RESUMO
The concentrations of circulating glucocorticoids are regulated by their synthesis and metabolism. Cytochrome P450 (CYP), primarily expressed in the liver, is one of the main metabolizers of glucocorticoids. Since glucocorticoids, as well as monoamines, are fundamental in stress, the link between hepatic glucocorticoid metabolism and central monoamine transmission might be important in pathophysiology of stress-related disorders. We had previously reported that CYP inhibition by proadifen (SKF525) led to the inhibition of central serotonin (5-HT) neurons. The aim of this study was to investigate the effect of SKF525 on the excitability of central catecholamine neurons. Adult male Wistar rats were administered SKF525 forty-eight, twenty-four, and one hour before electrophysiological assessments. Control animals were injected saline. Rats were anesthetized with chloral hydrate and glass electrodes were inserted into the locus coeruleus (LC) or ventral tegmental area (VTA). Noradrenaline neurons of the LC and dopamine of the VTA neurons were identified, and their firing activity was recorded. It was found that the SKF525 enhanced the excitability of noradrenaline and reduced the excitability of dopamine neurons. We suggest that corticosterone-induced inhibition of 5-HT neurons underlines, at least in part, the ability of SKF525 to stimulate noradrenaline neurons. The inhibitory effect of SKF525 on dopamine neurons might be in turn secondary to the stimulatory effect of this compound on noradrenaline neurons.
Assuntos
Catecolaminas , Proadifeno , Serotonina , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Catecolaminas/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Glucocorticoides/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Norepinefrina/metabolismo , Proadifeno/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Serotonina/metabolismoRESUMO
Colon cancer is the third most common cancer and the second leading cause of cancer-related death in the United States, emphasizing the need for the discovery of new cellular targets. Using a metabolomics approach, we report here that epoxygenated fatty acids (EpFA), which are eicosanoid metabolites produced by cytochrome P450 (CYP) monooxygenases, were increased in both the plasma and colon of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon cancer mice. CYP monooxygenases were overexpressed in colon tumor tissues and colon cancer cells. Pharmacologic inhibition or genetic ablation of CYP monooxygenases suppressed AOM/DSS-induced colon tumorigenesis in vivo. In addition, treatment with 12,13-epoxyoctadecenoic acid (EpOME), which is a metabolite of CYP monooxygenase produced from linoleic acid, increased cytokine production and JNK phosphorylation in vitro and exacerbated AOM/DSS-induced colon tumorigenesis in vivo. Together, these results demonstrate that the previously unappreciated CYP monooxygenase pathway is upregulated in colon cancer, contributes to its pathogenesis, and could be therapeutically explored for preventing or treating colon cancer. SIGNIFICANCE: This study finds that the previously unappreciated CYP monooxygenase eicosanoid pathway is deregulated in colon cancer and contributes to colon tumorigenesis.
Assuntos
Carcinogênese/efeitos dos fármacos , Neoplasias do Colo/prevenção & controle , Sistema Enzimático do Citocromo P-450/química , Eicosanoides/metabolismo , Inibidores Enzimáticos/farmacologia , Metabolômica , Animais , Antifúngicos/farmacologia , Apoptose , Azoximetano/toxicidade , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Proliferação de Células , Clotrimazol/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sistema Enzimático do Citocromo P-450/fisiologia , Sulfato de Dextrana/toxicidade , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proadifeno/farmacologia , RNA Interferente Pequeno/genética , Células Tumorais CultivadasRESUMO
Acetylshikonin is a biologically active compound with anti-cancer and anti-inflammatory activity, which is isolated from the roots of Lithospermum erythrorhizoma. An inhibitory effect of acetylshikonin against CYP2J2 activity was discovered recently. Based on this result, this study was expanded to evaluate the inhibitory effects of acetylshikonin against nine different cytochrome P450 (P450) isoforms in human liver microsomes (HLMs) using substrate cocktails incubation assay. Acetylshikonin showed a strong inhibitory effect against all P450s tested with IC50 values of 1.4-4.0 µ m. Pre-incubation of acetylshikonin with HLMs and NADPH did not alter the inhibition potency, indicating that acetylshikonin is not a mechanism-based inhibitor. SKF-525A, a widely used non-specific P450 inhibitor, had no inhibitory activity against CYP1A2, 2A6, 2E1 and 2J2, while it showed an inhibitory effect against CYP2B6, CYP2C19 and 2D6 with IC50 values of 2.5, 3.6 and 0.5 µ m, respectively. Our findings indicate that acetylshikonin may be a novel general P450 inhibitor, which could replace SKF-525A.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Antraquinonas/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Lithospermum/química , Microssomos Hepáticos/enzimologia , Proadifeno/farmacologiaRESUMO
The gonadal hormones testosterone (T) in adult males and estradiol (E2) in adult females have been reported to modulate behavioral effects of ∆9-tetrahydrocannabinol (THC). This study determined whether activational effects of T and E2 are sex-specific, and whether hormones modulate production of the active metabolite 11-hydroxy-THC (11-OH-THC) and the inactive metabolite 11-nor-9-carboxy-THC (THC-COOH). Adult male and female rats were gonadectomized (GDX) and treated with nothing (0), T (10-mm Silastic capsule/100g body weight), or E2 (1-mm Silastic capsule/rat). Three weeks later, saline or the cytochrome P450 inhibitor proadifen (25mg/kg; to block THC metabolism and boost THC's effects) was injected i.p.; 1h later, vehicle or THC (3mg/kg females, 5mg/kg males) was injected i.p., and rats were tested for antinociceptive and motoric effects 15-240min post-injection. T did not consistently alter THC-induced antinociception in males, but decreased it in females (tail withdrawal test). Conversely, T decreased THC-induced catalepsy in males, but had no effect in females. E2 did not alter THC-induced antinociception in females, but enhanced it in males. The discrepant effects of T and E2 on males' and females' behavioral responses to THC suggests that sexual differentiation of THC sensitivity is not simply due to activational effects of hormones, but also occurs via organizational hormone or sex chromosome effects. Analysis of serum showed that proadifen increased THC levels, E2 increased 11-OH-THC in GDX males, and T decreased 11-OH-THC (and to a lesser extent, THC) in GDX females. Thus, hormone modulation of THC's behavioral effects is caused in part by hormone modulation of THC oxidation to its active metabolite. However, the fact that hormone modulation of metabolism did not alter THC sensitivity similarly on all behavioral measures within each sex suggests that other mechanisms also play a role in gonadal hormone modulation of THC sensitivity in adult rats.
Assuntos
Analgésicos/farmacologia , Dronabinol/antagonistas & inibidores , Dronabinol/farmacologia , Estradiol/farmacologia , Caracteres Sexuais , Testosterona/farmacologia , Animais , Catalepsia/induzido quimicamente , Catalepsia/prevenção & controle , Dronabinol/análogos & derivados , Dronabinol/sangue , Interações Medicamentosas , Feminino , Masculino , Proadifeno/farmacologia , RatosRESUMO
Proadifen (SKF-525A) is a P450 monooxygenase inhibitor with potential anti-proliferative activity and the ability to potentiate the toxicity of hypericin-mediated photodynamic therapy and mitoxantrone via alteration of ABC transport proteins. Elevated expression of some ABC transporters may also determine the efficacy of cisplatin-based chemotherapy. Thus, the purpose of this study was to investigate the ability of proadifen to sensitize A2780 and A2780cis ovarian cancer cells to cisplatin (CDDP). Herein, we show for the first time that proadifen sensitized resistant ovarian cancer cells to CDDP-induced cell death. The chemosensitizing effect of proadifen on CDDP action was also confirmed by MTT assays in multicellular spheroids. The possible mechanisms responsible for the enhanced cytotoxicity of proadifen/CDDP combined treatment may be attributed to a decrease of reduced relative glutathione levels, downregulation of multidrug resistance-associated proteins 1 and 2 (MRP1, MRP2) and attenuation of survivin expression. Taken together, our results indicate that proadifen is a promising compound for further in vivo experiments related to overcoming multidrug resistance and sensitization of resistant ovarian carcinoma to CDDP.
Assuntos
Cisplatino/farmacologia , Proadifeno/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação para Baixo , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Membranas Mitocondriais/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Survivina , Regulação para Cima , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
PURPOSE: Mevalonate pathway inhibitors are potentially useful chemotherapeutic agents showing growth inhibition and pro-apoptotic effects in cancer cells. The effects of statins and bisphosphonates on cancer growth are attributed to a reduction in protein isoprenylation. Post-translational modification and activation of GTPase binding Ras superfamily permit the recruitment of these signal proteins to membranes where they mediate the cancer phenotype. Here, the effects of three inhibitors of the mevalonate pathway and one specific inhibitor of sterol regulatory element-binding proteins were studied in both an ER-negative, Ras-inactive breast (MDA-MB-231) and lung adenocarcinoma (CaLu-1) cells in vitro. METHODS: Treated cells were subject to genome-wide gene expression profiling. A gene subset was established so that the epithelial to mesenchymal transition (EMT) could be observed and compared with signalling protein shifts. RESULTS: Within the subset, some genes normally up-regulated during EMT were asymmetrically reduced by a Δ-24 DHCR inhibitor in the lung cells. Signalling proteins associated with caveolae were down-regulated by this oxidoreductase inhibitor, while those associated with membrane rafts were up-regulated. CONCLUSIONS: This study decouples isoprenylation effects from cholesterol events per se. The data support a hypothesis that caveolae are abolished by Δ-24 DHCR intervention and it is revealed that these microdomains are vital EMT signalling structures for lung cells but not ER- and Ras-negative breast cells. When signalling by extracellular signals is quenched by removal of the hydrophilic conduit provided by caveolae, the transcriptome responds by moving the cellular identity towards quiescence.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/secundário , Antineoplásicos/uso terapêutico , Cavéolas/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Ácido Mevalônico/antagonistas & inibidores , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Antineoplásicos/farmacologia , Cavéolas/fisiologia , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Metástase Neoplásica , Proteínas do Tecido Nervoso/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Fenótipo , Proadifeno/farmacologia , Proadifeno/uso terapêuticoRESUMO
The aim of this work was to investigate the effects of chronic treatment with the combination of ezetimibe and simvastatin on gluconeogenesis in rat liver. Rats were treated daily for 28 days with the combination of ezetimibe and simvastatin (10/40 mg/kg) by oral gavage. To measure gluconeogenesis and the associated pathways, isolated perfused rat liver was used. In addition, subcellular fractions, such as microsomes and mitochondria, were used for complementary measures of enzymatic activities. Treatment with the combination of simvastatin and ezetimibe resulted in a decrease in gluconeogenesis from pyruvate (-62%). Basal oxygen consumption of the treated animals was higher (+22%) than that of the control rats, but the resulting oxygen consumption that occurred after pyruvate infusion was 43% lower in animals treated with the combination of simvastatin and ezetimibe. Oxygen consumption in the livers from treated animals was completely inhibited by cyanide (electron transport chain inhibitor), but not by proadifen (cytochrome P450 inhibitor). Chronic treatment with ezetimibe/simvastatin decreased the activity of the key enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase by 59% and 45%, respectively, which is probably the major reason for the decreased gluconeogenesis seen in ezetimibe-/simvastatin-treated rats. It is also possible that part of the effect of this combination on gluconeogenesis and on the oxygen consumption is related to the impairment of mitochondrial energy transduction.
Assuntos
Ezetimiba/farmacologia , Gluconeogênese/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fígado/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Cianetos/farmacologia , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Frutose-Bifosfatase/metabolismo , Glucose-6-Fosfatase/metabolismo , Fígado/citologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Proadifeno/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Using Fura-2AM microfluorimetry the possible involvement of epoxygenase pathway of arachidonic acid metabolism in the effect of glutoxim and molixan on intracellular Ca2+ concentration in rat peritoneal macrophages was investigated. It was shown for the first time that preincubation of the macrophages with epoxygenase inhibitors, proadifen and econazole, significantly decreases the intracellular Ca2+ concentration increase induced by glutoxim and molixan. The addition of the epoxygenase inhibitors during the already developed store-dependent Ca(2+)-entry induced by glutoxim or molixan partially inhibits Ca(2+)-entry. The obtained data suggest the involvement of the products and/or enzymes of epoxygenase pathway of the arachidonic acid metabolism in the glutoxim and molixan effect on the Ca2+ signaling processes in macrophages.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Econazol/farmacologia , Inibidores Enzimáticos/farmacologia , Macrófagos Peritoneais/metabolismo , Oligopeptídeos/farmacologia , Proadifeno/farmacologia , Animais , Macrófagos Peritoneais/citologia , Masculino , Ratos , Ratos WistarRESUMO
Multidrug resistance caused by the overexpression of ABC transporter proteins in cancer cells remains a major obstacle limiting chemotherapy efficacy. Drugs inhibiting these transporters have been shown to increase the anti-proliferative properties of chemotherapeutics. As we previously described, proadifen, a P450 monooxygenase inhibitor, might also be able to inhibit some ABC transporters, including breast cancer resistance protein (BCRP). Because mitoxantrone (MTX) is a strong BCRP substrate and is often used in the treatment of leukemia, we investigated the effect of 24 h proadifen pre-treatment on the cytotoxicity of MTX in leukemic cell lines that are sensitive to MTX (HL-60) and MTX-resistant ABCG2-overexpressing subclone (cBCRP). We show for the first time that proadifen is able to enhance the cytotoxic properties of MTX in cBCRP cells, particularly through the inhibition of BCRP expression and activity. This proadifen-MTX synergism was also mediated by the inhibition of various cellular proteins engaged in apoptosis, including Mc-1, Bcl-xL, survivin and activation of procaspase-3. Proadifen also decreased the expression of γH2AX, which is involved in the recruitment of reparation proteins. Moreover, the inhibition of DNA damage repair proteins Ku86 and B23 after proadifen treatment indicate a possible role of proadifen in DNA repair blockage, thus suppressing the reparation rate of MTX-induced DSBs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Promielocítica Aguda/patologia , Mitoxantrona/farmacologia , Proadifeno/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fipronil is an insecticide used to control pests in animals and plants that can causes hepatotoxicity in animals and humans, and it is hepatically metabolized to fipronil sulfone by cytochrome P-450. The present study aimed to characterize the effects of fipronil (10-50µM) on energy metabolism in isolated perfused rat livers. In fed animals, there was increased glucose and lactate release from glycogen catabolism, indicating the stimulation of glycogenolysis and glycolysis. In the livers of fasted animals, fipronil inhibited glucose and urea production from exogenous l-alanine, whereas ammonia and lactate production were increased. In addition, fipronil at 50µM concentration inhibited the oxygen uptake and increased the cytosolic NADH/NAD⺠ratio under glycolytic conditions. The metabolic alterations were found both in livers from normal or proadifen-pretreated rats revealing that fipronil and its reactive metabolites contributed for the observed activity. The effects on oxygen uptake indicated that the possible mechanism of toxicity of fipronil involves impairment on mitochondrial respiratory activity, and therefore, interference with energy metabolism. The inhibitory effects on oxygen uptake observed at the highest concentration of 50µM was abolished by pretreatment of the rats with proadifen indicating that the metabolites of fipronil, including fipronil sulfone, acted predominantly as inhibitors of respiratory chain. The hepatoxicity of both the parent compound and its reactive metabolites was corroborated by the increase in the activity of lactate dehydrogenase in the effluent perfusate in livers from normal or proadifen-pretreated rats.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Canais de Cloreto/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Moduladores de Transporte de Membrana/toxicidade , Pirazóis/toxicidade , Animais , Biotransformação/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Glicogenólise/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Técnicas In Vitro , Inseticidas/metabolismo , Fígado/metabolismo , Masculino , Moduladores de Transporte de Membrana/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , Perfusão , Proadifeno/farmacologia , Pirazóis/metabolismo , Ratos Wistar , Ureia/metabolismoRESUMO
BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.
Assuntos
Acetoexamida/farmacologia , Neoplasias da Mama/tratamento farmacológico , Endotélio Linfático/efeitos dos fármacos , Isoxsuprina/farmacologia , Vasos Linfáticos/efeitos dos fármacos , Nifedipino/farmacologia , Proadifeno/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular , Quimiotaxia/efeitos dos fármacos , Técnicas de Cocultura , Sinergismo Farmacológico , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hipoglicemiantes/farmacologia , Metástase Linfática , Vasos Linfáticos/irrigação sanguínea , Vasos Linfáticos/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Vasodilatadores/farmacologiaRESUMO
Proadifen (SKF-525A) is a well-known inhibitor of cytochrome P450 monooxygenases. Besides the prevention of drug metabolism it affects the proliferation of cancer cells, although the mechanisms of possible anti-cancer activity of proadifen have not been fully understood yet. The aim of this study therefore was to evaluate the potential anti-proliferative effect of proadifen on HT-29 colon cancer cells. Our results show that proadifen inhibited the growth of HT-29 cells by the accumulation of cells in the G1 phase of the cell cycle, reduction of metabolic activity and colony formation and by the induction of apoptosis. Analyses of Western blots and flow cytometry revealed time- and dose-dependent phosphatidylserine externalization, caspase-3 activation and PARP cleavage. Intense upregulation of NAG-1 and ATF3 and downregulation of Mcl-1 and Egr-1 were also observed. Further investigation showed that NAG-1 gene silencing by siRNA had no effect on the pro-apoptotic action of proadifen. In contrast, we found that AR-A014418, the specific inhibitor of glycogen synthase kinase-3 ß (GSK-3ß), significantly decreased proadifen-induced apoptosis. Inactivation of GSK-3ß (phosphorylation at serine 9) resulted in changes in phosphatidylserine externalization and caspase-3 activation. These data suggest that GSK-3ß is an important factor in the induction of apoptosis in HT-29 colon cancer cells treated with proadifen.
Assuntos
Antineoplásicos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Proadifeno/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Ureia/análogos & derivados , Adenocarcinoma , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo , Glicogênio Sintase Quinase 3 beta , Células HT29 , Humanos , Ureia/farmacologiaRESUMO
Serine proteases are versatile signaling molecules and often exert this function by activating the proteinase-activated receptors (PAR(1)-PAR(4)). Our previous study on the mouse cecum has shown that the PAR(1)-activating peptide (AP) and PAR(2)-AP both induced electrogenic anion secretion. This secretion mediated by PAR(1) probably occurred by activating the receptor on the submucosal secretomotor neurons, while PAR(2)-mediated anion secretion probably occurred by activating the receptor on the epithelial cells. This present study was aimed at using trypsin to further elucidate the roles of serine proteases and PARs in regulating intestinal anion secretion. A mucosal-submucosal sheet of the mouse cecum was mounted in Ussing chambers, and the short-circuit current (I(sc)) was measured. Trypsin added to the serosal side increased I(sc) with an ED(50) value of approximately 100 nM. This I(sc) increase was suppressed by removing Cl(-) from the bathing solution. The I(sc) increase induced by 100 nM trypsin was substantially suppressed by tetrodotoxin, and partially inhibited by an NK(1) receptor antagonist, by a muscarinic Ach-receptor antagonist, and by 5-hydroxytryptamine-3 (5-HT(3)) and 5-HT(4) receptor antagonists. The I(sc) increase induced by trypsin was partially suppressed when the tissue had been pretreated with PAR(1)-AP, but not by a pretreatment with PAR(2)-AP. These results suggest that the serine protease, trypsin, induced anion secretion by activating the enteric secretomotor nerves. This response was initiated in part by activating PAR(1) on the enteric nerves. Serine proteases and PARs are likely to be responsible for the diarrhea occurring under intestinal inflammatory conditions.
Assuntos
Ânions/metabolismo , Ceco/metabolismo , Sistema Nervoso Entérico/fisiologia , Receptor PAR-1/metabolismo , Tripsina/farmacologia , Animais , Ceco/efeitos dos fármacos , Eicosanoides/fisiologia , Sistema Nervoso Entérico/efeitos dos fármacos , Indometacina/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Masoprocol/farmacologia , Camundongos , Oligopeptídeos/fisiologia , Proadifeno/farmacologia , Inibidores de Proteases/farmacologia , Serotonina/farmacologiaRESUMO
Studies of cancer chemoprevention with caffeic acid phenethyl ester (CAPE) in the resistant hepatocyte model of hepatocarcinogenesis have shown the participation of CYP drug metabolizing enzymes. To prevent neoplastic and preneoplasic lesions, we must specifically identify which CYP activities are modified in the mechanism of action of CAPE. Male Fischer-344 rats were pretreated with CAPE twelve hours before administration of diethylnitrosamine (DEN) and were sacrificed twelve hours after CAPE and twelve hours, twenty-four hours, twenty-four days, and twelve months after DEN. Other rats were treated with the CYP inhibitors α-naphthoflavone or SKF525A and sacrificed twenty-four hours and twenty-four days after DEN. Microsomes were obtained from livers to quantify protein using Western blot. Diethylnitrosamine metabolism was measured based on nitrite formation and liver histology using GGT histochemistry. Caffeic acid phenethyl ester diminished the protein levels of CYP1A2 and CYP2B1/2. The inhibition of CYP2B1/2 prevented the appearance of preneoplastic lesions. Microsomal assays demonstrated that CAPE interfered with DEN activation diminishing nitrites similar to SKF525A and probably mediated by CYP2B1/2 inhibition. A single dose of CAPE before DEN treatment reduced the appearance of tumors by 43%. These results confirmed that CAPE is a promising agent to confer chemoprotection in liver cancer and should be considered for human therapies.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Ácidos Cafeicos/farmacologia , Citocromo P-450 CYP2B1/metabolismo , Neoplasias Hepáticas Experimentais/prevenção & controle , Álcool Feniletílico/análogos & derivados , Esteroide Hidroxilases/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Benzoflavonas/farmacologia , Testes de Carcinogenicidade , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2B1/antagonistas & inibidores , Citocromos/antagonistas & inibidores , Citocromos/metabolismo , Dietilnitrosamina , Modelos Animais de Doenças , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Álcool Feniletílico/farmacologia , Proadifeno/farmacologia , Ratos , Ratos Endogâmicos F344 , Esteroide Hidroxilases/antagonistas & inibidoresRESUMO
OBJECTIVES: Of several enzymes metabolizing xenobiotics, cytochrome P450 (CYP) and peroxidase enzymes seem to be most important. One of the major challenges in studies investigating metabolism of xenobiotics is to resolve which of these two groups of enzymes is predominant to metabolize individual xenobiotic compounds. Utilization of selective inhibitors of CYP and peroxidase enzymes might be a useful tool to identify the contribution of these enzymes to metabolism of xenobiotics in samples, where both types of enzymes are present. The aim of this study was to investigate specificities of several known CYP inhibitors to these enzymes; whether they inhibit only the CYP enzymes and do not inhibit peroxidases. METHODS: Since the oxidation of o-anisidine catalyzed by a model peroxidase used, horseradish peroxidase (HRP), is a two-substrate reaction, the inhibition potential of tested chemicals was studied with respect to both peroxidase substrates, o-anisidine and hydrogen peroxide. Initial velocities of o-anisidine oxidation by HRP under various conditions were determined spectrophotometrically. RESULTS: The CYP inhibitors metyrapone, troleandomycine, disulfiram, sulfaphenazole, quinidine and 1-aminobenzotriazole do not inhibit o-anisidine oxidation catalyzed by HRP. In contrast, ketoconazole, diethyldithiocarbamate, ellipticine, α-naphtoflavone, proadifen SKF525A, piperonylbutoxide, were found to inhibit not only the CYPs, but also the HRP-mediated oxidation of o-anisidine. Interestingly, α-naphtoflavone inhibits oxidation of o-anisidine by HRP with respect to H2O2, but not with respect to o-anisidine. Diethyldithiocarbamate is the most potent peroxidase inhibitor of o-anisidine oxidation with Ki with respect to o-anisidine of 10 µM and Ki with respect to H2O2 of 60 µM, being even the better peroxidase inhibitor than the classical "peroxidase inhibitor" - propyl gallate (Ki with respect to o-anisidine of 60 µM and Ki with respect to H2O2 of 750 µM). CONCLUSIONS: The results of the present study demonstrate that 1-aminobenzotriazole, a potent inhibitor of various CYP enzymes, seems to be the best candidate suitable for utilization in studies evaluating participation of CYP enzymes in metabolism of xenobiotics in various complex biological materials containing both CYP and peroxidase enzymes. Moreover, precaution to prevent misinterpretation of results is necessary in cases when proadifen SKF525A, piperonylbutoxide, diethyldithiocarbamate, ketoconazole, α-naphtoflavone and ellipticine are used in similar studies (as CYP inhibitors in various complex biological materials containing both CYP and peroxidase enzymes), since these chemicals can except of CYP enzymes inhibit also peroxidase-mediated reactions.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Peroxidase do Rábano Silvestre/antagonistas & inibidores , Triazóis/farmacologia , Benzoflavonas/química , Benzoflavonas/farmacologia , Dissulfiram/química , Dissulfiram/farmacologia , Ditiocarb/química , Ditiocarb/farmacologia , Elipticinas/química , Elipticinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Cetoconazol/química , Cetoconazol/farmacologia , Metirapona/química , Metirapona/farmacologia , Butóxido de Piperonila/química , Butóxido de Piperonila/farmacologia , Proadifeno/química , Proadifeno/farmacologia , Quinidina/química , Quinidina/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Sulfafenazol/química , Sulfafenazol/farmacologia , Triazóis/química , Troleandomicina/química , Troleandomicina/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Gentiopicroside (GPS) is a secoiridoid glucoside isolated from the ethanol extract of Gentianae Radix with a content of 13%, which has been used for centuries in Chinese as a digestive aid. AIM OF THE STUDY: This study investigates the pharmacokinetics of GPS and its metabolic pathway for the liver ischemia/reperfusion (I/R) in rats. MATERIALS AND METHODS: The experimental animals were anesthetized intraperitoneally (i.p.) with a mixture of urethane (1.0 g/kg) and α-chloralose (0.1 g/kg). A midline laparatomy was performed and the liver hilum was gently exposed. All structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were occluded with silk thread for 30 min. Ischemia was followed by a sudden reperfusion after removing the occluding threads. After 60 min reperfusion, the rats received a single intravenous 5 mg/kg dose of GPS. RESULTS: The area under concentration curve (AUC) was significantly increased; however, the clearance (Cl) was significantly decreased in the liver I/R rats. Furthermore, after pretreated with SKF-525A (50 mg/kg, i.p.), a cytochrome P450 (CYP) inhibitor, AUC, elimination half-life (t(1/2)) and the mean residence time (MRT) of GPS in rat blood were significantly increased, suggesting that CYP was involved in the metabolism of GPS. For the group without liver I/R, GPS was administered at doses of 5 mg/kg and 100 mg/kg intravenously and orally, respectively. The pharmacokinetic results indicated that the AUC was 565±95.1 and 1163±273 min µg/mL and the t(1/2) of GPS was 71±9 and 106±17 min after intravenous and oral administration, respectively. The oral bioavailability of GPS was 10.3±2.4% in the rats. CONCLUSIONS: The status of I/R might prolong the disposition of GPS, and the plasma concentration of GPS in the liver I/R injury rats was significantly increased. The increased body exposure of GPS in the treatment of liver I/R may result from the decreased metabolism of GPS mediated by CYP in the liver.
Assuntos
Medicamentos de Ervas Chinesas/farmacocinética , Gentiana , Glucosídeos Iridoides/farmacocinética , Fígado/irrigação sanguínea , Fígado/metabolismo , Traumatismo por Reperfusão/metabolismo , Administração Oral , Animais , Área Sob a Curva , Disponibilidade Biológica , Biotransformação , Cromatografia Líquida de Alta Pressão , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Gentiana/química , Meia-Vida , Injeções Intravenosas , Glucosídeos Iridoides/administração & dosagem , Glucosídeos Iridoides/sangue , Glucosídeos Iridoides/isolamento & purificação , Fígado/efeitos dos fármacos , Masculino , Medicina Tradicional Chinesa , Taxa de Depuração Metabólica , Plantas Medicinais , Proadifeno/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/tratamento farmacológico , Reprodutibilidade dos Testes , Espectrofotometria UltravioletaRESUMO
dl-Praeruptorin A (Pd-Ia) is the major active constituent of the traditional Chinese medicine Peucedanum praeruptorum Dunn. Recently it has been identified as a novel agent in the treatment and prevention of cardiovascular diseases. Accordingly, we investigated the metabolism of Pd-Ia in rat liver microsomes. The involvement of cytochrome P450 (CYP) and CYP isoforms were identified using a CYP-specific inhibitor (SKF-525A), CYP-selective inhibitors (α-naphthoflavone, metyrapone, fluvastatin, quinidine, disulfiram, ketoconazole and ticlopidine) and CYP-selective inducers (phenobarbital, dexamethasone and ß-naphthoflavone). Residual concentrations of the substrate and metabolites were determined by HPLC, and further identified by their mass spectra and chromatographic behavior. These experiments showed that CYP450 is involved in Pd-Ia metabolism, and that the major CYP isoform responsible is CYP3A1/2, which acts in a concentration-dependent manner. Four Pd-Ia metabolites (M1, M2, M3, and M4) were detected after incubation with rat liver microsomes. Hydroxylation was the primary metabolic pathway of Pd-Ia, and possible chemical structures of the metabolites were identified. Further research is now needed to link the metabolism of Pd-Ia to its drug-drug interactions.
Assuntos
Fármacos Cardiovasculares/metabolismo , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Fármacos Cardiovasculares/farmacologia , Cromatografia Líquida de Alta Pressão , Cumarínicos/farmacologia , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Medicamentos de Ervas Chinesas , Indução Enzimática , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Hidroxilação , Isoenzimas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Estrutura Molecular , Proadifeno/metabolismo , Proadifeno/farmacologia , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
OBJECTIVE: To investigate the mechanism of enhanced large conductance calcium-activated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). METHODS: Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16, 17-epoxydocosapentaenoic acid (16, 17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. RESULTS: BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2 ± 2.7)% of total potassium currents (n = 20). DHA could activate BK channels, and its 50% effective concentration (EC(50)) was (0.23 ± 0.03) µmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16, 17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC(50) was (19.7 ± 2.8) nmol/L. CONCLUSION: DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.
Assuntos
Vasos Coronários/citologia , Ácidos Docosa-Hexaenoicos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Animais , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Ácidos Graxos Insaturados/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Proadifeno/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Eperisone, 4-ethyl-2-methyl-3-piperidinopropiophenone, is a centrally acting muscle relaxant widely used to relieve muscle stiffness and back pain. In this study, enantioselectivity for carbonyl reduction of eperisone was investigated in human liver microsomes, and the enzymes involved in the carbonyl reduction were characterised. Carbonyl reduction of eperisone predominantly occurred in microsomal fractions and 11ß-hydroxysteroid dehydrogenase type 1(11ß-HSD 1) played a major role in this reaction as judged by selective inhibition of the activity by BVT-14225 and KR-66344. The kinetic study with (+)-S- and (-)-R-eperisone showed that the formation of the carbonyl reduced metabolite (M5) from the (-)-R-isomer was more efficient than that from the (-)-S-isomer. As eperisone is a racemic compound with one chiral centre, the carbonyl reduced metabolite of eperisone (M5) may have four possible diastereoisomeric structures. Chiral separation of incubation mixtures of racemic eperisone with human liver microsome revealed that (1S, 2S)-M5 and (1R, 2R)-M5 were generated specifically from (+)-S- and (-)-R-eperisone, respectively. Selective formation of anti-diastereomers was further confirmed by incubation of individual enantiomer with microsomes. Carbonyl reduction of eperisone by microsomal 11ß-HSD 1 may significantly contribute to the metabolic disposition of eperisone in human and (-)-R-isomer is preferentially reduced by this enzyme.
Assuntos
Carbono/metabolismo , Microssomos Hepáticos/metabolismo , Propiofenonas/química , Propiofenonas/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Relaxantes Musculares Centrais/química , Relaxantes Musculares Centrais/isolamento & purificação , Relaxantes Musculares Centrais/metabolismo , Oxirredução/efeitos dos fármacos , Proadifeno/farmacologia , Propiofenonas/isolamento & purificação , Estereoisomerismo , Sulfonamidas/farmacologia , Tiazóis/farmacologiaRESUMO
Adverse reactions of acetaminophen have been associated to oxidative stress, which may be elicited by reactive oxygen species (ROS) and/or production of the metabolite NAPQI. Both phenomena would arise through the activity of liver cytochrome P450 (CYP450) system, but their contribution to this oxidative stress is yet to be clarified. A NADPH oxidase activity has been proposed in rat liver microsomes. This activity may be due to the presence of NAD(P)H oxidase (NOX) isoforms in liver endoplasmic reticulum. Both NOX and the CYP450 system activities can catalyze ROS generation using NADPH as a cofactor. Therefore, acetaminophen biotransformation, which requires NADPH, may promote ROS generation through either activity or both. To discriminate between these possibilities, rat liver microsomes were incubated with acetaminophen and NADPH in the presence or absence of specific inhibitors. Incubation with NADPH and acetaminophen elicited lipid peroxidation and decreased thiol content and glutathione-S-transferase (GST) activity. The NOX inhibitors apocynin and plumbagin prevented all these phenomena but the decrease in thiol content. In contrast, this decrease was completely prevented by the specific CYP450 system inhibitor SKF-525A. These data suggest that ROS generation following incubation of microsomes with acetaminophen and NADPH appears to be mainly caused by a NOX activity. In light of these data, toxicity of acetaminophen is discussed.