6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) from Wasabia japonica alleviates inflammatory bowel disease (IBD) by potential inhibition of glycogen synthase kinase 3 beta (GSK-3ß).

Inflammatory bowel disease (IBD) describes a set of disorders involving alterations to gastrointestinal physiology and mucosal immunity. Unravelling its complex pathophysiology is important since many IBD patients are refractory to or suffer adverse side effects from current treatments. Isothiocyanates (ITCs), such as 6-(methylsulfinyl)hexyl ITC (6-MITC) in Wasabia japonica, have potential anti-inflammatory activity. We aimed to elucidate the pathways through which 6-MITC alleviates inflammation by examining its role in the nuclear factor-kappa B (NF-κB) pathway through inhibition of glycogen synthase kinase 3 beta (GSK-3ß) using a chemically induced murine model of IBD, cell-based and in silico techniques. The effects of 6-MITC and two NF-κB inhibitors, sulfasalazine (SS), pyrrolidine dithiolcarbamate (PDTC) were investigated on a dextran sulfate sodium (DSS)-induced murine mouse model of acute and chronic colitis using macroscopic measurements and pro-inflammatory markers. The effect of 6-MITC on NF-κB induction was assessed using a murine macrophage cell line. Complexes of GSK-3ß-6-MITC and GSK-3ß-ATP were generated in silico to elucidate the mechanism of 6-MITC's direct inhibition of GSK-3ß. Changes in pro-inflammatory markers, inducible nitric oxide synthase (iNOS) (increased) and interleukin-6 (IL-6) (decreased) demonstrated that iNOS regulation occurred at the translational level. Intraperitoneal (ip) injection of 6-MITC to the colitis-induced mice ameliorated weight loss whereas oral administration had negligible effect. Fecal blood and colon weight/length ratio parameters improved on treatment with 6-MITC and the other NF-κB inhibitors. Levels of NF-κB decreased upon addition of 6-MITC in vitro while structural studies showed 6-MITC acts competitively to inhibit GSK-3ß at the ATP binding site. In this study we demonstrated that 6-MITC inhibits NF-κB signaling via GSK-3ß inhibition ameliorating fecal blood, colonic alterations and DSS-induced weight loss indirectly indicating reduced intestinal stress. Taken together these results suggest a role for 6-MITC in the treatment of IBD acting to alleviate inflammation through the GSK-3ß/NF-κB pathway. Furthermore, the GSK-3ß-6-MITC model can be utilized as a basis for development of novel therapeutics targeting GSK-3ß for use in other disorders including cancer.

Larval mesenchyme cell specification in the primitive echinoid occurs independently of the double-negative gate.

Echinoids (sea urchins) are divided into two major groups - cidaroids (a 'primitive' group) and euechinoids (a 'derived' group). The cidaroids are a promising model species for understanding the ancestral developmental mechanisms in echinoids, but little is known about the molecular mechanisms of cidaroid development. In euechinoids, skeletogenic mesenchyme cell specification is regulated by the double-negative gate (DNG), in which hesC represses the transcription of the downstream mesenchyme specification genes (alx1, tbr and ets1), thereby defining the prospective mesenchyme region. To estimate the ancestral mechanism of larval mesenchyme cell specification in echinoids, the expression patterns and roles of mesenchyme specification genes in the cidaroid Prionocidaris baculosa were examined. The present study reveals that the expression pattern and function of hesC in P. baculosa were inconsistent with the DNG model, suggesting that the euechinoid-type DNG is not utilized during cidaroid mesenchyme specification. In contrast with hesC, the expression patterns and functions of alx1, tbr and ets1 were similar between P. baculosa and euechinoids. Based on these results, we propose that the roles of alx1, tbr and ets1 in mesenchyme specification were established in the common ancestor of echinoids, and that the DNG system was acquired in the euechinoid lineage after divergence from the cidaroid ancestor. The evolutionary timing of the establishment of the DNG suggests that the DNG was originally related to micromere and/or primary mesenchyme cell formation but not to skeletogenic cell differentiation.

Intracellular accumulation of structurally varied isothiocyanates correlates with inhibition of nitric oxide production in proinflammatory stimuli-activated tumorigenic macrophage-like cells.

In the present study, we analyzed the intracellular accumulation of 6-(methylsulfinyl)hexyl isothiocyanate (6MITC) and its analogs in proinflammatory stimuli-activated J774.1 cells to predict the biological potencies of the ITCs. Our present analyses exhibited that the intracellular accumulation was in the order of 6MITC>2b>2e≈2c>2g>2d>2f>2h. Investigation of reactivity of the ITCs with glutathione (GSH) in the tumor cells revealed partial inhibition of GSH by the ITCs. Furthermore, the inhibition of nitric oxide (NO) production in the tumor cells was ascribed to the intracellularly accumulated ITCs. The NO suppression was correlated with the inhibition of tumor cell growth. Our present results suggest that the intracellular accumulation of the ITCs can be used to predict their biological potencies, such as inhibition of NO production that was correlated with suppression of tumor cell growth. To the best of our knowledge, this is the first report to predict the biological potency of 6MITC and its analogs with their intracellular accumulation.

Utilization of 6-(methylsulfinyl)hexyl isothiocyanate for sensitization of tumor cells to antitumor agents in combination therapies.

In the present study, we performed in silico and in vitro analyses to evaluate the chemosensitizing effects of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) on tumor cells. Our in silico analyses of the ligand-receptor interactions between 6-MITC and the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) revealed that 6-MITC possibly inhibited GCL enzyme activity, and that Cys-249 and Gln-251 were important residues for stable binding of ligands to GCLC. It was further found that 6-MITC interfered with the hydrogen bonds of the cysteinyl and glutamyl moieties of GSH with Cys-249 and Gln-251, respectively, and possibly overrode the feedback inhibition of GCL enzyme activity by GSH. To the best of our knowledge, this is the first in silico analysis to suggest an overriding effect of 6-MITC on GSH-induced feedback inhibition of GCL. In our in vitro analyses, combined treatment with 6-MITC and L-buthionine-S,R-sulfoximine (BSO) depleted GSH within 4 h in tumorigenic human c-Ha-ras and mouse c-myc-cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells, but did not deplete GSH in normal SFME cells. Furthermore, exposure to 6-MITC plus BSO for 4h, followed by glycyrrhetinic acid (GA) treatment for 3h, eradicated the tumor cells with minimal damage to the normal cells. The present findings suggest that 6-MITC in combination therapies could be used to sensitize tumor cells to antitumor agents, thereby leading to their eradication.

Homology modeling and structural analysis of human P-glycoprotein.

UNLABELLED: Homology modeling and structural analysis of human P-glycoprotein (hP-gp) were performed with a software package the Molecular Operating Environment (MOE). A mouse P-gp (mP-gp; PDB code: 3G5U) was selected as a template for the 3D structure modeling of hP-gp. The modeled hP-gp showed significant 3D similarities at the drug biding site (DBS) to the mP-gp structure. The contact energy profiles of the hP-gp model were in good agreement with those of the mP-gp structure. Ramachandran plots revealed that only 3.5% of the amino acid residues were in the disfavored region for hP-gp. Further, docking simulations between 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and the P-gp models revealed the similarity of the ligand-receptor bound location between the hP-gp and mP-gp models. These results indicate that the hP-gp model was successfully modeled and analyzed. To the best of our knowledge, this is the first report of a hP-gp model with a naturally occurring isothiocyanate, and our data verify that the model can be utilized for application to target hP-gp for the development of antitumor drugs. ABBREVIATIONS: ABC - ATP-binding cassette, ASE-Dock - alpha sphere and excluded volume-based ligand-protein docking, DBS - drug biding site, MDR - multidrug resistance, MOE - Molecular Operating Environment, ITC - isothiocyanate, P-gp - P-glycoprotein.

"Micromere" formation and expression of endomesoderm regulatory genes during embryogenesis of the primitive echinoid Prionocidaris baculosa.

Blastomere composition and expression profiles of wnt8 and hox11/13b orthologues were examined in the primitive indirect-developing echinoid Prionocidaris baculosa. We found that blastomere composition in the 16-cell-stage Prionocidaris embryos was different from that of the indirect-developing echinoids belonging to Euechinoidea, a derived group of the echinoids. The sizes of the blastomeres in the 16-cell-stage embryo varied, and no embryos formed a "micromere quartet," a group of four equal-sized micromeres. The smallest blastomere was usually located around the vegetal pole. We also found significant differences in early expression profiles of wnt8 orthologues of the Prionocidaris and euechinoids. Unlike euechinoids, the expression of wnt8 orthologue of Prionocidaris was not detected at the 16-cell stage; it began at the 32-cell stage in the broad area containing the vegetal pole. However, in later stages, the expression profiles of hox11/13b and wnt8 orthologues of Prionocidaris were similar to that of euechinoid orthologues. The present study suggests that there are considerable differences between Prionocidaris and euechinoids in early developmental mechanisms in the vicinity of the vegetal pole.

Structural insight into the ligand-receptor interaction between glycyrrhetinic acid (GA) and the high-mobility group protein B1 (HMGB1)-DNA complex.

UNLABELLED: Structural analysis of the high-mobility group protein B1 (HMGB1)-DNA complex and a docking simulation between glycyrrhetinic acid (GA) and the HMGB1-DNA complex were performed with a software package the Molecular Operating Environment (MOE). An HMGB1-DNA (PDB code: 2GZK) was selected for the 3D structure modeling of the HMGB1-DNA complex. The Site Finder module of the MOE identified 16 possible ligand-binding sites in the modeled HMGB1-DNA complex. The docking simulation revealed that GA possibly inhibits functions of HMGB1 interfering with Lys(90), Arg(91), Ser(101), Tyr(149), C(230) and C(231) in the HMGB1-DNA complex. To the best of our knowledge, this is the first report of an HMGB1-DNA complex with GA, and our data verify that the GA-HMGB1-DNA model can be utilized for application to target HMGB1 for the development of antitumor drugs. ABBREVIATIONS: ASE-Dock - alpha sphere and excluded volume-based ligand-protein docking, CNS - central nervous system, GA - glycyrrhetinic acid, GL - glycyrrhizin, HMGB1 - high-mobility group protein B1, LBS - ligand-biding site, MOE - Molecular Operating Environment, SRY - sex-determining region on the Y chromosome.

Molecular docking and structural analysis of cofactor-protein interaction between NAD⁺ and 11ß-hydroxysteroid dehydrogenase type 2.

Molecular docking and structural analysis of the cofactor-protein interaction between NAD(+) and human (h) or mouse (m) 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) were performed with the molecular operating environment (MOE). 11ßHSD1 (PDB code: 3HFG) was selected as a template for the 3D structure modeling of 11ßHSD2. The MOE docking (MOE-dock) and the alpha sphere and excluded volume-based ligand-protein docking (ASE-dock) showed that both NAD(+)-h11ßHSD2 and NAD(+)-m11ßHSD2 models have a similar binding orientation to the template cofactor-protein model. Our present study also revealed that Asp91, Phe94, Tyr232 and Thr267 could be of importance in the interaction between NAD(+) and 11ßHSD2. NADP(+) was incapable of entering into the cofactor-binding site of the 11ßHSD2 models. The present study proposes the latest models for 11ßHSD2 and its cofactor NAD(+), and to the best of our knowledge, this is the first report of a m11ßHSD2 model with NAD(+).

Mouse 11ß-hydroxysteroid dehydrogenase type 2 for human application: homology modeling, structural analysis and ligand-receptor interaction.

Mouse (m) 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) was homology-modeled, and its structure and ligand-receptor interaction were analyzed. The modeled m11ßHSD2 showed significant 3D similarities to the human (h) 11ßHSD1 and 2 structures. The contact energy profiles of the m11ßHSD2 model were in good agreement with those of the h11ßHSD1 and 2 structures. The secondary structure of the m11ßHSD2 model exhibited a central 6-stranded all-parallel ß-sheet sandwich-like structure, flanked on both sides by 3-helices. Ramachandran plots revealed that only 1.1% of the amino acid residues were in the disfavored region for m11ßHSD2. Further, the molecular surfaces and electrostatic analyses of the m11ßHSD2 model at the ligand-binding site exhibited that the model was almost identical to the h11ßHSD2 model. Furthermore, docking simulation and ligand-receptor interaction analyses revealed the similarity of the ligand-receptor bound conformation between the m11ßHSD2 and h11ßHSD2 models. These results indicate that the m11ßHSD2 model was successfully evaluated and analyzed. To the best of our knowledge, this is the first report of a m11ßHSD2 model with detailed analyses, and our data verify that the mouse model can be utilized for application to the human model to target 11ßHSD2 for the development of anticancer drugs.

Ligand-receptor interaction between triterpenoids and the 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme predicts their toxic effects against tumorigenic r/m HM-SFME-1 cells.

The present study deals with in silico prediction and in vitro evaluation of the selective cytotoxic effects of triterpenoids on tumorigenic human c-Ha-ras and mouse c-myc cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells. Ligand fitting of five different triterpenoids to 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) was analyzed with a molecular modeling method, and glycyrrhetinic acid (GA) was the best-fitted triterpenoid to the ligand binding site in 11ßHSD2. Analysis of antiproliferative effects revealed that GA, oleanolic acid, and ursolic acid had selective toxicity against the tumor cells and that GA was the most potent triterpenoid in its selectivity. The toxic activity of the tested triterpenoids against the tumor cells showed good correlations with the partition coefficient (logP) and polar surface area values. Time-lapse microscopy, fluorescence staining, and confocal laser scanning microscopic observation revealed that GA induced morphologic changes typical of apoptosis such as cell shrinkage and blebbing and also disrupted the cytoskeletal proteins. Furthermore, GA exhibited a strong inhibitory effect on 11ßHSD2 activity in the tumor cells. Our current results suggest that analysis of the ligand-receptor interaction between triterpenoids and 11ßHSD2 can be utilized to predict their antitumor effects and that GA can be used as a possible chemopreventive and therapeutic antitumor agent. To the best of our knowledge, this is the first report on in silico prediction of the toxic effects of triterpenoids on tumor cells by 11ßHSD2 inhibition.

Homology modeling and structural analysis of 11ß-hydroxysteroid dehydrogenase type 2.

11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) was homology-modeled by a Boltzmann-weighted randomized modeling procedure, using the X-ray crystal structure of 11ßHSD1 (PDB code: 3HFG) as a template. The model exhibited significant 3D similarities to 11ßHSD1. The contact energy profiles of the 11ßHSD2 model were in good agreement with that of the X-ray structure of 11ßHSD1. The secondary structure of the 11ßHSD2 model exhibited a central 6-stranded all-parallel ß-sheet sandwich-like structure, flanked on both sides by 3-helices. Ramachandran plots revealed that only 1.9% of the amino acid residues were in the disfavored region for 11ßHSD2. Furthermore, the ligand-binding site (LBS) volume was calculated to be 845 Å(3), which suggests that the LBS of 11ßHSD2 is sufficiently large to contain cofactors and substrates (ligands), such as NAD(+) and cortisol. The electrostatic analysis revealed that the 11ßHSD2 model had a positive potential at the LBS, which indicates that 11ßHSD2 possibly attracts negatively charged ligands at the LBS. These results indicate that the model was successfully evaluated and analyzed. Consequently, it is proposed that the 11ßHSD2 model in the present study will be suitable for further in silico structure-based de novo antitumor drug designing. To the best of our knowledge, this is the latest report of an accurate 11ßHSD2 model to target 11ßHSD2 for the development of anticancer drugs.

Novel effects of glycyrrhetinic acid on the central nervous system tumorigenic progenitor cells: induction of actin disruption and tumor cell-selective toxicity.

Licorice extracts are used worldwide in foods and medicines, and glycyrrhetinic acid (GA) is a licorice component that has been reported to induce various important biological activities. In the present study, we show that GA induces actin disruption and has tumor cell-selective toxic properties, and that its selectivity is superior to those of all the clinically available antitumor agents tested. The cytotoxic activity of GA and the tested antitumor agents showed better correlation with the partition coefficient (log P) values rather than the polar surface area (PSA) values. For selective toxicity against tumor cells, GA was most effective at 10 microM that was the same concentration as the previously reported maximum plasma GA level reached in humans ingesting licorice. These results suggest that GA could be utilized as a promising chemopreventive and therapeutic antitumor agent. The underlying mechanisms involved in the selective toxicity to tumor cells by GA are also preliminarily discussed.

Glycyrrhetinic acid induces anoikis-like death and cytoskeletal disruption in the central nervous system tumorigenic cells.

We analyzed the effects of glycyrrhetinic acid (GA), a licorice compound, on the induction of anoikis-like death and cytoskeletal disruption in the central nervous system (CNS) tumorigenic cells. GA was cytotoxic in time- and dose-dependent manners, and the tumorigenic cells shed floating cells upon the GA treatment and even some of the adherent cells were easily detached from the fibronectin-coated culture dish by gentle shaking and aspiration. Reculture of the detached cells revealed that the longer the duration of GA exposure, the less the number of the proliferatable cells. These results indicate that GA perturbs cell adhesion and induces anoikis-like cell death. Further, GA also induced morphologic changes and disturbed cytoskeletal proteins.

Selective cytotoxicity of glycyrrhetinic acid against tumorigenic r/m HM-SFME-1 cells: potential involvement of H-Ras downregulation.

With the intensive need for the development of more effective and safer agents for chemoprevention and therapy of human cancer, natural products from plants have been expected to play significant roles in creating new and better chemopreventive and therapeutic agents. Selectivity is also an important issue in cancer prevention and therapy. In the present study, normal serum-free mouse embryo (SFME) and tumorigenic human c-Ha-ras and mouse c-myc cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells were treated with various concentrations of clinically available antitumor agents or glycyrrhetinic acid (GA), and the antiproliferative effects of these compounds were determined by the MTT assay. Western blotting analysis, RT-PCR, fluorescence staining and confocal laser scanning microscopic observation were adopted to analyze H-Ras regulation. GA exhibited the tumor cell-selective toxicity through H-Ras downregulation, and its selectivity was superior to those of all the clinically available antitumor agents examined. For the selective toxicity of tumor cells, GA was most effective at 10 microM. Interestingly, this concentration was the same as the previously reported maximum plasma GA level reached in humans ingesting licorice. These results in the present study suggest that GA with its cytotoxic effects could be utilized as a promising chemopreventive and therapeutic antitumor agent.

Anti-nitric oxide production activity of isothiocyanates correlates with their polar surface area rather than their lipophilicity.

There is increasing demand for novel anti-inflammatory drugs with different mechanisms of action. We synthesized a series of isothiocyanates 2b-h based on 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) found in the pungent spice Wasabia japonica. Inhibitory activities against in-vitro growth of tumor cells and production of nitric oxide (NO) using the mouse macrophage-like cell line J774.1 were noted. All isothiocyanates were optimized by Hartree-Fock/3-21G model, and the logP values and the polar surface area (PSA) values were calculated. Substitution of the methylsulfinyl group (CH(3)S(O)-R) in 6-MITC with a formyl (CHO-R), a methylsulfanyl (CH(2)S-R) or a methyl (CH(3)-R) group reduced the activities of the parent isothiocyanate. Substitution with a formyl group resulted in lower lipophilicity (logP value) whereas substitution with a methylsulfanyl or methyl group resulted in a lower PSA value. The inhibitory activity of isothiocyanates showed better correlation with their PSA values rather than their partition coefficient (logP) values. Isothiocyanates with higher PSA values and some degree of logP value may have potent biological activity.

The augmenting activity of 6-(methylsulfinyl)hexyl isothiocyanate on cellular glutathione levels is less sensitive to thiol compounds than its cytotoxic activity.

We analyzed the effects of thiol compounds on the biological activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC). Thiol compounds abolished the cytotoxic activity of 6-MITC, but did not abolish its activity augmenting cellular total glutathione levels and gamma-glutamylcysteine ligase gene expression. Thiol compounds might play an important role in the augmentation of several significant biological activities by overcoming the inherent limitations of 6-MITC.

New cytotoxic phenolic derivatives from matured fruits of Magnolia denudata.

Magnolia species are widely cultivated in Japan as garden plants, and have been found to contain various compounds, including alkaloids, terpenoids, lignans, and neolignans. The constituents of the mature fruits of M. denudata were investigated, and two new phenolic derivatives, named denudalide and denudaquinol, were isolated and characterized, together with a known neolignan compound (denudatin A). Denudalide and denudaquinol showed cytotoxicity against the SFME and r/mHM-SFME-1 cell lines.

Machilin G and four neolignans from young fruits of Magnolia denudata show various degrees of inhibitory activity on nitric oxide (NO) production.

Denudatin A and B, denudadione B, fargesone A and machilin G were isolated from Magnolia denudata. These compounds showed inhibitory effects on nitric oxide (NO) production in the lipopolysaccharide plus interferon-gamma activated-murine macrophage cell line, J774.1. Some but not all of the inhibition of NO production by machilin G, and denudatin A and B was apparently through the decreased expression of the inducible NO synthase (iNOS) gene.

Differentiation of serum-free mouse embryo cells into an astrocytic lineage is associated with the asymmetric production of early neural, neuronal and glial markers.

Serum-free mouse embryo (SFME) cells, the astrocyte progenitor cells in the central nervous system (CNS), were exposed to 10 ng/ml leukemia inhibitory factor (LIF) and 10 ng/ml bone morphogenic protein 2 (BMP2) to induce differentiation, and expression of cell-type specific markers. Nestin, a marker of early neural lineage, betaIII-tubulin, a marker of neuronal lineage, oligodendrocyte marker O4 (O4), a marker of oligodendrocytic lineage and glial fibrillary acidic protein (GFAP), a marker of astrocytic lineage, were analyzed. Characteristics of SFME cells, as a CNS progenitor, were identified and a possible mechanism, underlying SFME cell specification into an astrocytic lineage upon differentiation, was investigated. These markers were present, both at the initial proliferative phase and after induction of differentiation. GFAP expression increased strongly upon differentiation, while expression of the other markers changed very little. These results indicate that astrocytic differentiation is associated with the asymmetric production of these markers, rather than through induction of astrocytic markers.

Detection of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and its conjugate with N-acetyl-L-cysteine (NAC) by high performance liquid chromatograpy-atmospheric pressure chemical ionization mass spectrometry (HPLC-MS/APCI).

A method using high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) was established for the detection of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and its conjugate with N-acetyl-L-cysteine (NAC). The optimal chromatographic conditions were obtained on an ODS column (150 x 4.6 mm, 3 microm) with the column temperature at 37 degrees C. The mobile phase consisted of a methanol-0.1% trifluoroacetic acid (TFA) mixture (50 : 50, v/v), and the flow rate was 0.3 ml/min. The detection wavelength was set at 220 nm. The identities of the peaks were accomplished by comparing retention times (tR), UV and mass data. All calibration curves showed good linear regression (correlation coefficients for 6-MITC and NAC>0.999) within test ranges. The developed method provided satisfactory precision calculated as percent coefficient of variation with overall intra-day and inter-day variations of less than 5% (4.1 and 4.9% for 6-MITC; 4.2 and 4.9% for NAC). Both 6-MITC and NAC had good responses in the positive APCI and formed strong [M+H]+ ions in the full scan spectra at an m/z of 206 and 164, respectively. The presence of the [M+H]+ ion for the 6-MITC/NAC conjugate was also observed at an m/z of 369. To our best knowledge, this is the first report that describes the formation of the 6-MITC/NAC conjugate and its detection method by HPLC-MS.