NMR spectroscopy. This study is focused on structural characterization of these C18 monoepoxy FAMEs using techniques in NMR spectroscopy including 1H, 13C, 1H-1H correlated spectroscopy (COSY) and 1H-13C heteronuclear correlation (HETCOR). The proton and carbon NMR chemical shifts of the epoxide, the double bonds, and the interrupted methylenes are assigned. Also discussed is an interpretation of the 1H and 13C NMR spectra of these monoepoxides including the changes in the 13C resonance of the olefinic carbons on the neighboring double bonds resulting from epoxide formation.