The interaction between MK-801, a model of psychosis and KCl-induced depolarization or electroconvulsive shock (ECS), a therapeutic model of electroconvulsive therapy (ECT), was investigated in SH-SY5Y cells and the rat frontal cortex.SH-SY5Y cells were pretreated with 1 µM MK-801 for 15 min, followed by cotreatment with 100 mM KCl for 5 min. MK-801 was reintroduced after the KCl was washed out, and the samples were incubated before harvesting. For the experiments in rats, male Sprague-Dawley rats were treated with MK-801 followed by ECS. Immunoblot analyses of glycogen synthase kinase 3β (GSK3β) (Ser9), AKT (Ser473) and extracellular legulated kinase (ERK)1/2 in SH-SY5Y cells and the rat frontal cortex were performed.KCl-induced neuronal depolarization resulted in the transient dephosphorylation of AKT (Ser473) and GSK3β (Ser9), followed by increased phosphorylation of the enzymes in SH-SY5Y cells. Cotreatment with MK-801 and KCl inhibited the initial dephosphorylation of AKT and GSK3β produced by KCl-induced neuronal depolarization. Similarly, ECS resulted in the transient dephosphorylation of AKT (Ser473) and GSK3β (Ser9), whereas cotreatment with MK-801 inhibited the initial dephosphorylation of AKT (Ser473) and GSK3β (Ser9) produced by ECS in the rat frontal cortex. No significant interaction was observed between MK-801 and KCl in the dephosphorylation of ERK1/2.These results suggest that an antagonistic interplay between MK-801 and neuronal depolarization by KCl or ECS is involved the regulation of AKT (Ser473) and GSK3β (Ser9) phosphorylation. Intracellular signal interference may play a substantial role in the development of neuropathological processes in psychosis, thus the elimination of the cause of the interference may be a potential target in the treatment of psychosis. Complex arrays of intracellular signal transduction pathways lead to changes in cellular function. To determine the relationship between pathological processes and recovery mechanisms, it is necessary to clarify the effects of the interaction between pathogenic and therapeutic stimuli on intracellular signal transduction pathways.7MK-801 is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that is commonly used to induce a psychosis-like state that is characterized by both negative and positive symptoms of schizophrenia. Rodents treated with MK-801 displayed psychotomimetic behavior and behaviors that correspond to the core symptoms of psychosis.12in vitro model of neuronal activity and ECS.20Electroconvulsive therapy (ECT) has been proven to be very effective in managing psychotic or depressive symptoms that respond poorly to pharmacotherapy.To better understand the interplay between the signaling pathways involved in the pathogenesis and therapeutic mechanisms, we tried to explore the possibility of a cellular model. Thus, we investigated the interaction between MK801, a model of psychosis, and KCl-induced depolarization, a therapeutic model of ECT in SH-SY5Y human neuroblastoma cells. The aim of this study was to analyze the interaction between MK-801 and KCl-induced depolarization and investigate its role in the regulation of AKT-GSK3β and ERK1/2 in SH-SY5Y cells. The second aim of this study was to investigate whether the interactions observed in these cell lines were reproducible in the rat frontal cortex after MK801 treatment followed by ECS.2·KCl and NaCl (Sigma-Aldrich) were dissolved in DMEM. MK-801  was dissolved in normal saline. Before treatment, SH-SY5Y cells were washed and incubated overnight in fresh DMEM containing 10% FBS. Neuronal depolarization was induced by treating the cells with 100 mM KCl in complete medium for 5 min. The cells were then immediately washed with fresh medium and reintroduced into fresh medium. For treatment with MK-801, 1 µM MK-801 was added, and the cells were incubated for 15 min, after which the medium was replaced with fresh medium containing 1 µM MK-801. The dissociation value (KD) of MK-801 was 5.7 nM for the whole brain and 8.1 nM for the spinal cord.SH-SY5Y cells were purchased from American Type Culture Collection . SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  and 100 U/ml penicillin/streptomycin (Sigma-Aldrich) and incubated at 37℃ in 5% COAnimals were treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH). Male Sprague-Dawley rats (150-200 g) were housed for 2 weeks before the experiment and maintained under 12 h light/12 h dark conditions with food and water available ad libitum. The rats were divided into three groups of three each. The groups were given the following treatments: group 1, intraperitoneal injection of normal saline followed by sham treatment; group 2 intraperitoneal injection of normal saline followed by a single ECS; and group 3, intraperitoneal injection of 1 mg/kg MK-801, followed by a single ECS. Previous experiments have shown that AKT and GSK phosphorylation increased after treatment with MK-801 at concentrations up to 1 mg/kg in the rat frontal cortex but decreased at higher dosages.3VO4, 1 mM NaF, 20 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, a protease inhibitor mixture , and 1% Triton X-100. The cells were lysed by incubating the suspension on ice for 30 min. Rat cortical tissue was homogenized in 10 v/w of icecold homogenization buffer . Homogenates were centrifuged, and supernatants were boiled with Laemmli's sample buffer. The samples were then separated by SDS-PAGE.SH-SY5Y cells were harvested with a cell scraper in lysis buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 0.1 mM EDTA, 1 mM NaAntibodies against GSK3β, ERK1/2 were purchased from Santa Cruz Biotechnology , and antibodies against phosphorylated GSK3β , phosphorylated AKT , phosphorylated ERK1/2  and AKT (1:3000) were obtained from New England BioLab's Cell Signaling Group . Horseradish peroxidase-conjugated goat antimouse and goat anti-rabbit IgG antibodies (1 : 3000) were obtained from Zymed . The blots were incubated with enhanced chemiluminescence substrate mix  and exposed to radiographic film  in order to obtain images. The densities of the bands were quantified using an image analyzer .All analyses were performed using SPSS 13.0 for Windows , and the data are presented as the means±S.D. The nonparametric Mann-Whitney U test was used for non-normal data to analyze mean values, and statistical significance was established at p<0.05 (two-tailed).The phosphorylation of AKT (Ser473) was significantly decreased at 0 min after the cells were treated with 100 mM KCl for 5 min . The phosphorylation of AKT (Ser473) increased between 5 and 30 min after the initial dephosphorylation, peaked at 15 min, and then returned to baseline at 60 min. No significant changes in the phosphorylation of AKT (Ser473) were observed after treatment with MK-801 alone, though some fluctuations were observed. When the cells were treated with KCl and MK-801, the phosphorylation of AKT (Ser473) showed a pattern of change similar to that observed after treatment with KCl alone, except that no immediate dephosphorylation was observed at 0 min . There was a significant difference in phosphorylation at 0 min between the KCl-treated cells and the MK-801- and KCl-treated cells , indicating that MK-801 blocked the KCl-induced dephosphorylation of AKT (Ser473) at 0 min. The total amount of AKT did not change significantly under any of the conditions .GSK3β (Ser9) phosphorylation was decreased at 0 min after KCl treatment, as compared with the vehicle-treated controls , and then increased slightly before returning to a basal level. The phosphorylation of GSK3β (Ser9) also decreased at 0 min after MK-801 treatment and recovered slowly to a basal level at 30-60 min. After treatment with KCl and MK-801, the phosphorylation of GSK3β (Ser9) showed a pattern of change similar to that observed after treatment with KCl alone, except that no immediate dephosphorylation was observed at 0 min . There was a significant difference in phosphorylation at 0 min between cells treated with KCl alone and those treated with MK-801 and KCl . The total amount of GSK3β did not change significantly under any of the conditions .The phosphorylation of ERK1/2 at Thr202/Tyr204 was decreased to below basal level at 0 min after treatment with KCl alone , peaked at 15 min, and then returned to baseline at 60 min. After treatment with MK-801 alone, the phosphorylation of ERK1/2 (Thr202/Tyr204) was decreased at 0 min  and then slowly recovered to basal level between 5-60 min. After cotreatment with both KCl and MK-801, the phosphorylation of ERK1/2 (Thr202/Tyr204) showed a pattern of change similar to that observed after treatment with KCl alone, exhibiting dephosphorylation at 0 min . The total amount of ERK1/2 did not change significantly under any of the conditions .The depolarizing and osmotic effects of KCl were evaluated. SH-SY5Y cells were treated with 100 mM of KCl or NaCl for 5 min, and the effects of these treatments on the phosphorylation of AKT (Ser473), GSK3β (Ser9), and ERK1/2 (Thr202/Tyr204) were compared. The phosphorylation of AKT (Ser473) was decreased in cells treated with KCl in comparison with the vehicle-treated controls , but unchanged in cells treated with NaCl  was decreased at 0 and 5 min in the frontal cortex of saline-ECS-treated rats in comparison to saline-sham-treated rats . After the initial dephosphorylation, the phosphorylation of AKT (Ser473) showed a tendency to increase and then return to baseline around 30 min in the frontal cortex of saline-ECS-treated rats. In MK-801-ECS-treated animals, the phosphorylation of AKT (Ser473) was increased at 0 min in comparison to the saline-sham-treated samples, and it remained increased until 30 minutes after ECS, with statistical significance at 0 min and 10 min . At 0 and 5 min after ECS, the phosphorylation of AKT at Ser 473 was af-fected by pretreatment with either saline or MK-801 . There was no difference between the saline-ECS- and MK-801-ECS-treated groups at 10 or 30 min after ECS .The phosphorylation of GSK3β (Ser9) was also decreased at 0 min in the frontal cortex of saline-ECS-treated rats in comparison to saline-sham-treated rats . After the initial dephosphorylation, the phosphorylation of GSK3β (Ser9) increased and then returned to baseline after approximately 30 min in the saline-ECS-treated rat frontal cortex. In the MK-801-ECS-treated animals, the phosphorylation of GSK3β (Ser9) was increased at 0 min in comparison to the saline-sham-treated samples, remained increased until 10 min after ECS and then returned to baseline at 30 minutes after ECS . At 0 min after ECS, phosphorylation of GSK3β (Ser9) was affected by pretreatment with either saline or MK-801 . There was no difference between the saline-ECS-treated and MK-801-ECS-treated groups at 5,10 or 30 min after ECS .In this study, the interaction between MK-801 and KCl-induced depolarization and its role in the regulation of AKT-GSK3β and ERK1/2 in SH-SY5Y cells were investigated. We found that MK-801 inhibited the immediate KCl-induced dephosphorylation of AKT (Ser473) and GSK3β (Ser9) in SH-SY5Y neuroblastoma cells. MK-801 had no effect on the KCl-induced immediate dephosphorylation of ERK1/2 (Thr202/Tyr204). We also observed that ECS resulted in immediate transient dephosphorylation of AKT (Ser473) and GSK3β (Ser9), whereas pretreatment with MK-801 inhibited the immediate dephosphorylation of AKT (Ser473) and GSK3β (Ser9) produced by ECS in the rat frontal cortex. These findings suggest that the AKT-GSK3β pathway may be involved in the antagonistic interplay between MK-801 and KCl-induced depolarization/ECS.2+ flow via VGCC, whereas a primary effect of MK-801 is to block the flow of intracellular Ca2+ via the NMDA receptor.2+ influx caused by MK-801 may inhibit the KCl-induced activation of phosphatases, which may result in the inhibition of dephosphorylation of AKT (Ser473) and GSK3β (Ser9).A primary effect of KCl is to induce intracellular CaThe time series changes in the phosphorylation of AKT (Ser473) and GSK3β (Ser9) after treatment with MK-801 were previously reported by the authors and are therefore not shown in this study.Moreover, we found that, for AKT (Ser473) and GSK3β (Ser9), the interaction between ECS and MK-801 in the rat frontal cortex was similar to the interaction between KCl-induced depolarization and MK-801 in SH-SY5Y cells. These findings also suggest that the depolarizing effect of KCl may share a similar mechanism with the immediate response of ECS. In addition, the similarity in the findings in SH-SY5Y neuroblastoma cells and rat frontal cortex suggests the possibility of using SH-SY5Y as a cellular model for investigating the mechanism of interaction between MK-801 and ECS in future studies.Our result also indicated that there is no significant interaction between MK-801 and KCl related to the dephosphorylation of ERK1/2. Furthermore, the dephosphorylation of ERK1/2 (Thr202/Tyr204) was not caused by the depolarizing effect of KCl but rather by its osmotic effect because the effect of NaCl was similar to that of KCl. Our findings show that the depolarizing effect of KCl contributes to the dephosphorylation of AKT and GSK3β and that the osmotic effect of KCl contributes to the dephosphorylation of ERK1/2 (Thr202/Tyr204). These findings are consistent with previous reports demonstrating that ECS, which is said to result in massive transient depolarization of neurons,23ERK1/2 has been found to be activated by glutamate receptor stimulation and deactivated by NMDA receptor antagonists, including MK-801, in primary cortical cultures.The roles of AKT-GSK3β and ERK1/2 in psychiatric disorders have been discussed in numerous reports on bipolar disorder and schizophrenia.


The gastrointestinal peptide hormone gastrin is known to regulate various cellular processes including proliferation, migration and metastasis in gastrointestinal (GI) cells. The studies described here were undertaken to elucidate in detail the signaling pathways mediating the migratory responses of amidated gastrin (G17) and to understand the involvement of the serine/threonine kinase Glycogen Synthase Kinase-3 beta (GSK3β) in this.Ser9 phosphorylation, indicative of an inhibition of the kinase. Pretreatment with a pharmacological inhibitor of PI3Kinase pathway (Wortmannin) was unable to antagonize G17-induced GSK3βSer9 phosphorylation, suggesting that this might involve PI3Kinase-independent pathways. Treatment with G17 was also associated with increased Snail expression, and β-catenin nuclear translocation, both of which are GSK3β downstream targets. Pretreatment with a pharmacological inhibitor of GSK3β (AR-A014418) augmented Snail expression and β-catenin nuclear translocation in the absence of G17, whereas overexpression of a phosphorylation deficient mutant of GSK3β (S9A) abrogated Snail promoter induction. These suggested that G17 modulates Snail and β-catenin pathways via inhibiting GSK3β. In addition, overexpression of GSK3β wild type (WT) or S9A mutant inhibited G17-induced migration and MMP7 promoter induction. G17 studies designed following small interference RNA (siRNA)-mediated knockdown of Snail and β-catenin expression indicated a significant reduction of G-17-induced migration and MMP7 promoter induction following combined knockdown of both proteins.Our results indicate that incubation of gastric cancer cells overexpressing CCK2 receptor (AGSE cells) with G17 results in a dose and time dependent increase of GSK3βOur studies indicate that inhibition of GSK3β is necessary to activate G17-induced migratory pathways in gastric cancer cells. Inhibition of GSK3β leads to an induction of Snail expression and β-catenin nuclear translocation, both of which participate to promote G17-induced migration. Helicobacter pylori (H. pylori) infection, diet as well as genetic background [Helicobacter infection leading to the development of invasive gastric cancer [Gastric cancer is the second leading cause of cancer-related deaths worldwide , and areckground ,4. Interckground ,6. Resulckground , which cc cancer . Prolongc cancer  and mighc cancer . Recent c cancer . In addic cancer . All theH. pylori infection [Glycogen Synthase Kinase-3 beta (GSK3β) is a ubiquitously expressed serine/threonine kinase, which is active in resting epithelial cells . Phosphonfection .in vivo metastasis. Our earlier studies in gastric cancer cells have demonstrated that G17-induced migration involves an activation of the Mixed-Lineage-Kinase 3/JNK1 signaling axis [Ser9 phosphorylation in a transient manner, which was also associated with a corresponding increase in the expression and promoter activation of Snail and an increase in the nuclear translocation of β-catenin. Inhibition of GSK3β via a pharmacological inhibitor resulted in increased Snail expression and β-catenin nuclear translocation in the absence of G17 and overexpression of a phosphorylation deficient mutant of GSK3β (S9A) antagonized G17-mediated induction of Snail promoter. Similarly, ectopic overexpression of Wild type (WT) or S9A mutant of GSK3β antagonized G17-induced migration and MMP7 promoter induction. Our studies also indicate that, combined knockdown of Snail and β-catenin by small interference RNA (siRNA) significantly attenuated G17-induced migration and MMP7 transcription. These studies indicate that G17 modulates Snail and β-catenin pathways via inhibiting GSK3β, both of which in turn participate to mediate G17-induced migration.Despite an apparent connection of gastrin in gastric cancer progression, the detailed mechanism by which gastrin mediates its effects is still unclear. In addition to stimulating proliferation, recent studies have shown that G17 as well as its unprocessed forms can activate migration as well as invasion -33 whiching axis . Due to Ser9 panel) and dose-dependent  corresponding to the time of GSK3βSer9 phosphorylation, indicating a simultaneous activation of AKT. However, pretreatment of the cells with PI3Kinase inhibitor (Wortmannin) was unable to antagonize G17-induced GSK3βSer9 phosphorylation , although it completely antagonized AKT phosphorylation (pAKTSer473 panel). In addition, treatment of another gastric cancer cell line (MKN45) with G17 showed an increase in GSK3βSer9 phosphorylation without any increase in AKT phosphorylation  . These ident Fig  increasetion Fig , indicattion Fig . These sTo understand the consequences of G17-mediated inhibition of GSK3β, G17 studies were performed to determine changes in the expression of GSK3β-downstream target Snail. Incubation of AGSE cells with G17 resulted in an increase in Snail protein expression in a time Fig  and doseIn order to determine whether G17 increased Snail expression via inhibiting GSK3β, G17 studies were performed following pretreatment of the cells with a pharmacological inhibitor of GSK3β (AR-A014418) . These sTo understand whether G17-induced inhibition of GSK3β was critical to induce migration, wound-healing assays were carried out following overexpression of either wild-type or mutant forms of GSK3β. As shown in Fig Since GSK3β inhibition in these cells was enough to induce Snail expression and β-catenin nuclear translocation Figs , and inhTo elucidate further the mechanism by which G17/GSK3β axis mediated migration, studies were also designed with the MMP7 promoter which was shown to be induced by G17 in our earlier studies . TreatmeThe GI peptide hormone gastrin (G17 and its unprocessed forms) can regulate various cellular processes involved in cancer ,38. The Ser9 phosphorylation , was unable to antagonize G17-induced GSK3βSer9 phosphorylation , despite a complete inhibition of AKTSer473 phosphorylation (pAKTSer473 panel). In addition, treatment of another gastric cancer cell line (MKN45) with G17 produced an increase in GSK3βSer9 phosphorylation without any effect on AKT phosphorylation  induced Snail expression and transcription in the absence of G17 , we observed no increase in β-catenin total protein expression at any time point following G17 treatment as reported earlier [The detailed mechanism by which GSK3β regulates migration is still unknown and might involve specific downstream targets. Since Snail and β-catenin are both downstream targets of GSK3β ,26, whicG17 Figs . In addiG17 Figs . The oth earlier . However earlier . The sec earlier . Recent  earlier . It is t earlier  and G17  earlier , G17 stuTo understand whether Snail and β-catenin were specific downstream targets of GSK3β to mediate G17-induced migration, G17 studies were performed following siRNA-mediated knockdown of endogenous Snail or β-catenin expression. Knockdown of either protein alone produced a partial inhibition of G17-induced migration Fig  as well The present study demonstrates that G17-induced migration and MMP7 promoter induction in gastric cancer cells involve an inhibition of GSK3β pathway. G17-induced inhibition of GSK3β leads to an increase in Snail expression and β-catenin nuclear translocation, both of which collectively mediate the migratory response. Thus β-catenin and Snail serve as two downstream targets of GSK3β in G17-induced migration pathway. However, this G17/GSK3β/Snail-β-catenin axis seems to be independent of a previously identified G17/MLK3/JNK1 axis  and bothSer9, AKT, pAKTSer473, pc-Jun, c-Jun, Snail and Lamin A/C , GAPDH , β-catenin , HA.11 . The CCK2R antagonist YM 022 was from Tocris Bioscience , PI3 Kinase inhibitor Wortmannin and GSK3β inhibitor AR-A014418 from EMD Biosciences  and JNK inhibitor SP600125 from Alexis Biochemicals, Axxora . The AGSE cells were obtained from Dr. Timothy C. Wang  as described earlier [DMEM, LipofectAMINE 2000 and β-Galactosidase assay kit were purchased from Invitrogen ; amidated gastrin (G17) from Bachem  and the luciferase assay kit from Promega . The antibodies utilized were obtained from the following sources: GSK3β, pGSK3β earlier ,36. The  earlier  and the  earlier .The AGSE cells used in these studies were derived from AGS cells stably overexpressing CCK2R as described previously . These c3 LB 960) and a plate reader  respectively. The results obtained were calculated as the ratio of relative light units (RLU) to β-Gal values  and expressed as % increase compared to controls.Subconfluent populations of cells were transiently transfected with Snail-luciferase  or MMP7-3VO4, supplemented with protease inhibitors) and rotated at 4°C for 30 minutes. This was followed by centrifugation at 13,000 rpm for 15 minutes to pellet nuclei, washing the nuclear pellet once with wash buffer (containing lysis buffer + 25% glycerol) and centrifugation again. To obtain nuclear extract, the nuclear pellet was lysed in nuclear lysis buffer (wash buffer + 300 mM NaCl), sonicated and incubated on ice for 30 minutes followed by centrifugation. Western Blot analysis was performed with equal amounts of nuclear protein with the indicated antibodies. To determine the purity of the nuclear preparation, they were blotted with antibodies against GAPDH (cytoplasmic protein) and Lamin A/C (nuclear protein).Whole cell extracts were prepared from cells treated with G17 by RIPA extraction buffer, and equal amounts of total protein were subjected to Western Blot analysis utilizing procedures described previously ,36. NuclThe β-catenin siRNA  was syntThese were performed following procedures described earlier . ConflueH. pylori: Helicobacter pylori; JNK: c-Jun NH2-terminal Kinase; MLK3: Mixed lineage kinase-3; MMPs: matrix metalloproteinases; PAK1: p21-activated kinase 1; PBS: Phosphate buffer saline; PI3Kinase: phosphatidylinositol-3 kinase; RLU: relative light units; siRNA: small interference RNA; TCF/LEF: T cell factor/lymphoid enhancer factor; WT: Wild Type;APC: Adenomatous Polyposis Coli; β-Gal: β-Galactosidase; CCK2R: CCK2 receptor; EV: Empty Vector; EMT: epithelial-to-mesenchymal transition; G-17: amidated gastrin; G-Gly: glycine-extended gastrin; GI: Gastrointestinal; GSK3β: Glycogen Synthase Kinase 3beta; The authors declare that they have no competing interests.PM performed Western blot analysis, Luciferase analysis, overexpression and migration experiments. SS performed some of the gastrin-related migration and Western experiments in various gastrointestinal cells, obtained photographs of migration and contributed to the editing of the manuscript. AR helped with the analysis of the GSK3β overexpression experiments and provided intellectual input in this collaborative study. BR contributed to the overall study design, interpretation of the results during all phases and drafted/edited the final manuscript. All authors read and approved the final manuscript.


Vanda species. A preliminary biological screening revealed that Vanda coerulea (Griff. ex. Lindl) crude hydro-alcoholic stem extract displayed the best DPPH /•OH radical scavenging activity and in vitro inhibition of type 2 prostaglandin (PGE-2) release from UVB (60 mJ/cm2) irradiated HaCaT keratinocytes.In our investigations towards the isolation of potentially biologically active constituents from Orchidaceae, we carried out phytochemical and biological analyses of 1) methoxycoelonin (2) gigantol (3) flavidin (4) and coelonin (5). Stilbenoids (1–3) were the most concentrated in crude hydro-alcoholic stem extract and were considered as Vanda coerulea stem biomarkers. Dihydro-phenanthropyran (1) and dihydro-phenanthrene (2) displayed the best DPPH/•OH radical scavenging activities as well as HaCaT intracellular antioxidant properties  compared to bibenzyle (3) (IC50 20.6 µM). In turn, the latter showed a constant inhibition of PGE-2 production, stronger than stilbenoids (1) and (2) . Western blot analysis revealed that stilbenoids (1–3) inhibited COX-2 expression at 23 µM. Interestingly, stilbenoids (1) and (2) but not (3) were able to inhibit human recombinant COX-2 activity.Bio-guided fractionation and phytochemical analysis led to the isolation of five stilbenoids: imbricatin (1–3) from Vanda coerulea stems displayed an inhibition of UVB-induced COX-2 expression. Imbricatin (1) and methoxycoelonin (2) were also able to inhibit COX-2 activity in a concentration-dependent manner thereby reducing PGE-2 production from irradiated HaCaT cells. Our studies suggest that stilbenoids (1–3) could be potentially used for skin protection against the damage caused by UVB exposure.Major antioxidant stilbenoids ( B (290–320 nm) radiations trigger the production of reactive oxygen species (ROS), increasing oxidative stress in irradiated skin. ROS are an inherent part of the anabolism and catabolism of tissues, including skin. Cellular enzymes and controlled metabolic processes ordinarily keep oxidative damage to cells at a minimum. In times of increased ROS production  protective controls may not be adequate anymore resulting in an oxidant-antioxidant imbalance, defined as oxidative stress. It can cause many adverse effect (ageing) and pathological conditions  via the modulation of biochemical, genetic and signal transduction pathways Skin is continuously exposed to ultraviolet (UV) radiations from sunlight and to environmental pollution. UVB radiations induce cyclo-oxygenase 2 (COX-2) expression and subsequently increase the production of prostaglandin E2 (PGE-2) in keratinocytes. COX-2 is a rate limiting enzyme for the generation of prostaglandin metabolites and its expression has been linked to the pathophysiology of inflammation and cancer B increase oxidative stress and the generated reactive oxygen intermediate are most likely involved in the up-regulation of COX-2 UVB-induced inflammatory responses, partly due to the suppression of COX-2 expression and/or its enzymatic activity Plants have also to protect themselves from solar radiation. Thus, they synthetize natural chromophores like polyphenolic compounds  that are able to absorb UV radiations and display antioxidant properties Dendrobium, Bletilla, Cymbidium, Gastrodia and Vanda occurred mostly in tropical areas C-glycosides Vanda have not been well studied considering their biological activities on human cells and their chemical composition. Only two kinds of phenanthropyran have been described in the species V. parvifloraV. tesselataVanda coerulea Griff. ex Lindl, also known as the “Blue Orchid”. A preliminary biological screening on stem, root and leaf crude hydro-alcoholic extracts revealed that V. coerulea stem extract displayed best DPPH /OH radical scavenging activity and in vitro inhibition of PGE-2 production from irradiated HaCaT . Bio-guided fractionation combined with phytochemical analysis allowed us to isolate and identify five stilbenoids (1), and flavidin (4), two 9,10 dihydro-phenanthrenes methoxycoelonin (2) and coelonin (5) and one bibenzyle gigantol (3). Additional compounds identified were carbohydrates and terpenoids such as sesquiterpenes and phytosterols.The Orchidaceae is subdived in five subfamilies. Genera form the Epidendroideae subfamily such as Vanda coerulea stems display radical scavenging activities, attenuate intracellular ROS formation and reduce both UVB-induced PGE-2 production on HaCaT cells and COX-2 activity. Comparison of their radical scavenging properties led us to a first structure-activity relation (SAR) study performed with orchid's stilbenoids. Biological analyses on HaCaT cells were performed with the three most concentrated stilbenoids (1–3). We evaluated here for the first time the potential interest of stilbenoids from Orchidaceae as new skin photoprotecting agents.In the present work, we examine whether stilbenoids from Quercetin was obtained from Extrasynthese. Luminol was obtained from Fluka Biochemika. Indomethacin, tocopherol and all other chemicals like dimethylsulfoxyde (DMSO), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH DA) probe, were from Sigma. Keratinocytes serum free medium (KSFM), foetal bovine serum (FBS) for HaCaT culture, penicillin/streptomycin, glutamine and phosphate buffer serum (PBS) were from Invitrogen. Cell proliferation kit II reagent (XTT) was provided by Roche Diagnostic. Bicinchoninic acid (BCA) assay kit for protein dosage was obtained from Uptima Interchim. Enzyme immunoassay (EIA) kits for PGE-2 were obtained from R&D Systems and Cayman Chemical. Antibodies anti β-actin (sc-130300) and anti COX-2 (sc-19999) as well as CCD-1064SK cell lysate were provided by Santa Cruz Biotechnology Inc. Amersham Hybond ECL nitrocellulose membrane, horseradish peroxidase-conjugated secondary antibody (NIF-825) and enhanced chemiluminescence (ECL) kit were obtained from GE Healthcare Europe GmbH.Vanda coerulea Griff. ex Lindl. was identified by Dr. Josef Margraf in Thailand. A voucher specimen (No HITBC128192) was deposited in the Herbarium of the Xishuangbanna Tropical Botanical Garden (XTBG). Stems from flowering specimens were collected in October 2006. Cut stems were dried by sunshine on hanging basket to avoid contamination by soil and prevent fungus development. After reception in France, dried cut stems were finally reduced to powder in the ultra-centrifuge crusher (Retsch ZM 200) just before extraction. Supply of stems was carried out in accordance with CITES  regulations.V. coerulea stems (100 g) were exhaustly extracted with EtOH-H2O (9∶1) under reflux and filtered to provide a red brown crude hydro-alcoholic extract (8.9 g). Dried stems (100 g) were also successively extracted by cyclohexane (C6H12), dichloromethane (CH2Cl2) and methanol (MeOH) using an automatisable Soxtec (Avanti 2055), to yield C6H12 (1.0 g), CH2Cl2 (1.0 g), and MeOH (5.8 g) extracts, respectively. An aliquot of CH2Cl2 extract (0.64 g), which concentrated stilbenoids, was suspended in MeOH-CH2Cl2 (8∶2) and subjected to Sephadex LH20 column (H: 27 cm d: 2.5 cm) with MeOH-CH2Cl2 (8∶2) elution and collection of 25 fractions of 10 mL. Fractions with similar TLC (Thin Layer Chromatography) profiles were gathered. Fractions 3 to 6, rich in stilbenoids, were further purified by semi preparative reversed-phase HPLC to give compounds 1 (70 mg), 2 (35 mg) 3 (19 mg), 4 (8 mg) and 5 (8 mg).Dried 2O+0.1% HCO2H) using a linear gradient at a flow rate of 1 mL.min−1, starting from 20% up to 47% MeOH in 13.5 min, maintaining 47% MeOH during 10 min and finally from 47% to 100% MeOH in 10 min. The fractions were monitored between 200 and 400 nm. Semi-preparative HPLC purifications of stilbenoids were carried out on another chromatograph equipped with a Gilson 322 pump and a UV-VIS 151 Gilson wavelength detector, using a Nucleodur C18ec column  eluted with MeOH-(H2O+0.1% HCO2H) under the following conditions: 47% MeOH during 10 min and to 100% MeOH in 30 min at a flow rate of 15 mL.min−1. The fractions were monitored at 210 nm.Extract and fractions were analyzed by RP-HPLC using a Nucleodur C18ec column  in a Varian chromatograph equipped with a Prostar 230 pump and a Prostar 330 diode-array detector. The column was eluted with MeOH- and confirmed by comparison with those reported in literature Structures of these stilbenoids were elucidated by spectral analyses (Imbricatin (1): yellow gum, UV (in MeOH) λmax (log ξ) 217 (4.4), 285 (4), and 307 (4) nm characteristic of a dihydro-phenanthrene skeleton 1H NMR : δ = 6.29 , 6.31 , 6.73 , 2.79-2.78  and 5.19 , 3.77 .13C NMR data  δ = 108.7 (C-1), 155.0 (C-2), 101.8 (C-3), 120.1 (C-4a), 113.1 (C-4b), 153.0 (C-4), 121.0 (C-5), 141.0 (C-6), 147.6 (C-7), 114.4 (C-8), 129.8 (C-8a), 136.0 (C-10a), 28.0 (C-10), 27.5 (C-9) 63.8 (C- O--CH2--Ar), 62.2 (C-OMe); HR-ESI-MS ion [M-H]- at m/z = 269.0815 .Methoxycoelonin (2): yellow brown gum, UV (in MeOH) λmax (log ξ) 216 (4.3), 285 (3.9), and 307 (3.8) nm characteristic of dihydro-phenanthrene skeleton 1H NMR : δ = 6.30 , 6.37 , 7.81 , 6.72 , 2.69  and 3.86 , 3.82 . For 13C NMR data : δ =  107.8 (C-1), 154.1 (C-2), 97.8 (C-3), 124.3 (C-4a), 116.3 (C-4b), 156.9 (C-4), 110.8 (C-5), 143.6 (C-6), 142.9 (C-7), 112.7 (C-8), 130.9 (C-8a), 140.8 (C-10a), 28.6 (C-9), 30.4 (C-10), 55.3 (C-OMe), 54.9 (C-OMe); HR-ESI-MS ion [M-H]- at m/z = 271.0968 .Gigantol (3): colorless gum, UV (in MeOH) λmax (log ξ) 206 (4.1), 281 (3) nm 1H-NMR : δ = 6.22 , 6.21 , 6.66 , 6.28 , 6.81 , 6.61 , 2.79 , 3.82 , 3.73 . For 13C NMR data  δ =  107.6 (C-1), 156.1 (C-2), 98.0 (C-3), 160.4 (C-4), 120.5 (C-4a), 106.4 (C-4b), 114.0 (C-5), 144.1 (C-6), 145.8 (C-7), 110.7 (C-8), 133.8 (C-8a), 143.3 (C-10a), 37.2 (C-9), 38.2 (C-10), 55.4 (C-OMe), 54.8 (C-OMe); HR-ESI-MS ion [M-H]- at m/z = 273.1123 .Flavidin (4): colorless gum UV (in MeOH) λmax (log ξ) 214 (4.4) 286 (4.1), 300 (4.0) nm. 1H-NMR : δ = 6.30 , 6.19  6.42 , 6.55 , 5.01 , and 2.78 .13C NMR data  δ = 109.6 (C-1), 158.4 (C-2), 103.0 (C-3), 120.2 (C-4b), 113.0 (C-4a), 154.6 (C-4), 130.7 (C-5), 110.0 (C-6), 157.6 (C-7), 115.0 (C-8), 136.6 (C-8a), 135.3 (C-10a), 29.0 , 69.4 (C- O--CH2--Ar). HR-ESI-MS ion [M+H] + at m/z = 241.0861 .Coelonin (5): colorless gum UV (in MeOH) λmax (log ξ) 322 (4.6), 280 (4.3), 291 (4.2) nm1H-NMR  δ = 6.31 , 6.41 , 8 , 6.62 , 6.62 , 2.63 , 3.80 . For 13C NMR data  δ = 107.9 (C-1), 157.7 (C-2), 99.3 (C-3), 126.0 (C-4b), 117.0 (C-4a), 159.0 (C-4), 130.2 (C-5), 113.7 (C-6), 156.0 (C-7), 115.0 (C-8), 141.4 (C-10a), 140.6 (C-8a), 31.3 (C-10), 31.9 (C-9), 56.0 (C-OMe). HR-ESI-MS ion [M+H] + at m/z = 243.1022 .δ = chemical shift in ppmHaCaT Keratinocytes were provided by LVMH recherche. Cells were cultivated in keratinocytes serum free medium (KSFM) complemented with 0.25% human recombinant EGF , 2.5% pituitary bovine extract, and 5% fetal bovine serum (FBS). All biological analyses were realized at passages 15 to 20.in vitro radical-scavenging experiments  and IC50 calculations were assayed as previously described by Parejo et al50. Serial dilutions of 1–5, and quercetin (used as standard reference) were prepared in MeOH.All Vanda coerulea stem biomarkers, HaCaT cells were seeded in black 96-well microplates (Greiner Bio One 655090) at a concentration of 103 cells/well in complemented KSFM with 5% FBS. Microplates were incubated during 24 h  before UVB irradiation at 60 mJ/cm2 and treatment. For western blot analysis HaCaT cells were seeded at a density of 106 cells/ 100 mm dishes and incubated during 48 h before irradiation with UVB at 60 mJ/cm2  and treatment. All experiments were performed at 90% cell confluency. Cell viability was previously evaluated thanks to XTT analysis according to Roehm NW et al1–3, were prepared in DMSO . The first higher concentration tested was not cytotoxic (data not shown).For measurement of reactive oxygen species neutralization and PGE-2 quantification with 1–3 and quercetin (standard reference) after 24 h incubation. Treated microplates were further incubated during 24 h . HaCaT cells were washed with phosphate buffer saline (PBS), and DCFH-DA (0.05 mM) was added to the cells. 45 min after incubation, cells were stimulated by a H2O2 solution (100 µM in PBS) to generate an oxidative stress. Immediately after F(T0) and 40 min after stimulation F(T40), DCFH fluorescence was detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm, using a SpectraFluor Plus spectrofluorometer (Tecan). Two kinds of assay controls were used: negative control was non-stimulated cells and positive control was H2O2 stressed cells (DMSO 0.1% v/v). Results were expressed in percentage of fluorescence inhibition  calculated by comparison with H2O2 stimulated cells.The DCFH-DA method was used to detect the levels of intracellular ROS F(T40)-F(T0)] sample/[F(T40)-F(T0)]positive control] ×100.  =  50 values  and Hill coefficients (nH) were determined by non-linear regression analysis of the inhibition curves generated with mean replicate values using Hill equation curve fitting.The ICIn vitro experiments were performed three times independently. All data were expressed as means ± standard deviations (sd). Treatment groups were compared by using one way analysis of variance ANOVA post-hoc test. Each stilbenoid effect was compared to positive control using student t tests. Statistical significance (*) was set at p<0.05.Figure S1.Confirmation of PGE-2 inhibitory effect on human normal epidermal keratinocytes is provided as Vanda coerulea stems revealed that a stilbenoid-enriched CH2Cl2 extract displayed a better in vitro DPPH/ •OH radical scavenging activity and inhibition of PGE-2 release from (UVB 60 mJ/cm2) irradiated HaCaT than crude hydro-alcoholic extract. These observations led us to the isolation and identification of the five stilbenoids described in 1) was first identified in the orchid Pholidota imbricata4) in BulbophyllumCoelogyne and Pholidota species 2) in Agrostophyllum callosum and in Coelogyne flaccida5) was isolated from two species of Coelogyne: C. ochracea and C. elata3) was identified for the first time in Cymbidium giganteum5) and methoxycoelonin (2) in Cymbidium aloifoliumBio-guided fractionation performed on 1–5 were only present in Vanda coerulea stems. The most concentrated (1–3) were considered as V. coerulea stem biomarkers.A comparison of phytochemical profiles of root, stem and leaf crude extracts showed that stilbenoids Vanda coerulea stems was already described for its radical scavenging properties mostly concerning DPPH neutralization et al had evaluated radical scavenging properties of gigantol (3) using the DPPH (IC50 56.4 µM) and ORAC assays et al and Guo XY et al indicated that coelonin (5) and imbricatin (1) neutralized DPPH radical with IC50 of 16.7 and 8.8 µM, respectively 4) were evaluated using different antioxidant assays, including DPPH and hydrogen peroxide neutralization  1 to 5 were not compared so far. Here, we aimed to propose a structure-activity relationship (SAR) relative to DPPH and •OH radical scavenging activities.Each stilbenoid isolated from  •OH radicals are among the most aggressive forms implied in cellular oxidative stress 1, 4) and dihydro-phenanthrenes  displayed better •OH but also DPPH radicals scavenging properties than bibenzyle (3). Activities of imbricatin (1) and methoxycoelonin (2) were quite similar to the standard quercetin on •OH radical scavenging activities, while flavidin (4) and coelonin (5) were globally less active. : 1≈2>4≈5>3 for •OH radical scavenging activities.SAR on ortho-methoxy-hydroxyl substitution. Thus, a classification could be proposed as followed:We also observed a tendency for dihydro-phenanthropyran structures to better neutralize DPPH radical. Scavenging activities were better on structures without 4 >1≈5≥2>3 for DPPH radical scavenging capacities.50 of imbricatin (1) calculated for DPPH scavenging activities was in accordance with those indicated in literature 50 values were obtained with gigantol (3) and coelonin (5) than those previously described by Zhang X et al and Wang J et al , respectively In comparison with already published data, the IC2, 5) or phenanthropyrans  are planar structures compared with the bibenzyle gigantol (3). Thus, electron delocalization and structure stabilization is better on phenanthrenes than on bibenzyle. As explained by Jayaprakasha GK et al, the antioxidant capacities of 9,10 dihydro-phenanthrenes or phenanthropyrans may be attributed to hydrogen donating ability, the product phenoxyl radical being stabilized by resonance delocalization of the unpaired electron to the other position in the ring The 9,10 dihydro-phenanthrenes (1–3), considered as Vanda coerulea stem biomarkers, could neutralize intracellular ROS in H2O2 stressed HaCaT cells.The excessive production of intracellular ROS plays an important role in the signalling events leading to gene activation 2O2 (100 µM) stressed HaCaT. The intracellular fluorescence measurement using this probe mainly reflects the ability of stilbenoids to inhibit intracellular •OH radical 1) (IC50 8.8 µM) and methoxycoelonin (2) (IC50 9.4 µM) showed better ROS neutralization than gigantol (3) (IC50 20.6 µM) and even than quercetin (IC50 13.8 µM).  and the dihydro-phenanthrene (2).The probe 2′,7′-dichlorofluorescin is used as an indicator of ROS formation in H.8 µM). ,Table 2B-induced COX-2 expression 1–3) reduce the generation of intracellular ROS partly due to direct radical scavenging activities.Intracellular ROS produced in excess contribute to inflammatory response triggered by UV radiations and have been suggested to be involved in UVPGE-2 is known to play an important role in UV-induced skin inflammation. Its production is mediated by UVs and ROS induction of COX-2 expression. Moreover, it has been reported that keratinocytes proliferation and apoptosis are also regulated by COX-2 after acute UV radiations 3) and coelonin (5) only, were already described in the scientific literature and referred mostly to the neutralization of nitric oxide production (and PGE-2 production for gigantol) on murine macrophages RAW 264.7 V. coerulea antioxidant stem biomarkers as skin anti-inflammatory agents. Their effects were evaluated on PGE-2 production and on inhibition of COX-2 expression in UVB irradiated HaCaT cells. As shown in 1–3) inhibited PGE-2 release from UVB (60 mJ/cm2) irradiated HaCaT cells. To compare biomarker activities the same range of concentrations was tested for each stilbenoid. Concentration-dependent effect was observed for imbricatin (1) (IC50 12.2 µM) and methoxycoelonin (2) (IC50 19.3 µM), but not for gigantol (3). The results revealed that even at the lowest concentration tested, gigantol (3) strongly inhibited PGE-2 production. We deduced from these data that these three major stilbenoids were principally responsible for the antioxidant and the reduction of PGE-2 production previously observed with V. coerulea crude stem extract. Bibenzyle gigantol (3) displayed better PGE-2 inhibition than dihydro-phenanthropyran (1) and dihydro-phenanthrene (2), but without any concentration-dependant effect at the tested concentrations.Anti-inflammatory properties for gigantol  and methoxycoelonin (2) but not gigantol (3) were able to inhibit human recombinant COX-2 enzyme , suggesting that dihydro-phenanthropyran (1) and dihydro-phenanthrene (2) but not bibenzyle (3) could reduce PGE-2 production through COX-2 inhibition. Their effect on human recombinant COX-2 enzyme was comparable to the activity of indomethacin (IC50 1.6 µM), a classical non-steroidal COX inhibitor.Interestingly, ,Table 350 calculated for inhibition of PGE-2 production on HaCaT are different from those calculated for COX-2 inhibition. Indeed, for the first analysis we have measured stilbenoid activities on the whole cell, determining a global response leading to the release of PGE-2. For the second analysis, we have directly determined stilbenoids effects on human recombinant and isolated COX-2 enzyme. Because of these differences between cellular and enzymatic analysis the IC50 calculated in both experiments were not identical.The ICet al 2006 have demonstrated that gigantol (3) was able to inhibit PGE-2 production on murine macrophages RAW 264.7 as a result of inhibition of COX-2 expression but also of NF-κB expression 3) on HaCaT PGE-2 production could be explained by a strong reduction of UVB-induced COX-2 enzyme expression.Wong J.H. B (60 mJ/cm2) irradiated HaCaT cell lysates. Two concentrations of each stilbenoids were tested (23 and 2.87 µM) on HaCaT. As shown in B-induced COX-2 expression at 23 µM. Best results were obtained with methoxycoelonin (2) and gigantol (3). They did not inhibit COX-2 expression at the lowest concentration tested (2.87 µM). Gigantol demonstrated strong inhibition of PGE-2 production even at 2.87 µM but did not inhibit COX-2 expression and activity at this concentration. Gigantol (3) may therefore act through other mechanisms to exert its inhibitory effect on PGE-2 production and COX-2 enzyme may not be its only biological target.Western blot analysis was performed with UVB exposure induces ROS accumulation in human skin and is involved in its ageing process as well as its inflammatory response V. coerulea stems. Our results revealed that the dihydro-phenanthrene methoxycoelonin (2) and the dihydro-phenanthropyran imbricatin (1) displayed the best intracellular antioxidant activities on HaCaT keratinocytes and interesting anti-inflammatory properties due to a concentration-dependant inhibition of COX-2 activity and expression. The bibenzyle gigantol (3) better inhibited HaCaT PGE-2 production compared to methoxycoelonin (2) and imbricatin (1) at each concentration tested. Its activity could be partly attributed to an attenuation of UVB-induced COX-2 expression. No concentration-dependent inhibitory effect was observed to allow IC50 calculation for gigantol (3) effect on PGE-2 production. Wong JH et al also demonstrated that this compound (3) was able to reduce NF-κB expression on murine macrophages RAW 264.7 3) displayed a 16-fold stronger inhibition of PGE-2 production on human HaCaT than on murine macrophage RAW 264.7 UV1–3), considered as Vanda coerulea stem biomarkers, provided chromophore structures absorbing UVB radiations. Applied locally these polyphenolic compounds may act as a sunscreen hitherto obtained showed that imbricatin (1), methoxycoelonin (2) and gigantol (3) displayed antioxidant properties on HaCaT by means of intracellular ROS neutralization. The three stilbenoids were able to reduce UVB-induced COX-2 expression, thereby reducing PGE-2 production on HaCaT keratinocytes. These activities could be partly linked to the reduction of intracellular oxidative stress, to a reduction of COX-2 expression and, for imbricatin (1) and methoxycoelonin (2) to a direct inhibition of COX-2 enzyme activity. Globally, we observed complementary properties between the dihydro-phenanthropyran imbricatin (1) the dihydro-phenanthrene methoxycoelonin (2) and the bibenzyle gigantol (3). The first displayed better antioxidant activities whereas the latter showed strong inhibition of PGE-2 production. Anti-inflammatory property of orchid's stilbenoids was here described for the first time on HaCaT keratinocytes. Vanda coerulea stem biomarkers could be considered as potential new natural skin photoprotecting agents.In summary, major stilbenoids  on PGE-2 release from irradiated (UVB 60 mJ/cm2) normal human epidermal keratinocytes (NHEK). Histogram represents the quantity of PGE-2 produced  according to different treatments. Untreated irradiated (UVB+) and non irradiated (UVB-) NHEK were used as positive and negative controls, respectively. Indomethacin (Indomet.) at 2.79 µM represented the positive reference (86% inhibition of PGE-2 production). Inhibition percentages of PGE-2 release were calculated by comparison with the positive control (UVB+). Values represen mean ± sds calculated with three independent experiments. Treatment groups were compared by using one way analysis of variance ANOVA post-hoc test. Student paired t test was used to compare each stilbenoid effect to the positive control. Statistical significance (*) was set at p<0.05. Significant differences were observed between each biomarker effect. IC50 were calculated with more points than those represented here on the graph.(0.82 MB TIF)Click here for additional data file.


Results indicated that MdOS, a novel marine-derived oligosaccharide sulfate, exhibited a broad-spectrum PTK inhibitory action. At an enzymatic level, MdOS inhibited HER2, EGFR, VEGFR, PDGFR, c-Kit, FGFR1 and c-Src, with little impact on FGFR2. In cellular settings, MdOS inhibited phosphorylation of PTKs, exemplified by HER2, EGFR and VEGFR2, and downstream molecules of Erk1/2 and AKT. Further studies demonstrated that MdOS acted as an ATP-competitive inhibitor via directly binding to the residues of entrance rather than those of the ATP-binding pocket. Furthermore, MdOS inhibited proliferation and tube formation of HMECs, arrested microvessel outgrowth of rat aortic rings and hindered the neovascularization of chick allantoic membrane. Taken together, results presented here indicated that MdOS exhibited anti-angiogenic activity in a PTK-dependent manner and make it a promising agent for further evaluation in PTK-associated cancer therapy.Despite the emerging success of multi-targeted protein tyrosine kinase (PTK) inhibitors in cancer therapy, significant side effects and resistance concerns seems to be avoided unlikely. The aim of the present study was to identify novel multi-targeting PTK inhibitors. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay (ELISA). The antiproliferative activities in human microvascular endothelial cells (HMECs) were evaluated by sulforhodamine (SRB) assay. The phosphorylation of kinases and their downstream molecules was probed by western blot analysis. The binding mode between MdOS and PTKs was profiled by surface plasmon resonance (SPR) approach and molecular simulation. Tube formation assay, rat aortic ring method and chicken chorioallantoic membrane assay were combined to illustrate the  Protein tyrosine kinases (PTKs) play crucial roles in signal transduction pathways that regulate a number of cellular functions, such as cell proliferation, differentiation, migration and apoptosisOver the past years, there has been a proliferation of agents designed to inhibit single PTK in tumor, including those directed against Bcr-Abl (e.g. imatinib mesylate)Since small molecule inhibitors may not only possess potent cytotoxicity and poor solubility, they may also increase the likelihood of development of resistance The oligosaccharides bear unique backbone totally different from that of small molecules that have never been challenged in this setting. Enzyme-linked immunosorbent assay (ELISA), a sensitive and specific assay for the detection and quantification of antigens or antibodies, has been widely used in tyrosine kinase related drug discovery research due to easy-handling and free from radio-contamination, in particular comparing to 32P incorporation 50 values of 0.13, 0.28 and 1.8 µg/ml, respectively. Moreover, MdOS moderately inhibited those of PDGFRβ, c-Kit, c-Src and FGFR1, but had little effect on that of FGFR2. MdOS is, therefore, a multi-targeted tyrosine kinase inhibitor.The effects of MdOS on the activities of various tyrosine kinases were evaluated using enzyme-linked immunosorbent assays (ELISAs) with purified recombinant proteins. As shown in Given the relative high inhibitory potency of MdOS against HER-2, EGFR and VEGFR2, together with the fact that HER-2, EGFR and VEGFR2 are the most verified targets in cancer therapy, we took these three kinases as representatives to probe MdOS -driven PTKs-associated events.We next intended to investigate the kinase inhibitory activity of MdOS at cellular level. For this, both naturally and genetically kinase expressing cell lines were selected.The activity of MdOS against HER-2 was followed by measuring receptor autophosphorylation in naturally HER-2-overexpressing SK-OV-3 cells and a newly generated NIH-3T3 cells lines stably expressing HER-2 (NIH-3T3/neu cells). MdOS dose-dependently dephosphorylated HER-2, with a complete abolishment obtained at 100 µg/ml . Erk1/2 The effects of MdOS on EGFR were measured in naturally EGFR-overexpressing A431 and MDA-MB-468 cells. As shown in We explored the kinase-inhibitory effects of MdOS on VEGFR2 using HMEC cells that naturally overexpress VEGFR2 and a newly generated NIH-3T3 cells lines stably expressing VEGFR2 (NIH-3T3/flk1 cells). Interestingly, MdOS was noted to be more effective against VEGFR2 in cellular assay compared with the inhibitory potency against HER-2 and EGFR. As shown in Taken together, MdOS can inhibit phosphorylation of HER-2, EGFR and VEGFR2 intracellularly, with potency against VEGFR2 relatively higher than the other two.Consensus view holds that oligosaccharides act extracellularly by inhibiting the interactions of growth factors to their receptors. In the aforementioned study, MdOS was interestingly and distinctly identified to arrest the kinase activity of growth factor receptors mainly via its intracellular action. To further confirm the MdOS-driven intracellular events, we first examined the cell entry of MdOS by using a FITC-labeled MdOS  probe in SK-OV-3 cellsTo provide more direct evidence and rule out contributions of extracellular activity, we then carried out another subsequent experiments, by withdrawing the added MdOS prior to the exposure of growth factors and by depriving the growth factors from the experimental settings as well. As expected, MdOS inhibited the phosphorylation of HER-2 in SK-OV-3 cells in the MdOS-withdrawal sample similar to that in MdOS-unwithdrawal sample . LikewisTogether with our previous data in D) of 5.76×10−7 M  and particularly suppressed VEGFR2 phosphylation and subsequent downstream signaling in HMECs . In addiTo address this issue, we first defined the appropriate concentrations and duration that is irrelevant to the cytotoxic effect of MdOS. For this, both MTT and BrdU incorporation assays were selected. MdOS demonstrated little inhibitory impact on cell viability within 12 h, even at concentration of 100 µg/ml (data not shown). Accordingly, the concentrations no more than 100 µg/ml and exposure time within 12- h were selected for the subsequent experiments.50 values of 48.3 µg/ml, 61.0 µg/ml, 56.2 µg/ml and 62.7 µg/ml, respectively), whereas with less potency against FBS-mediated events   . These fAs tube formation represents one of the late stage of angiogenesis, we next evaluated the effects of MdOS on tube formation of HMECs on a Matrigel substratumWe further evaluated the anti-angiogenic effects of MdOS in an ex vivo aorta sprout outgrowth assay in vivo, we extended our studies to a CAM model To determine whether anti-angiogenic effects of MdOS could be recapitulated The high frequency of genetic alterations in cancer cells resulting in permanent activation of protein tyrosine kinases has led to the positioning of these kinases as therapeutic targetsversus toxicity of multi-targeted PTK inhibitors, these benefits emerge to weight against the potential limitation, for instance, the mutations in residues of ATP-binding cleft disable the active access of the established inhibitors to the ATP-binding domain, thereby offering the inevitable resistance With an increasing understanding of SAR-based efficacy in vivo anti-angiogenic efficacies of MdOS should be the synergistic outcomes of this broad-spectrum inhibitory action. Notably, the unique binding mode by the simulation exercise that MdOS hydrogen bonded to the residues of the entrance rather than those of ATP-binding pocket helps, from a theoretical view, to understand the likely distinct pharmacological profiles from small molecules in general and, the potential value against small molecule-evoked resistance in particular. Of course, much hope lies on further investigation.MdOS is structurally defined as a 1,4-linked β-D-mannuronate of pyranohexuronic acid residues with the reducing end oxidized to mannurarate, bearing an average of 1.5 sulfates per sugar residue at the 2-hydroxyl, partial 3-hydroxyl with partial esterification at 6-carboxyl groups. This structure offers MdOS appreciable negative charges, which allows MdOS to afford a high-affinity interaction with PTK in a broad spectrum manner. Our ELISA-based kinase inhibitory outcomes that MdOS broadly inhibited PTKs, together with our cellular assays that MdOS inhibited PTKs phosphorylation and downstream signaling, substantiate this story. The concomitant Although electrostatic charges are viewed to initiate attachments between sugars and proteins, the structure-based conformation likely dominates the functions. The alternative findings that some other marine-derived oligosaccharides, even bearing the same negative charges but with different sugar compositions, did not generate the likelihood of functions as MdOS did (data not shown), help reinforce this view. The structural components responsible for interactions between PTK and sulfated oligosaccharides have not been well characterized, but the nature of the sugar scaffold seems to be critical, and distribution of sulfation seems essential.In summary, this is the first time to disclose that MdOS, a marine-derived oligosaccharide, functions as a broad spectrum of tyrosine kinase inhibitor. It is canonically viewed that oligosaccharides act extracellularly by inhibiting the interactions of growth factors to their receptors 3H in formamide after partially esterified at the 6-carboxyl groups. Briefly, oligomannuronate was mixed with propylene oxide at a certain ration, and the reaction was catalyzed by NaOH at 45°C for 3 h. Then the esterified products were added to sulfating reagents containing formamide and ClSO3H, and reacted for 3 h. The pH of the products was adjusted to 7.0 with NaOH, and desalted using Sephadex G-10. The product peak was pooled and freeze-dried. The molecular weights of MdOS and its precursor were analyzed by High Performance Gel Permeation Chromatography (HPGPC) using a TSK G3000PWxl column (300 mm×7.8 mm) . Stock solutions were prepared in saline solution at 10 mg/ml and then diluted to the desired concentration with saline solution.The oligomannuronic acid was degraded by hydrogen peroxide from polymannuronate blocks obtained from sodium alginate. The MdOS compound was further obtained by semi-synthesis following sulfate modification by reacting its precursor with ClSO2 at 37°C.Human squamous cancer cells A431, human ovarian cancer cells SK-OV-3, human micro-vascular endothelial cells (HMECs) and mouse fibroblast cells NIH-3T3 were obtained from the American Type Culture Collection . NIH-3T3/neu and NIH-3T3/flk-1 were established as described previouslySprague-Dawley rats (6-week-old) were provided by Shanghai Laboratory Animal Center, Chinese Academy of Sciences. The use of animals was approved by the Institute animal reviews boards of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with confirm adherence to the ethical guidelines for the care and use of animals.2, 0.1 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added to each well. Various concentrations of MdOS diluted in 10 µl buffered saline were added to each reaction well. 10 µl buffered saline was used as the vehicle control. The kinase reaction was performed in triplicate and initiated by adding purified tyrosine kinase proteins diluted in 40 µl of kinase reaction buffer. After incubation for 60 min at 37°C, the plate was washed three times with Phosphate Buffered Saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 µl anti-phosphotyrosine (PY99) antibody  diluted in T-PBS containing 5 mg/ml BSA was added and the plate was placed at at 37°C for 30 min. After the plate was washed three times, 100 µl Horseradish peroxidase-conjugated goat anti-mouse IgG  diluted in T-PBS containing 5 mg/ml BSA was added and the plate was reincubated at 37°C for 30 min. The plate was washed, then 100 µl citrate buffer  containing 0.03% H2O2 and 2 mg/ml o-phenylenediamine was added and samples were incubated at room temperature until color emerged. The reaction was terminated by adding 50 µl of 2 M H2SO4, and the plate was read using a multiwell spectrophotometer  at 492 nm. The inhibition rate (%) was calculated with the formula: [1-(A492/A492 control)]×100%. IC50 values were calculated from the inhibitory curves. For ATP competitive assay, conditions were the same as above except that varying concentrations of ATP were added in the absence or presence of a single concentration of MdOS to generate ATP concentration curves.The kinase domain of EGFR, HER-2, VEGFR2, c-Kit, FGFR1, FGFR2 and c-Src were expressed using the Bac-to-Bac™ baculovirus expression system  and purified on Ni-NTA columns  as previously describedCells were grown to half confluence in six-well plates, starved in serum-free medium for 24 h, and then exposed to MdOS at concentrations of 2, 10, 50 and 100 µg/ml for 6 h. For analysis of receptor tyrosine kinase phosphorylation and downstream signal transduction pathways, cells were stimulated with 50 ng/ml EGF or VEGF  for 15 min at 37°C at the end of MdOS treatment. Western blot analyses were subsequently performed as previous describedMdOS was labeled with fluorescein isothiocyanate (FITC) as previously described5 cells/ml in DMEM medium containing 10% FCS. The medium was replaced with serum-free DMEM containing 100 µg/ml FITC-MdOS (green) or normal saline for 6 h. Fixed preparations were obtained by exposing cells on culture slides to 4% paraformaldehyde in PBS for 10 min at 25°C, followed by washing three times with PBS containing 0.2% Triton X-100 for 5 min. For HER-2 proteins staining, cells were blocked with 5% bovine serum albumin for 30 min, then incubated with HER-2 antibody in 3% BSA (1∶200) for 30 min, and followed by coincubation with Alexa 633-conjugated secondary antibody diluted in 3% BSA (1∶ 500) for 30 min. Then 1 µg/ml DAPI was added for visualization of nuclear acid. coverslips were slightly dried for a few minutes and mounted on the slides with anti-fade agent, and pictures were obtained using Leica TCS confocal microscope .For fluorescence staining, SK-OV-3 cells were plated on glass culture slides at a density of 1×10The kinetics and specificity of the binding reactions between MdOS and VEGFR2 or HER-2 or EGFR or 6×his-tag were carried out with the BIAcore×surface plasmon resonance apparatus. Briefly, MdOS was immobilized on CM5 sensor chips according to the Ligand Thiol protocol described in the application Handbook. To assess real-time binding capacity, 35 µl of soluble VEGFR2 or HER-2 or EGFR or 6×his-tag was injected over the sensor chip surface with the immobilized MdOS, followed by washed with HBS-EP buffer for 5 min. The sensor chip surface was regenerated using 60 µl NaCl (2 M). All binding experiments were performed at 25°C with a constant flow rate of 10 µl/min HBS-EP. For binding affinity assessment, the association phase was allowed to proceed to equilibrium. To correct for nonspecific binding and bulk refractive index change, a blank channel (FC2) without oligosaccharide was employed as a control for each experiment. Sensorgrams for all binding interactions were recorded in real time and analyzed after subtracting that from the blank channel. Changes in mass due to the binding response were recorded as resonance units (Ru). Binding kinetics and affinities were determined by SPR using BIACORE software 3.1.http://www.mdl.com). The initial structures were minimized using molecular mechanics with the Tripos force field The crystal structure of VEGFR2 kinase in complex with a known inhibitor was retrieved from the Brookhaven Protein Data Bank (PDB entry 1YWN). The crystal structure of EGFR kinase in complex with lapatinib was retrieved from the Brookhaven Protein Data Bank (PDB entry 1XKK). The molecular geometry of MdOS disaccharide was modeled using the ISIS draw 2.5 program  were added. Cells were further cultured for 2 days. Cell proliferation was determined using sulforhodamine B  assay.4/well) were seeded on the Matrigel and cultured in MCDB-131 with various concentrations of MdOS for 8 h at 37°C and 5% CO2. Images were recorded by an inverted phase contrast microscope , the enclosed networks of complete tubes from five randomly chosen fields were counted and photographed.96-well plates were coated with 60 µl liquid Matrigel  per well and incubated at 37°C for 1 h to promote gelling. HMECs  saturated with compounds or normal saline was placed on areas between preexisting vessels and the embryos were further incubated for 48 h. The neovascular zones beside the glass coverslips were photographed under a stereomicroscope  and printed out as 5×7 in2 prints. Vessel branches <3 mm long in five random 1×1 in2 zones per pictures were counted and five eggs were chosen from each group.Groups of 10 fertilized chicken eggs were incubated in a humidified egg incubator  maintained at 37°C and 50% humidity and allowed to grow for 7 days. Gentle suction was applied at the hole located at the broad end of the egg to create a false air sac directly over the chicken chorioallantoic membrane, and a 1-cmAll data are represented as mean±SD. The significance of differences was assessed by Student's t test. P-values of <0.05 were regarded as statistically significant.


Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway.The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases.ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected.These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins. The MEKK1 protein kinase is a critical upstream mediator in signaling pathways that control the response of cells to stress stimuli. It directly phosphorylates and activates the SEK1 protein kinase, leading to activation of the stress activated protein kinase/jun N terminal kinase (SAPK/JNK) . By virtMEKK1 is a large protein kinase  with actThe reactive cysteine in the ATP binding pocket of MEKK1 is quite unique among protein kinases. In an effort to identify cysteine reactive compounds that might likewise inhibit MEKK1 by targeting this residue, we considered physiologic agents that could result in protein modification on cysteine. One such group of compounds is the isothiocyanate (ITC) class of dietary chemopreventives, that have established roles in apoptosis and prevention of cancer, , proCancer chemoprevention by ITCs has been attributed to their ability to induce gene expression of a family of enzymes involved in detoxification and excretion of carcinogens, the Phase 2 genes . HoweverThe nature of the direct molecular target(s) of ITCs within cells has not been resolved. We reasoned that ITCs, being electrophilic compounds, could decorate cell regulatory proteins through stable covalent modification of cysteine or lysine residues, and that these targets could be identified using an affinity-labeled variant of nutritional ITCs. We tested this hypothesis on MEKK1, since the SAPK/JNK pathway that is regulated by MEKK1 has been implicated in apoptosis and cell growth, processes that are also impacted by ITCs.We describe here results that indicate that nutritional isothiocyanates are able to covalently and irreversibly inhibit MEKK1. This inhibition is specific to MEKK1 and not to a closely related kinase, and occurs through covalent modification requiring a single cysteine in ATP binding pocket.N,N-dimethylformamide (DMF) and tetrahydrofuran (THF) were obtained from OptiDry canisters . THF was passed through an activated alumina (activity I) column before use. DMF, MeCN , and chloroform were used without further drying. Norbiotinamine hydrochloride , EZ-Link™ 5-(biotinamido)-pentylamine  were purchased and used as obtained. All other reagents, including 1,1'-thiocarbonyldi-2,2'-pyridone (TCDP), were purchased from Aldrich and used as obtained.Unless otherwise specified, all chemicals were obtained from Sigma-Aldrich. The Streptavidin-HRP detection reagent was from Zymed. MEKK1 and ASK1 plasmids and the C1238V mutant have been previously described .LNCaP cells were grown in RPMI with 10% fetal bovine serum and penicillin/streptomycin antibiotics. For analysis of endogenous MEKK1, cells were serum starved for 18 hours prior to treatment. HeLa and CV-1 cells were grown in DMEM with 10% calf serum and penicillin/streptomycin antibiotics. For immunoblotting of active MAPKs, the cells were serum starved for 18 hours prior to treatment. Expression of proteins was accomplished using the T7-polymerase-driven vaccinia virus overexpression system  as previ32P-ATP. One μg of bacterially-expressed GST-tagged catalytically inactive SEK (GST SEK-KR) was used as substrate and the reactions were incubated for 30 minutes at room temperature. Kinase assays were stopped by addition of an equal volume of 2× Laemmli sample buffer , 20% Glycerol, 150 mM DTT, Bromophenol Blue), and then boiled and electrophoresed on SDS-PAGE (10%). Gels were transferred to PVDF (Immobilon) and imaged for quantification using a Packard Instant Imager. Equivalent protein was confirmed by immunoblotting with anti-MEKK1 (Santa Cruz Biotechnology).Following treatment, cells were lysed in MOPS lysis buffer  supplemented with 2.5 μg/ml aprotinin, 2.5 μg/ml leupeptin, 10 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 10 mM β glycerolphosphate. Lysates were clarified at 14,000 × g for 5 minutes in a microcentrifuge. CBD-tagged proteins were purified using chitin beads (New England Biolabs). The beads were washed 3 times with MLB, then once with 50 mM Tris/HCl (pH7.4). In some experiments, purified proteins were treated with isothiocyanates in vitro, in 50 mM Tris/HCl (pH7.4). Beads were then washed to remove isothiocyanates prior to kinase assay. Kinase assays were carried out in a buffer containing 50 mM Tris (pH7.4), 10 mM magnesium chloride, and 7.5 μM ATP in the presence of 10 μCi gamma(g). A solution of TCDP  in chloroform (0.40 mL) was added to the previously prepared solution. Following 15 minutes of continued stirring, one drop of triethylamine was added. The yellow-orange color was diminished upon addition. After 45 minutes, an additional drop of triethylamine was added and the reaction's progress was followed by thin layer chromatography (TLC). After four hours, the starting material was consumed and the reaction was concentrated to a crude material that was purified by normal phase preparative-TLC  using 1:9 methanol/chloroform as an eluent. Following separation, the desired band was carefully isolated from the plate and washed through a fritted funnel with multiple portions of ethyl acetate. The organic solvent was concentrated to 7 mG (70%) of the title compound as a off-white powdery solid. Rf (10% MeOH in CHCl3) = 0.22. 1H NMR : 4.50 , 4.33 , 3.58 , 2.95 , 2.72 , 1.83–1.52  ppm. 13C NMR : 168.30, 125.85, 63.41, 61.63, 56.90, 41.05, 31.00, 29.08, 27.33 ppm. MS (ESI+) m/z 258 [M+H]+.A solution of norbiotinamine hydrochloride  dissolved in acetonitrile (2 mL) and chloroform (0.40 mL) was prepared in a flame dried flask under ArUntreated or PEITC treated cells were lysed in Tris lysis buffer  supplemented with 2.5 μg/ml aprotinin and 2.5 μg/ml leupeptin. Lysates were clarified by centrifugation at 14,000 × g for 5 minutes. Clarified lysates were reacted with 500 μM Bio-ITC for 30 minutes at room temperature. Samples were removed for in vitro kinase assay, cell lysate immunoblots, and/or chitin bead purification and immunoblot analysis, as indicated in the figure legends.HeLa cells were seeded in 6 well plates and then serum starved overnight. PEITC at concentrations of 200 μM to 12.5 μM  was added to the media and the cells incubated for 7 minutes at 37°C. Sorbitol was then added to 400 mM to induce hyperosmotic stress, and the incubation continued for an additional 20 minutes. Cells were lysed in 500 μL MLB, and 200 μL used to prepare a whole cell lysate by adding 50 μL of 5× Laemmli sample buffer. Samples were analyzed by immunoblotting using antibodies directed against phosphorylated forms of various MAPK family members as described in the figure legend.in vitro kinase assay using a bacterially-expressed inactive mutant of SEK (SEK-KR) as substrate. Treatment of intact cells with PEITC inhibited MEKK1 in a dose dependent fashion within 30 minutes of exposure  tag to allow purification of the protein by binding to chitin beads. In parallel, we expressed the related related MAPK3K, the Apoptosis Signal-regulating Kinase (ASK1), bearing the same carboxy-terminal CBD tag. The cells were treated with increasing concentrations of phenethyl isothiocyanate (PEITC), and each kinase was purified by affinity chromatography using chitin beads followed by We previously identified a cysteine residue in the ATP binding pocket of MEKK1 that is glutathionylated during oxidative stress, resulting in inhibition of the kinase . We compin vitro. CBD-tagged wild type MEKK1 or the C1238V mutant were expressed in cells, purified on chitin beads and then treated with PEITC in vitro. As observed in experiments with intact cells, wild type MEKK1 was inhibited by PEITC treatment in a dose dependent manner, while the C1238V mutant was completely resistant to PEITC treatment . As before, wild type MEKK1 but not C1238V MEKK1 was inhibited in vitro by Bio-ITC , and wild type MEKK1 (but not C1238V MEKK1) was covalently labeled with Bio-ITC . These results suggest that both Bio-ITC and PEITC inhibit MEKK1 by direct modification in a manner that requires the cysteine at position 1238.We used a blocking strategy to demonstrate the specificity of C Figure . Pretreain vitro kinase assay. Hyperosmotic shock, induced by addition of 0.3 M sorbitol, strongly activated MEKK1 . HoweveIn vitro inhibition of MEKK1 by PEITC also supports a direct effect on catalytic activity, rather than a more complicated mechanism of regulation involving protein degradation or other regulators of MEKK1 function. Use of the Bio-ITC probe conclusively establishes the mechanism for this inhibition as covalent modification of C1238 (but not other cysteines) of MEKK1.Inhibition of MEKK1 activity within 30 minutes of PEITC addition indicates that inhibition is a specific and acute effect that is not due to protein loss that might accompany measurements taken at later time points after apoptosis had commenced. This is supported by equivalent recovery of MEKK1 protein from the treated and untreated samples. in vitro, and no evidence was presented to support the ability of sulforaphane to modify Keap1 in intact cells.Covalent modification of cellular proteins by ITCs has been hypothesized previously. One group used indirect assays to conclude that the Keap1 protein transiently reacts with sulforaphane . Since KWhile the Keap1 study predicted that ITCs should make readily-reversible reaction products with protein sulfhydryls, our results with MEKK1 demonstrate stable products that are resistant to heat, denaturants, and SDS used in protein electrophoresis. While transient interaction of ITCs with some protein thiols (as well as with glutathione) likely occur in intact cells, stable covalent modifications of proteins are more likely to represent functional targets, particularly at low ITC concentration. Our identification of MEKK1 as a functional target of ITCs points the way to an understanding of a general mechanism of ITCs and other bioactive electrophiles. It remains unproven whether modification and inhibition of MEKK1 plays a singular role in the effects of ITCs in apoptosis or cancer chemoprevention. We suspect that MEKK1 is one of several or many cellular targets that contribute to the multiple effects of ITCs. Identification of these targets and characterization of their relative role(s) in the transcriptional, cell growth and apoptosis pathways induced by ITCs is an important goal for future studies. We are currently using our Bio-ITC reagent and affinity chromatography to identify other stably modified proteins that could, as a whole, explain the means through which these dietary constituents prevent cancer.After the completion of this work, reports were published that ITCs and other thiol reactive electrophilic compounds are able to covalently modify and activate the TRPA1 ion channel involved in pain sensing ,35. The Our results provide new insights into the molecular actions of dietary isothiocyanates. ITCs directly and covalently modify at least one cell signaling protein within cells, resulting in the functional silencing of the MEKK1 protein kinase and blockade of SAPK/JNK signaling. Our results do not preclude ITCs having other functional targets in cells that may contribute to the pleiotropic functions of these nutritional compounds. However, we predict that all cellular targets of ITCs will parallel the mechanism of MEKK1 inactivation, i.e. through stable covalent modification resulting in altered protein function.ITC(s), isothiocyanate(s); MEKK1, MAPK/ERK kinase 1; SEK, SAPK/ERK kinase; SAPK/JNK, Stress Activated Protein Kinase/jun N-terminal kinase; CBD, chitin binding domain; MLB, MOPS lysis buffer; GST, glutathione S-transferase; Bio-ITC, Biotin-ITC; TLB, Tris lysis buffer; PEITC, phenethylisothiocyanate; ERK, extracellular signal regulating kinase; ARE, Antioxidant Response Element; ASK1, Apoptosis signal regulating kinase 1.The author(s) declare that they have no competing interests.JVC and DJT designed and interpreted the experiments. JVC performed most of the experiments. JMR assisted with design/execution of some experiments. FWF, Jr. and TLM perfected and executed the synthesis of Bio-ITC. JVC and DJT wrote the manuscript. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


When mononuclear cells were preincubated for 30 min with the selective PKA inhibitor, H89 (10−4 M), but not with the selective PKG inhibitor, Rp-8-pCPT-cGMPs (10−4 M), a significant reduction of the inhibitory effect of db-cAMP was noted. Thirty min incubation of mononuclear cells with Rp-8-pCPT-cGMPs induced a significant reduction of the inhibitory activities of both rolipram and Ro 20-1724 (10−9 to 10−5 M) on LPS-induced TNF-α release, whereas H89 elicited a moderate, but significant inhibition. The present data indicate that db-cAMP inhibits TNF-α release from human mononuclear cells through a PKA-dependent mechanism. In contrast, PDE 4 inhibitors elicit their in vitro anti-inflammatory activities via a PKG-dependent rather than PKA-dependent activation.We investigated the effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type 4 inhibitors and of the cell permeable analogue of cAMP, db-cAMP on LPS-induced TNF-α release from human mononuclear cells. Incubation from 30 min of mononuclear cells with dbcAMP (10


Such cell lines consist in suitable and evolving models to investigate additional factors involved in molecular pathways leading to whole Tau aggregation.The role of Tau phosphorylation in neurofibrillary degeneration linked to Alzheimer's disease remains to be established. While transgenic mice based on FTDP-17 Tau mutations recapitulate hallmarks of neurofibrillary degeneration, cell models could be helpful for exploratory studies on molecular mechanisms underlying Tau pathology. Here, “human neuronal cell lines” overexpressing Wild Type or mutated Tau were established. Two-dimensional electrophoresis highlights that mutated Tau displayed a specific phosphorylation pattern, which occurs in parallel to the formation of Tau clusters as visualized by electron microscopy. In fact, this pattern is also displayed before Tau pathology onset in a well established mouse model relevant to Tau aggregation in Alzheimer's disease. This study suggests first that pathological Tau mutations may change the distribution of phosphate groups. Secondly, it is possible that this molecular event could be one of the first Tau modifications in the neurofibrillary degenerative process, as this phenomenon appears prior to Tau pathology in an  Two studies with specific Tau constructs showed an abnormal Tau behaviour in cells. The first study based on overexpression of N-terminal half truncated Tau bearing ΔK280 mutation showed an increase in Tau aggregation Tau (tubulin associated unit) is a microtubule-associated protein. In the human brain, there are six Tau isoforms generated by alternative splicing. They differ by the combination of 0, 1 or 2 amino-terminal inserts and 3- or 4-microtubule-binding repeats (3R or 4R) encoded by exons 2,3 and 10 respectively. Tau aggregation is one of the key features common to Tauopathies, a group of neurodegenerative diseases including Alzheimer's disease (AD). Even though Tau is always found aggregated and hyperphosphorylated in these pathologies, the precise role of phosphorylation in Tau aggregation process is still debated. In the same way, physiopathological significance of Tau aggregation remains to be established. The discovery of Tau mutations associated with Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17), has allowed for generating several animal models and especially Tau transgenic mice that display a Tau pathology characterized by abnormal phosphorylation and Tau aggregation S305N Tau cells compared to WT and P301S cell lines . Interestingly, while WT 4RTau isovariants are displayed over a wide range of isoelectric point (pI) at mainly L1 molecular weight, isovariants for P301S and S305N mutations are found proportionally at less acidic pI and at higher molecular weight (L2 and L3). In fact, P301S and S305N isovariants within the basic pI range displayed a molecular weight shift. This shift, which is characterised by an increase in isovariants number on high molecular weight levels  electrophoresis was performed, followed by an immunostaining with Tau-exon10 antibody to reveal total exogenous 4RTau . AnalysiWT and mutated 4RTau are displaced into the most basic pI range , which is abolished after in vitro dephosphorylation. This “shift” could be due to specific mode of Tau phosphorylation so called “state I” described by Lindwall and col. in vivo relevance of the molecular shift displayed in mutated 4RTau cell lines, we performed the same overall approach with a Tau transgenic mice model, which exhibits a quite well defined Tau pathology associated to memory deficits In the present study, Tau phosphorylation was analysed by 2D electrophoresis. FTDP-17 mutated 4RTau were found differentially phosphorylated compared to P301S and S305N 4RTau mutants exist under a particular phosphorylation state. Our data suggest that pathological Tau mutations may both change distribution of phosphate groups and generate “clusters of phosphorylation”. This work could raise a common mechanism for several FTDP-17 mutations, which could act through the dysregulation of Tau phosphorylation status ultimately leading to conformational changes. Finally, our data underline the possibility that this molecular event could be one of the first Tau modifications during the neurofibrillary degeneration cascade since this phenomenon appears prior to Tau pathology in an in vivo model and is linked to early step of Tau nucleation in Tau mutants cell lines. Such cell lines consist in suitable and evolving models to investigate additional factors involved in molecular pathways leading to whole Tau aggregation.In summary, 2D approach reveals that P301S or the 4RTau S305N mutants. Wild-type and mutated cDNAs were sequenced and inserted in the tetracycline inducible mammalian expression T-REX vector pcDNA4/TO © .The Tau cDNA coding for the human 412 amino acids 4-Repeat (4R) isoform was a kind gift from M. Goedert . A polymerase chain reaction (PCR)-based site-directed mutagenesis  was used to generate two distinct constructs coding for the 4RTau WT or mutated). For differentiation, cells were maintained for 7 days in DMEM/F12 medium supplemented with 2 mM L-glutamine, 50 U/mL penicillin/streptomycin, 7 µg/mL progesterone, 1% Insulin/Transferrin/selenium (INVITROGEN) and 5 ng/ml β-NGF  Generation of human SH-SY5Y cell line overexpressing tetracycline repressor has been described elsewhere 2, 0.5 M EGTA, 1 mM NaCl, pH 6.5) with complete protease inhibitors  and phosphatase inhibitor: 125 nM okadaic acid . Cells were lysed using a dounce homogenizer and then collected on perlodion coated nickel-200 mesh grids. After 10 mn blocking with 1% gelatine-PBS at room Temperature, grids were incubated with TauCter 1902 antibody according to 2O. Observations were done with a Zeiss 9025 electron microscope.Cells were harvested in MES buffer  gel electrophoresis 100 µg of total proteins were dissolved in 400 µl of 2D electrophoresis buffer  Triton X-100, 20 mM dithithreitol and 0.6% (v/v) pharmalytes). Samples were loaded on immobilized pH gradient ReadyStrip IPG strip 3–10 , isoelectrofocused with the Protean IEF cell (BIORAD) by applying a total of 75 kV/h, according to the manufacturer's instructions . The IPG strips were then equilibrated three times (15 mn each) in a Laemmli Buffer  and were layered onto a 10% Tris-Glycine Polyacrylamide Gel. SDS-PAGE was performed with a Protean II Xi Cell (BIORAD) and blotted onto nitrocellulose or polyvinylidene difluoride membranes with Criterion Blotter (BIORAD) as recommended by the manufacturer. Membranes were then incubated with either anti-E10tau antibody or anti human tau antibody .2, NEB reaction buffer 1×) as recommended by the manufacturer  Cells were harvested into an ice-cold NP-40 lysis buffer  supplemented with protease inhibitors. Amount of total proteins was determined using the BCA kit and 100 µg of total proteins, with protease inhibitors to avoid any protein degradation, were incubated at 37°C, for 80 minutes with λ-protein phosphatase (EC 3.1.3.16) in reaction buffer (2 mM MgCl


Myristica fragrans Houtt. (Myristicaceae) on certain enzymes in the nervous tissue of freshwater snail Lymnaea acuminata Lamarck (Lymnaeidae). In vivo and in vitro treatments of trimyristin and myristicin  significantly inhibited the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activities in the nervous tissue of Lymnaea acuminata. The inhibition kinetics of these enzymes indicates that both the trimyristin and myristicin caused competitive noncompetitive inhibition of AChE. Trimyristin caused uncompetitive and competitive/noncompetitive inhibitions of ACP and ALP, respectively whereas the myristicin caused competitive and uncompetitive inhibition of ACP and ALP, respectively. Thus results from the present study suggest that inhibition of AChE, ACP, and ALP by trimyristin and myristicin in the snail Lymnaea acuminata may be the cause of the molluscicidal activity of Myristica fragrans.This study was designed to investigate the effects of molluscicidal components of  Lymnaea acuminata Lamarck (Lymnaeidae) is the vector of liver flukes, Fasciola gigantica Cobbold (Fascioliodae) and Fasciola hepatica Linnaeus (Fascioliodae) which are responsible for endemic fascioliasis in cattle population of northern India [ Myristica fragrans (Myristicaceae) seed (nutmeg) and aril (mace) have potent molluscicidal activity against Lymnaea acuminata [Lymnaea acuminata. The snail rn India , 2. One rn India , 4. Rececuminata . The actcuminata . The mecTrimyristin  catalog no. T 5141, and myristicin (4-methoxy-6-(2-propenyl)-1,3-benzodioxole), catalog no. M 9411, were purchased from Sigma Chemical Co., USA.Lymnaea acuminata  were collected locally from Ramgarh Lake located almost adjacent to this University campus. Snails were acclimatized to laboratory conditions for 72 hours and used as experimental animals. Twenty experimental snails were kept in a glass aquarium containing 3 L of dechlorinated tap water at 22–24°C. Six aquaria were set up for each concentration. Each set of experimental snails was exposed to sublethal concentrations −40 and 80% of 24 h and 96 h LC50 of trimyristin  and myristicin  for 24 and 96 hours. These concentrations were based on 24 and 96 hours LC50 values earlier reported by us [Adult In vitro experiments were performed by dissolving the molluscicides in ether and an appropriate volume containing 3, 5, 7, and 9 μg of trimyristin and 0.3, 0.5, 0.7, and 1.0 μg of myristicin  was added to 10 mm path length cuvette separately. Ether was then allowed to evaporate. Molluscicides were preincubated for 15 minutes at 25°C with an enzyme source and then enzyme activity was determined. The control cuvette contained ether only. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of different enzyme inhibitions were calculated by nonlinear regression. Lineweaver-Burk plots for the hydrolysis of different concentrations of substrate by the treated (0.09 mM trimyristin and 0.03 mM myristicin) and untreated enzymes were plotted to observe mode of enzyme inhibition [50 values of myristicin and trimyristin were calculated in between the negative log concentration of inhibitor versus relative activity between inhibited and uninhibited enzymes.hibition . IC50 vaLymnaea acuminata taken around the buccal mass was homogenized in 1.0 mL of 0.1 M phosphate buffer pH 8.0 for 5 minutes in an ice bath and centrifuged at 1000 g for 30 minutes at 4°C. The supernatant was used as an enzyme source. Enzyme activity was measured in a 10 mm path length cuvette using an incubation mixture consisting of 0.1 mL of enzyme source, 2.9 mL of 0.1 M buffer pH 8, 0.1 mL of chromogenic agent DTNB , and 0.02 mL of freshly prepared ATChI (aetylthiocholine iodide) solution in distilled water. The change in optical density at 412 nm was recorded for 3 minutes after every 30 s interval at 25°C. Enzyme activity has been expressed as μmole “SH” hydrolyzed min−1 mg−1 protein. For the estimation of kinetic constants of AChE, in vitro inhibition of the enzyme was carried out at different concentrations  of the substrate acetylthiocholine iodide. Acetylcholinesterase (AChE) activity was measured by the method of Ellman et al.  as modifLymnaea acuminata was measured by the method of Bergmeyer [μmole substrate hydrolyzed 30 min−1 mg−1 protein. For the determination of kinetic constants of acid phosphatase, in vitro inhibition of the enzyme was carried out at different concentrations  of the substrate 4-nitrophenyl phosphate.Acid phosphatase (ACP) activity in the nervous tissue of ergmeyer  as modifergmeyer . Tissue Lymnaea acuminata was measured by the method of Bergmeyer [2.6H2O, 165 mg 4-nitrophenyl phosphate disodium salt in 42 mL of 0.1 N NaOH and a mixture was made up to 100 mL with double distilled water). The incubation mixture was mixed thoroughly and incubated for 30 minutes at 37°C. 10 mL of 0.02 N NaOH was then added to the incubation mixture. The yellow colour, developed due to the formation of 4-nitrophenol, was determined colorimetrically at 420 nm. Standard curves were drawn with different concentrations of 4-nitrophenol. The ALP activity has been expressed as μmole substrate hydrolyzed 30 min−1 mg−1 protein. For the determination of kinetic constants of alkaline phosphatase, in vitro inhibition of the enzyme was carried out at different concentrations  of the substrate 4-nitrophenyl phosphate. Alkaline phosphatase (ALP) activity in the nervous tissue of ergmeyer  as modifergmeyer . Tissue Protein estimation was carried out by the method of Lowry et al.  using bo t-test was applied between control and treated groups to locate significant (P < .05) variations [Each experiment was replicated at least six times and results were expressed as mean ±SE of six replicates. Student'sriations . Lymnaea acuminata of control group was 0.73 μmole “SH” hydrolyzed min−1 mg−1 protein. In vivo treatment of 40 and 80% of 24 and 96 h LC50 of trimyristin and myristicin caused significant (P < .05) inhibition in AChE activity in the nervous tissue of Lymnaea acuminata. Maximum inhibition of AChE activity (49% of control) was observed in snails exposed to 80% of 96 h LC50 of myristicin (risticin .Lymnaea acuminata of control group was 32.3 μmole substrate hydrolyzed 30 min−1 mg−1 protein (In vivo treatment of 40 and 80% of 24 and 96 hours LC50 of trimyristin and myristicin caused significant (P < .05) inhibition in ACP activity in the nervous tissue of Lymnaea acuminata. Maximum inhibition of ACP activity (44% of control) was observed in snails exposed to 80% of 96 hours LC50 of myristicin (The acid phosphatase activity in the nervous tissue of  protein . In vivoristicin .Lymnaea acuminata of control group was 30.0 μmole substrate hydrolyzed 30 min−1 mg−1 protein (In vivo treatment of 40 and 80% of 24 and 96 h LC50 of trimyristin and myristicin caused significant (P < .05) inhibition in ALP activity in the nervous tissue of Lymnaea acuminata. Maximum inhibition of ALP activity (52% of control) was observed in snails exposed to 80% of 96 h LC50 of myristicin  dose dependent inhibition in AChE, ACP, and ALP activities  on the Y axis of Lineweaver-Burk plots. Inhibition of ALP by trimyristin is also competitive noncompetitive. The kinetic study clearly indicates that the inhibition of AChE by trimyristin and myristicin is competitive noncompetitive, as Myristica fragrans kill the snails by inhibiting these enzymes in different ways.Inhibition of AChE, ACP, and ALP by trimyristin and myristicin indicate, different types of inhibition kinetics. It seems that the molluscicidal components of