ABSTRACT
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.
Subject(s)
Catecholamines , Proadifen , Serotonin , Animals , Brain/drug effects , Brain/metabolism , Catecholamines/metabolism , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Glucocorticoids/metabolism , Male , Neurons/drug effects , Norepinephrine/metabolism , Proadifen/pharmacology , Rats , Rats, Sprague-Dawley , Rats, Wistar , Serotonin/metabolismABSTRACT
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.
Subject(s)
Carcinogenesis/drug effects , Colonic Neoplasms/prevention & control , Cytochrome P-450 Enzyme System/chemistry , Eicosanoids/metabolism , Enzyme Inhibitors/pharmacology , Metabolomics , Animals , Antifungal Agents/pharmacology , Apoptosis , Azoxymethane/toxicity , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Proliferation , Clotrimazole/pharmacology , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cytochrome P-450 Enzyme System/physiology , Dextran Sulfate/toxicity , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Proadifen/pharmacology , RNA, Small Interfering/genetics , Tumor Cells, CulturedABSTRACT
The aim of this study was to investigate the effect an inhibitor of cytochrome-P450, proadifen hydrochloride (SKF525), on the excitability of serotonin 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 stereotaxically inserted into the dorsal raphe nucleus (DRN). Serotonin neurons were identified and their firing activity was recorded. It was found that the SKF525 inhibits the excitability of 5-HT neurons. We suggest that corticosterone might play a key role in the SKF525-induced inhibition of 5-HT neurons.
Subject(s)
Brain , Animals , Cytochrome P-450 Enzyme System , Male , Neurons , Proadifen , Rats , Rats, Wistar , SerotoninABSTRACT
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.
Subject(s)
Anthraquinones/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Microsomes, Liver/drug effects , Anthraquinones/administration & dosage , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents, Phytogenic/administration & dosage , Cytochrome P-450 Enzyme Inhibitors/administration & dosage , Cytochrome P-450 Enzyme System/drug effects , Humans , Inhibitory Concentration 50 , Lithospermum/chemistry , Microsomes, Liver/enzymology , Proadifen/pharmacologyABSTRACT
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.
Subject(s)
Analgesics/pharmacology , Dronabinol/antagonists & inhibitors , Dronabinol/pharmacology , Estradiol/pharmacology , Sex Characteristics , Testosterone/pharmacology , Animals , Catalepsy/chemically induced , Catalepsy/prevention & control , Dronabinol/analogs & derivatives , Dronabinol/blood , Drug Interactions , Female , Male , Proadifen/pharmacology , RatsABSTRACT
The cytochrome P450 (CYP) inhibitor SKF-525A is commonly used to study drug metabolism and toxicity, particularly hepatotoxicity. By using Western blot and immunofluorescence staining, we unexpectedly found that SKF-525A at 2-20 µM caused remarkable accumulation of microtubule-associated protein light chain 3 II (LC3-II) in primary rat hepatocytes at 1, 4 and 24 h, indicating that autophagy was disrupted. SKF-525A showed no effects on chloroquine induced LC3-II accumulation, suggesting that autophagic flux was blocked, which is further supported by the increased level of the p62 protein after SKF-525A treatment. SKF-525A did not affect proteasome activities or gene expression of LC3-II or p62. Immunofluorescence of green fluorescent protein fused lysosomal-associated membrane protein 1 (LAMP1, a specific protein marker for lysosomes) and LC3-II showed that co-localization of these two proteins was partially abolished by SKF-525A, indicating that autophagosome-lysosome fusion was blocked. The other five CYP inhibitors, metyrapone, 1-aminobenzotriazole, alpha-naphthoflavone, ticlopidine, and ketoconazole, showed no effects in parallel experiments. These findings provide novel insights into the mechanisms by which various CYP inhibitors differentially affect a same drug's toxicity in hepatocytes. The data also indicate that SKF-525A is not an ideal chemical inhibitor for probing the relation between CYP mediated metabolism and toxicity in primary hepatocytes.
Subject(s)
Autophagy/drug effects , Cytochrome P-450 Enzyme Inhibitors/toxicity , Hepatocytes/drug effects , Proadifen/toxicity , Animals , Autophagosomes/drug effects , Autophagosomes/metabolism , Autophagosomes/pathology , Cells, Cultured , Hepatocytes/metabolism , Hepatocytes/pathology , Lysosomes/drug effects , Lysosomes/metabolism , Lysosomes/pathology , Male , Membrane Fusion/drug effects , Microtubule-Associated Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Cisplatin/pharmacology , Proadifen/pharmacology , Apoptosis/drug effects , Carcinoma, Ovarian Epithelial , Cell Line, Tumor , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Down-Regulation , Female , Humans , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Mitochondrial Membranes/metabolism , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Neoplasms, Glandular and Epithelial/drug therapy , Ovarian Neoplasms/drug therapy , Survivin , Up-Regulation , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
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.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/secondary , Antineoplastic Agents/therapeutic use , Caveolae/drug effects , Enzyme Inhibitors/therapeutic use , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mevalonic Acid/antagonists & inhibitors , Adenocarcinoma/metabolism , Adenocarcinoma of Lung , Antineoplastic Agents/pharmacology , Caveolae/physiology , Caveolin 1/genetics , Caveolin 1/metabolism , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Neoplasm Metastasis , Nerve Tissue Proteins/antagonists & inhibitors , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Phenotype , Proadifen/pharmacology , Proadifen/therapeutic useABSTRACT
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.
Subject(s)
Ezetimibe/pharmacology , Gluconeogenesis/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Liver/drug effects , Oxygen Consumption/drug effects , Simvastatin/pharmacology , Animals , Cyanides/pharmacology , Drug Combinations , Enzyme Inhibitors/pharmacology , Fructose-Bisphosphatase/metabolism , Glucose-6-Phosphatase/metabolism , Liver/cytology , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Mitochondria, Liver/drug effects , Mitochondria, Liver/enzymology , Proadifen/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Calcium Signaling/drug effects , Econazole/pharmacology , Enzyme Inhibitors/pharmacology , Macrophages, Peritoneal/metabolism , Oligopeptides/pharmacology , Proadifen/pharmacology , Animals , Macrophages, Peritoneal/cytology , Male , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Drug Resistance, Neoplasm/drug effects , Leukemia, Promyelocytic, Acute/pathology , Mitoxantrone/pharmacology , Proadifen/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/drug effects , Apoptosis Regulatory Proteins/metabolism , Blotting, Western , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Down-Regulation , Drug Synergism , Enzyme Inhibitors/pharmacology , Flow Cytometry , Humans , Membrane Potential, Mitochondrial/drug effects , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Chloride Channels/antagonists & inhibitors , Energy Metabolism/drug effects , Insecticides/toxicity , Liver/drug effects , Membrane Transport Modulators/toxicity , Pyrazoles/toxicity , Animals , Biotransformation/drug effects , Chemical and Drug Induced Liver Injury/enzymology , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Electron Transport/drug effects , Gluconeogenesis/drug effects , Glycogenolysis/drug effects , Glycolysis/drug effects , In Vitro Techniques , Insecticides/metabolism , Liver/metabolism , Male , Membrane Transport Modulators/metabolism , Mitochondria, Liver/drug effects , Mitochondria, Liver/enzymology , Oxygen Consumption/drug effects , Perfusion , Proadifen/pharmacology , Pyrazoles/metabolism , Rats, Wistar , Urea/metabolismABSTRACT
(+)-Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non-isoenzyme-selective inhibitor SKF-525A (20 µM), or the CYP1A inhibitor alpha-naphthoflavone (10 µM), or the CYP3A inhibitor ketoconazole (25 µM), the cytotoxicity of UA at 3~6 µM after 3~20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA-induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 µM, showed no effects in parallel experiments. These data demonstrate that UA is bio-transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
Subject(s)
Benzofurans/adverse effects , Cytochrome P-450 Enzyme Inhibitors/chemistry , Cytochrome P-450 Enzyme System/metabolism , Hepatocytes/drug effects , Animals , Benzoflavones/chemistry , Cells, Cultured , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathology , Dietary Supplements , Enzyme Inhibitors/chemistry , Ketoconazole/chemistry , Liver/drug effects , Liver/pathology , Male , Mitochondria/drug effects , Mitochondria/metabolism , Proadifen/chemistry , Rats , Rats, Sprague-Dawley , Ticlopidine/chemistryABSTRACT
Thelephoric acid is an antioxidant produced by the hydrolysis of polyozellin, which is isolated from Polyozellus multiplex. In the present study, the inhibitory effects of polyozellin and thelephoric acid on 9 cytochrome P450 (CYP) family members (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were examined in pooled human liver microsomes (HLMs) using a cocktail probe assay. Polyozellin exhibited weak inhibitory effects on the activities of all 9 CYPs examined, whereas thelephoric acid exhibited dose- and time-dependent inhibition of all 9 CYP isoforms (IC50 values, 3.2-33.7 muM). Dixon plots of CYP inhibition indicated that thelephoric acid was a competitive inhibitor of CYP1A2 and CYP3A4. In contrast, thelephoric acid was a noncompetitive inhibitor of CYP2D6. Our findings indicate that thelephoric acid may be a novel, non-specific CYP inhibitor, suggesting that it could replace SKF-525A in inhibitory studies designed to investigate the effects of CYP enzymes on the metabolism of given compounds.
Subject(s)
Humans , Cytochrome P-450 CYP1A2 , Cytochrome P-450 CYP2D6 , Cytochrome P-450 CYP2E1 , Cytochrome P-450 Enzyme System , Hydrolysis , Metabolism , Microsomes, Liver , Proadifen , Protein IsoformsABSTRACT
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.
Subject(s)
Acetohexamide/pharmacology , Breast Neoplasms/drug therapy , Endothelium, Lymphatic/drug effects , Isoxsuprine/pharmacology , Lymphatic Vessels/drug effects , Nifedipine/pharmacology , Proadifen/pharmacology , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Antineoplastic Combined Chemotherapy Protocols , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/drug therapy , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Ductal, Breast/pathology , Cell Adhesion/drug effects , Cell Movement , Chemotaxis/drug effects , Coculture Techniques , Drug Synergism , Endothelium, Lymphatic/cytology , Endothelium, Lymphatic/metabolism , Enzyme Inhibitors/pharmacology , Female , Humans , Hypoglycemic Agents/pharmacology , Lymphatic Metastasis , Lymphatic Vessels/blood supply , Lymphatic Vessels/pathology , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Spheroids, Cellular/metabolism , Tumor Cells, Cultured , Vasodilator Agents/pharmacologyABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Proadifen/pharmacology , Protein Kinase Inhibitors/pharmacology , Thiazoles/pharmacology , Urea/analogs & derivatives , Adenocarcinoma , Apoptosis/drug effects , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Colonic Neoplasms , Glycogen Synthase Kinase 3 beta , HT29 Cells , Humans , Urea/pharmacologyABSTRACT
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.
Subject(s)
Anions/metabolism , Cecum/metabolism , Enteric Nervous System/physiology , Receptor, PAR-1/metabolism , Trypsin/pharmacology , Animals , Cecum/drug effects , Eicosanoids/physiology , Enteric Nervous System/drug effects , Indomethacin/pharmacology , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Male , Masoprocol/pharmacology , Mice , Oligopeptides/physiology , Proadifen/pharmacology , Protease Inhibitors/pharmacology , Serotonin/pharmacologyABSTRACT
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.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Caffeic Acids/pharmacology , Cytochrome P-450 CYP2B1/metabolism , Liver Neoplasms, Experimental/prevention & control , Phenylethyl Alcohol/analogs & derivatives , Steroid Hydroxylases/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Benzoflavones/pharmacology , Carcinogenicity Tests , Cytochrome P-450 CYP1A2 , Cytochrome P-450 CYP2B1/antagonists & inhibitors , Cytochromes/antagonists & inhibitors , Cytochromes/metabolism , Diethylnitrosamine , Disease Models, Animal , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Male , Phenylethyl Alcohol/pharmacology , Proadifen/pharmacology , Rats , Rats, Inbred F344 , Steroid Hydroxylases/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Horseradish Peroxidase/antagonists & inhibitors , Triazoles/pharmacology , Benzoflavones/chemistry , Benzoflavones/pharmacology , Disulfiram/chemistry , Disulfiram/pharmacology , Ditiocarb/chemistry , Ditiocarb/pharmacology , Ellipticines/chemistry , Ellipticines/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Horseradish Peroxidase/metabolism , Humans , Ketoconazole/chemistry , Ketoconazole/pharmacology , Metyrapone/chemistry , Metyrapone/pharmacology , Piperonyl Butoxide/chemistry , Piperonyl Butoxide/pharmacology , Proadifen/chemistry , Proadifen/pharmacology , Quinidine/chemistry , Quinidine/pharmacology , Structure-Activity Relationship , Substrate Specificity/drug effects , Sulfaphenazole/chemistry , Sulfaphenazole/pharmacology , Triazoles/chemistry , Troleandomycin/chemistry , Troleandomycin/pharmacologyABSTRACT
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.