Identification of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 as functional targets of the anti-inflammatory and anti-carcinogenic garcinol

Garcinol (camboginol) from the fruit rind of Guttiferae species shows anti-carcinogenic and anti-inflammatory properties, but the underlying molecular mechanisms are unclear. Here we show that garcinol potently interferes with 5-lipoxygenase (EC 7.13.11.34) and microsomal prostaglandin (PG)E₂ synthase (mPGES)-1 (EC 5.3.99.3), enzymes that play pivotal roles in inflammation and tumorigenesis. In cell-free assays, garcinol inhibited the activity of purified 5-lipoxygenase and blocked the mPGES-1-mediated conversion of PGH₂ to PGE₂ with IC₅₀ values of 0.1 and 0.3 μM, respectively. Garcinol suppressed 5-lipoxygenase product formation also in intact human neutrophils and reduced PGE₂ formation in interleukin-1β-stimulated A549 human lung carcinoma cells as well as in human whole blood stimulated by lipopolysaccharide. Moreover, garcinol interfered with isolated cyclooxygenase (COX)-1 (EC 1.14.99.1, IC₅₀ = 12 μM) and with the formation of COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and thromboxane B₂ in human platelets. In contrast, neither Ca²⁺-ionophore (A23187)-induced arachidonic acid release in neutrophils nor COX-2 activity in A549 cells or whole blood, measured as formation of 6-keto PGF_1α, or isolated human recombinant COX-2 were significantly affected by garcinol (≤30 μM). Together, the high potency of garcinol to selectively suppress PGE₂ synthesis and 5-lipoxygenase product formation provides a molecular basis for the anti-inflammatory and anti-carcinogenic effects of garcinol and rationalizes its therapeutic use.     

In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450

The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC₅₀) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC₅₀ = 412.68 ± 15.44 μM) and CYP3A4 (IC₅₀ = 343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC₅₀ = 539.04 ± 14.18 μM) and CYP3A4 (IC₅₀ = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (K_i = 385.24 ± 8.75 μM) and CYP3A4 (K_i = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (K_i = 109.62 ± 6.14 μM) and CYP3A4 (K_i = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.     

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H₂O₂ (750 μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8 μM) and the non-selective agonist NECA (EC₅₀ 3.9 μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66 nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.     

Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatin derivatives against human cancer cell lines and primary human hepatocytes

A series of seven platinum(II) cyclobutane-1,1-dicarboxylato (cbdc) complexes {[Pt(cbdc)(L_n)₂], 1-7}, derived from carboplatin by a substitution of two NH₃ molecules for two 2,6,9-trisubstituted 6-benzylaminopurine-based N-donor ligands (L_n), was studied by the MTT assay for their in vitro cytotoxic activity against seven human cancer cell lines, i.e. lung carcinoma (A549), cervix epithelioid carcinoma (HeLa), osteosarcoma (HOS), malignant melanoma (G361), breast adenocarcinoma (MCF7), ovarian carcinoma (A2780) and its cisplatin-resistant analogue (A2780cis), and against two primary cultures of human hepatocytes (LH31 and LH32). The prepared complexes were cytotoxic against several cancer cells, in some cases even more than cisplatin. The best results were achieved for complexes 1 (IC₅₀ = 17.4 ± 2.0 μM) and 2 (IC₅₀ = 14.8 ± 2.1 μΜ) against HOS cells, 1 (IC₅₀ = 15.1 ± 6.8 μM), 2 (IC₅₀ = 13.6 ± 5.2 μM) and 6 (IC₅₀ = 19.0 ± 6.6 μM) against MCF7, 6 (IC₅₀ = 6.4 ± 0.1 μM) against A2780, and 1-6 (IC₅₀ = 15.6 ± 4.0, 12.9 ± 3.7, 15.8 ± 3.8, 16.6 ± 5.5, 22.1 ± 2.5, and 5.6 ± 1.7 μM, respectively) against A2780cis. Viability of human hepatocytes was not declined by the tested complexes up to the concentration of 50 μM (for 1, 3-7) and 20 μM (for 2; caused by lower solubility of this complex).     

Liquid chromatography/tandem mass spectrometry study of anti-inflammatory activity of Plantain (Plantago L.) species

To evaluate anti-inflammatory activity of selected Plantago species (P. lanceolata L. and P. major L.) an optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken. By using intact cell system (platelets) as a source of COX-1 and 12-LOX enzymes and highly sensitive and specific LC–MS/MS technique for detection of main arachidonic acid metabolites formed by COX-1 and 12-LOX, this test provides efficient method for evaluation of anti-inflammatory potential of plant extracts and isolated compounds. Our results validated the well-known COX-1 inhibitory activity of P. lanceolata and P. major methanol extracts (concentration required for 50% inhibition (IC₅₀) was 2.00 and 0.65 mg/ml, respectively). Furthermore, 12-LOX inhibitory activity of examined extracts was reported for the first time (IC₅₀ = 0.75 and 1.73 mg/ml for P. lanceolata and P. major, respectively). Although renowned inhibitors, such as acetylsalicylic acid and quercetin showed higher activity, this study verifies P. lanceolata and P. major as considerable anti-inflammatory agents.     

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.     

A COX-2 inhibitor nimesulide analog selectively induces apoptosis in Her2 overexpressing breast cancer cells via cytochrome c dependent mechanisms

Epidemiological and animal model studies have suggested that non-steroidal anti-inflammatory drugs (NSAIDs) can act as chemopreventive agents. The cyclooxygenase-2 (COX-2) inhibitor nimesulide shows anti-cancer effect in different type of cancers. In the current study, five breast carcinoma cell lines were used to explore the anti-cancer mechanisms of a nimesulide derivative compound 76. The compound dose dependently suppressed SKBR-3, BT474 and MDA-MB-453 breast cancer cell proliferation with IC₅₀ of 0.9 μM, 2.2 μM and 4.0 μM, respectively. However, it needs much higher concentrations to inhibit MCF-7 and MDA-MB-231 breast cancer cell growth with IC₅₀ at 22.1 μM and 19.6 μM, respectively. Further investigation reveals that compound 76 induced apoptosis in SKBR-3 and BT474 cells. Since these cells are Her2 overexpressing cells, the Her2 intracellular signaling pathways were examined after the treatment. There was no significant changing of kinase activity. However, the cytochrome c release assay indicated that the apoptosis induced by the compound was mediated by the mitochondria. These results suggest that compound 76 selectively induce apoptosis in Her2 overexpressing breast cancer cells through the mitochondria, and could be used as a lead to design more potent derivatives.     

Generic systems for the enantioseparation of basic drugs in NACE using single-isomer anionic CDs

The enantioseparation of 10 basic drugs was evaluated in NACE systems using heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)-β-CD (HMAS-β-CD). For this purpose, a D-optimal design with 21 experimental points was applied. Four antifungal agents (econazole, isoconazole, miconazole, sulconazole), three local anesthetics (bupivacaine, mepivacaine and prilocaine), two sympathomimetics (salbutamol and terbutaline) and one β-blocker (carvedilol) were selected as basic model analytes. The influence on the enantiomeric resolution of anionic CD and BGE anion concentrations as well as the BGE anion nature was investigated. For all studied analytes, the enantiomeric resolution was shown to be significantly influenced by the CD concentration. Based on the observed results, a generic NACE system was recommended, namely 20 mM HMAS-β-CD and 10 mM ammonium camphorSO₃⁻ in methanol acidified with 0.75 M formic acid. Moreover, this NACE system was compared to previous conditions with heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) or heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD). Finally, two generic systems using either HDAS-β-CD or HMAS-β-CD were proposed and evaluated for the enantioseparation of ketamine and norketamine after incubation of ketamine in phenobarbital-induced male rat liver microsomes systems.     

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and l-amino acid oxidase

Some proteins present in snake venom possess enzymatic activities, such as phospholipase A₂ and l-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA₂ (BmarPLA₂) and LAAO (BmarLAAO) on strains of bacteria, yeast, and Leishmania sp. The BmarTV was isolated by Protein Pack 5PW, and several fractions were obtained. Reverse phase HPLC showed that BmarPLA₂ was isolated from the venom, and N-terminal amino acid sequencing of sPLA₂ showed high amino acid identity with other lysine K49 sPLA₂s isolated from Bothrops snakes. The BmarLAAO was purified to high molecular homogeneity and its N-terminal amino acid sequence demonstrated a high degree of amino acid conservation with others LAAOs. BmarLAAO was able to inhibit the growth of P. aeruginosa, C. albicans and S. aureus in a dose-dependent manner. The inhibitory effect was more significant on S. aureus, with a MIC = 50 μg/mL and MLC = 200 μg/mL. However, the BmarTV and BmarPLA₂ did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L. chagasi and L. amazonensis, with an IC₅₀ = 2.55 μg/mL and 2.86 μg/mL for L. amazonensis and L. chagasi, respectively. BmarTV also provided significant inhibition of parasitic growth, with an IC₅₀ of 86.56 μg/mL for L. amazonensis and 79.02 μg/mL for L. chagasi. BmarPLA₂ did not promote any inhibition of the growth of these parasites. The BmarLAAO and BmarTV presented low toxicity at the concentrations studied. In conclusion, whole venom as well as the l-amino acid oxidase from Bothrops marajoensis was able to inhibit the growth of several microorganisms, including S. aureus, Candida albicans, Pseudomonas aeruginosa, and Leishmania sp.     

Elucidation of the molecular mechanism and the efficacy in vivo of a novel 1,4-benzoquinone that inhibits 5-lipoxygenase

Background and Purpose

1,4-Benzoquinones are well-known inhibitors of 5-lipoxygenase (5-LOX, the key enzyme in leukotriene biosynthesis), but the molecular mechanisms of 5-LOX inhibition are not completely understood. Here we investigated the molecular mode of action and the pharmacological profile of the novel 1,4-benzoquinone derivative 3-((decahydronaphthalen-6-yl)methyl)-2,5-dihydroxycyclohexa-2,5-diene-1,4-dione (RF-Id) in vitro and its effectiveness in vivo.

Experimental Approach

Mechanistic investigations in cell-free assays using 5-LOX and other enzymes associated with eicosanoid biosynthesis were conducted, along with cell-based studies in human leukocytes and whole blood. Molecular docking of RF-Id into the 5-LOX structure was performed to illustrate molecular interference with 5-LOX. The effectiveness of RF-Id in vivo was also evaluated in two murine models of inflammation.

Key Results

RF-Id consistently suppressed 5-LOX product synthesis in human leukocytes and human whole blood. RF-Id also blocked COX-2 activity but did not significantly inhibit COX-1, microsomal PGE₂ synthase-1, cytosolic PLA₂ or 12- and 15-LOX. Although RF-Id lacked radical scavenging activity, reducing conditions facilitated its inhibitory effect on 5-LOX whereas cell stress impaired its efficacy. The reduced hydroquinone form of RF-Id (RED-RF-Id) was a more potent inhibitor of 5-LOX as it had more bidirectional hydrogen bonds within the 5-LOX substrate binding site. Finally, RF-Id had marked anti-inflammatory effects in mice in vivo.

Conclusions and Implications

RF-Id represents a novel anti-inflammatory 1,4-benzoquinone that potently suppresses LT biosynthesis by direct inhibition of 5-LOX with effectiveness in vivo. Mechanistically, RF-Id inhibits 5-LOX in a non-redox manner by forming discrete molecular interactions within the active site of 5-LOX.     

Regioselective sulfonation of dopamine by SULT1A3 in vitro provides a molecular explanation for the preponderance of dopamine-3-O-sulfate in human blood circulation

SULT1A3 is an enzyme that catalyzes the sulfonation of many endogenous and exogenous phenols and catechols. The most important endogenous substrate is dopamine (DA), which is often used as a probe substrate for SULT1A3. We developed a new method for analyzing the SULT1A3 reaction products by high-performance liquid chromatography (HPLC) with electrochemical detection. The sulfonate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS), DA and the two dopamine sulfates, DA-3-O-sulfate and DA-4-O-sulfate, can be separated within 3 min. This enables quantitation of the sulfates without radioactive PAPS or the precipitation of unreacted PAPS. Both sulfates were synthesized as reference substances and characterized by ¹H and ¹³C nuclear magnetic resonance (NMR), mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The purity of the dopamine sulfates was estimated by HPLC using a diode array detector. We determined the enzyme kinetic parameters for formation of DA-3-O-sulfate and DA-4-O-sulfate using purified recombinant human SULT1A3. The reactions followed Michaelis-Menten kinetics up to 50 μM DA concentration, and strong substrate inhibition was observed at higher concentrations. The apparent K_m values for sulfonation at both hydroxy groups were similar (2.21 ± 0.764 and 2.59 ± 1.06 μM for DA-4-O-sulfate and DA-3-O-sulfate, respectively), but the V_max was approximately six times higher for the formation of the 3-O-sulfate (344 ± 139 nmol/min/mg protein) than the 4-O-sulfate (45.4 ± 16.5 nmol/min/mg protein). These results are in accordance with the observation that DA-3-O-sulfate is more abundant in human blood than DA-4-O-sulfate and that in the crystal structure of SULT1A3 with dopamine bound to the active site, the 3-hydroxy group is aligned to form hydrogen bonds with catalytic residues of the enzyme.     

Simultaneous quantitation of levodopa and 3-O-methyldopa in human plasma by HPLC-ESI-MS/MS: application for a pharmacokinetic study with a levodopa/benserazide formulation

A sensitive and simple method was developed for the quantitation of levodopa and its metabolite 3-O-methyldopa, in human plasma, after oral administration of tablet formulations containing levodopa (200 mg) and benserazide (50 mg). The analytes were extracted by a protein precipitation procedure, using carbidopa as an internal standard. A mobile phase consisting of 0.2% formic acid and acetonitrile (94:6, v/v) was used and chromatographic separation was achieved using ACE C₁₈ column (50 mm × 4.6 mm i.d.; 5 μm particle size). Selected reaction monitoring was performed using the fragmentation transitions m/z 198 → m/z 107, m/z 212 → m/z 166 and m/z 227 → m/z 181 for levodopa, 3-O-methyldopa and carbidopa, respectively. Calibration curves were constructed over the range 50.0-6000.0 ng/mL for levodopa and 25.0-4000.0 ng/mL for 3-O-methyldopa. The method shown to be specific, precise, accurate and provided recovery rates higher than 85% for all analytes. No matrix effect was detected in the samples. The validated method was applied in a pharmacokinetic study with a levodopa/benserazide tablet formulation in healthy volunteers.     

The Rhizomes of Acorus gramineus and the Constituents Inhibit Allergic Response In vitro and In vivo

The rhizomes of Acorus gramineus have frequently been used in traditional medicine mainly for sedation as well as enhancing brain function. In this study, the anti-allergic activity of A. gramineus was investigated. The 70% ethanol extract of the rhizomes of A. gramineus was found to inhibit the allergic response against 5-lipoxygenase (5-LOX)-catalyzed leukotriene (LT) production from rat basophilic leukemia (RBL)-1 cells and β-hexosaminidase release from RBL-2H3 cells with IC₅₀’s of 48.9 and >200 μg/ml, respectively. Among the 9 major constituents isolated, β-asarone, (2R,3R,4S,5S)-2,4-dimethyl-1,3-bis (2'',4'',5''-trimethoxyphenyl)tetrahydrofuran (AF) and 2,3-dihydro-4,5,7-trimethoxy-1-ethyl-2-methyl-3-(2,4,5-trimethoxyphenyl)indene (AI) strongly inhibited 5-LOX-catalyzed LT production in {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-treated RBL-1 cells, AI being the most potent (IC₅₀=6.7 μM). Against β-hexosaminidase release by antigen-stimulated RBL-2H3 cells, only AI exhibited strong inhibition (IC₅₀=7.3 μM) while β-asarone and AF showed 26.0% and 39.9% inhibition at 50 μM, respectively. In addition, the ethanol extract of A. gramineus showed significant inhibitory action against the hapten-induced delayed hypersensitivity reaction in mice by oral administration at 200 mg/kg. Therefore, it is suggested that A. gramineus possesses anti-allergic activity and the constituents including β-asarone and AI certainly contribute to the anti-allergic activity of the rhizomes of A. gramineus.     

Simultaneous determination of vitexin-4″-O-glucoside and vitexin-2″-O-rhamnoside from hawthorn leaves flavonoids in rat plasma by HPLC method and its application to pharmacokinetic studies

The present study was to investigate the pharmacokinetics of the two similar flavonoid glycosides, vitexin-4″-O-glucoside (VGL) and vitexin-2″-O-rhamnoside (VRH) in rats after intravenous administration of hawthorn leaves flavonoids (HLF). Blood samples were collected via tail vein at time intervals after drug administration and the plasma concentrations of the studied ingredients were analyzed by HPLC after the plasma protein was precipitated directly with methanol. VGL and VRH were successfully separated using a C₁₈ column with a UV detection at 330 nm and a mobile phase of methanol–acetonitrile–tetrahydrofuran–0.5% acetic acid (1:1:19.4:78.6, v/v/v/v). The assay linearities of VGL and VRH were confirmed over the range 0.23–138.42 and 0.36–218.49 μg/ml, respectively. The accuracy and precision of the two analytes at high, medium and low concentration were within the range of −3.13% to 3.51% and below 4%, the mean assay recoveries of them (n = 5) ranged from 96.87% to 101.75% and 96.88% to 103.51% for intra- and inter-day assays and the mean extraction recoveries of them (n = 5) varied from 92.68% to 95.74% for VGL and 93.45% to 99.26% for VRH, respectively. After intravenous administration of HLF to rats over the doses range of 10–40 mg/kg, the plasma concentration–time curves of VGL and VRH were both conformed to the three-compartment open pharmacokinetic model and linear pharmacokinetic characteristics.     

Inhibitory effect of ethanol extract of Magnolia officinalis and 4-O-methylhonokiol on memory impairment and neuronal toxicity induced by beta-amyloid

The components of the herb Magnolia officinalis have exhibited antioxidant and neuroprotective activities. In this study, we investigated effects of ethanol extract of M.officinalis and its major component 4-O-methylhonokiol on memory dysfunction and neuronal cell damages caused by Aβ. Oral pretreatment of ethanol extract of M. officinalis (2.5, 5 and 10 mg/kg) and 4-O-methylhonokiol (1 mg/kg) into drinking water for 5 weeks suppressed the intraventricular treatment of Aβ_1-42 (0.5 μg/mouse, i.c.v.)-induced memory impairments. In addition, 4-O-methylhonokiol prevented the Aβ_1-42-induced apoptotic cell death as well as β-secretase expression. 4-O-methylhonokiol also inhibited H₂O₂ and Aβ_1-42-induced neurotoxicity in cultured neurons as well as PC12 cells by prevention of the reactive oxygen species generation. 4-O-methylhonokiol also directly inhibited β-secretase activity and Aβ fibrilization in vitro. Thus, ethanol extract of M. officinalis may be useful for prevention of the development or progression of AD, and 4-O-methylhonokiol may be a major active component.     

Organic cation transporter 1 mediates the uptake of monocrotaline and plays an important role in its hepatotoxicity

Monocrotaline (MCT) is a kind of toxic retronecine-type pyrrolizidine alkaloids (PAs) from plants of Crotalaria, which can be bio-activated by cytochrome P450 (CYP) enzymes in liver and then induce hepatotoxicity. Since CYPs are localized in the endoplasmic reticulum, the influx of MCT to the liver is the key step for its hepatotoxicity. The objective of the present study was to investigate the role of organic cation transporter 1 (OCT1), a transporter mainly expressed in liver, in the uptake of MCT and in hepatotoxicity induced by MCT. The results revealed that MCT markedly inhibited the uptake of 1-methyl-4-phenylpyridinium (MPP⁺), an OCT1 substrate, in Madin–Darby canine kidney (MDCK) cells stably expressing human OCT1 (MDCK-hOCT1) with the IC₅₀ of 5.52 ± 0.56 μM. The uptake of MCT was significantly higher in MDCK-hOCT1 cells than in MDCK-mock cells, and MCT uptake in MDCK-hOCT1 cells followed Michaelis–Menten kinetics with the K_m and V_max values of 25.0 ± 6.7 μM and 266 ± 64 pmol/mg protein/min, respectively. Moreover, the OCT1 inhibitors, such as quinidine, d-tetrahydropalmatine (d-THP), obviously inhibited the uptake of MCT in MDCK-hOCT1 cells and isolated rat primary hepatocytes, and attenuated the viability reduction and LDH release of the primary cultured rat hepatocytes caused by MCT. In conclusion, OCT1 mediates the hepatic uptake of MCT and may play an important role in MCT induced-hepatotoxicity.     

Novel Alexa Fluor-488 labeled antagonist of the A2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay

Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A_2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A_2AAR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K_i value of 111 ± 16 nM in radioligand binding using [³H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A_2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A_2AAR antagonist. MRS5346 did not show any effect on A₁ and A₃ ARs in binding or the A_2BAR in a cyclic AMP assay at 10 μM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A_2AAR binding. The FP signal was optimal with 20 nM MRS5346 and 150 μg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K_d value of MRS5346 calculated from kinetic parameters was 16.5 ± 4.7 nM. In FP competition binding experiments using MRS5346 as a tracer, K_i values of known AR agonists and antagonists consistently agreed with K_i values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.     

Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1

OATP1A2 and OATP2B1 are uptake transporters of the human organic anion transporting polypeptide (OATP) family with a broad substrate spectrum including several endogenous compounds as well as drugs such as the antihistaminic drug fexofenadine and HMG-CoA reductase inhibitors. Both transporters are localized in the apical membrane of human enterocytes. Flavonoids, abundantly occurring in plants, have previously been shown to interact with drug metabolizing enzymes and transporters. However, the impact of flavonoids on OATP1A2 and OATP2B1 transport function has not been analyzed in detail. Therefore, HEK293 cell lines stably expressing OATP1A2 and OATP2B1 were used to investigate the influence of the Ginkgo flavonoids apigenin, kaempferol, and quercetin on the transport activity of OATP1A2 and OATP2B1. K_i values of all three flavonoids determined from Dixon plot analyses using BSP as substrate indicated a competitive inhibition with quercetin as the most potent inhibitor of OATP1A2 (22.0 μM) and OATP2B1 (8.7 μM) followed by kaempferol (OATP1A2: 25.2 μM, OATP2B1: 15.1 μM) and apigenin (OATP1A2: 32.4 μM OATP2B1: 20.8 μM). Apigenin, kaempferol, and quercetin led to a concentration-dependent decrease of the OATP1A2-mediated fexofenadine transport with IC₅₀ values of 4.3 μM, 12.0 μM, and 12.6 μM, respectively. The OATP1A2- and OATP2B1-mediated transport of atorvastatin was also efficiently inhibited by apigenin (IC₅₀ for OATP1A2: 9.3 μM, OATP2B1: 13.9 μM), kaempferol (IC₅₀ for OATP1A2: 37.3 μM, OATP2B1: 20.7 μM) and quercetin (IC₅₀ for OATP1A2: 13.5 μM, OATP2B1: 14.1 μM). These data indicate that modification of OATP1A2 and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food-drug or drug-drug interactions in humans.     

Quercetin 3-O-methyl ether protects FL83B cells from copper induced oxidative stress through the PI3K/Akt and MAPK/Erk pathway

Quercetin is a bioflavonoid that exhibits several biological functions in vitro and in vivo. Quercetin 3-O-methyl ether (Q3) is a natural product reported to have pharmaceutical activities, including antioxidative and anticancer activities. However, little is known about the mechanism by which it protects cells from oxidative stress. This study was designed to investigate the mechanisms by which Q3 protects against Cu^2 +-induced cytotoxicity. Exposure to Cu^2 + resulted in the death of mouse liver FL83B cells, characterized by apparent apoptotic features, including DNA fragmentation and increased nuclear condensation. Q3 markedly suppressed Cu^2 +-induced apoptosis and mitochondrial dysfunction, characterized by reduced mitochondrial membrane potential, caspase-3 activation, and PARP cleavage, in Cu^2 +-exposed cells. The involvement of PI3K, Akt, Erk, FOXO3A, and Mn-superoxide dismutase (MnSOD) was shown to be critical to the survival of Q3-treated FL83B cells. The liver of both larval and adult zebrafish showed severe damage after exposure to Cu^2 + at a concentration of 5 μM. Hepatic damage induced by Cu^2 + was reduced by cotreatment with Q3. Survival of Cu^2 +-exposed larval zebrafish was significantly increased by cotreatment with 15 μM Q3. Our results indicated that Cu^2 +-induced apoptosis in FL83B cells occurred via the generation of ROS, upregulation and phosphorylation of Erk, overexpression of 14-3-3, inactivation of Akt, and the downregulation of FOXO3A and MnSOD. Hence, these results also demonstrated that Q3 plays a protective role against oxidative damage in zebrafish liver and remarked the potential of Q3 to be used as an antioxidant for hepatocytes.