Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function.

Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.

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.

11ß-Hydroxysteroid dehydrogenases control access of 7ß,27-dihydroxycholesterol to retinoid-related orphan receptor γ.

Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways. However, recent research has emphasized the roles of oxysterols in essential physiologic processes and in various diseases. Despite these discoveries, the metabolic pathways leading to the different oxysterols are still largely unknown and the biosynthetic origin of several oxysterols remains unidentified. Earlier studies demonstrated that the glucocorticoid metabolizing enzymes, 11ß-hydroxysteroid dehydrogenase (11ß-HSD) types 1 and 2, interconvert 7-ketocholesterol (7kC) and 7ß-hydroxycholesterol (7ßOHC). We examined the role of 11ß-HSDs in the enzymatic control of the intracellular availability of 7ß,27-dihydroxycholesterol (7ß27OHC), a retinoid-related orphan receptor γ (RORγ) ligand. We used microsomal preparations of cells expressing recombinant 11ß-HSD1 and 11ß-HSD2 to assess whether 7ß27OHC and 7-keto,27-hydroxycholesterol (7k27OHC) are substrates of these enzymes. Binding of 7ß27OHC and 7k27OHC to 11ß-HSDs was studied by molecular modeling. To our knowledge, the stereospecific oxoreduction of 7k27OHC to 7ß27OHC by human 11ß-HSD1 and the reverse oxidation reaction of 7ß27OHC to 7k27OHC by human 11ß-HSD2 were demonstrated for the first time. Apparent enzyme affinities of 11ß-HSDs for these novel substrates were equal to or higher than those of the glucocorticoids. This is supported by the fact that 7k27OHC and 7ß27OHC are potent inhibitors of the 11ß-HSD1-dependent oxoreduction of cortisone and the 11ß-HSD2-dependent oxidation of cortisol, respectively. Furthermore, molecular docking calculations explained stereospecific enzyme activities. Finally, using an inducible RORγ reporter system, we showed that 11ß-HSD1 and 11ß-HSD2 controlled RORγ activity. These findings revealed a novel glucocorticoid-independent prereceptor regulation mechanism by 11ß-HSDs that warrants further investigation.

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.

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.

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.

Hypoxia enhances gene expression of inducible nitric oxide synthase and matrix metalloproteinase-9 in ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions.

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma; 100 U/ml) and/or lipopolysaccharide (LPS; 0.5 microg/ml) for 24 h to simulate inflammatory and infectious conditions and investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA, nitric oxide (NO) and matrix metalloproteinase-9 (MMP-9). In addition, aminoguanidine (AG; 1 mM), a NOS inhibitor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 10-200 microM), an NO donor or (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4; 10-200 microM), an NO donor, were added to analyze possible associations of NO with MMP-9. Tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also measured to analyze possible relationships of NO with the MMP-9/TIMP balance. Furthermore, the cells were treated with 1% O2 under the simulated inflammatory and infectious conditions and the mRNA expressions of iNOS and MMP-9 were analyzed to investigate the possible effects of hypoxia on the expression of genes involved in tumor malignant progression and distant metastasis. Co-treatment with IFN-gamma and LPS increased the expression levels of iNOS mRNA, NO and MMP-9, but NO may not be directly associated with MMP-9 or the MMP-9/TIMP balance. Treatment with 1% O2 markedly increased the gene expression levels of iNOS and MMP-9, indicating that ras/myc SFME cells alter the expression levels of tumor-associated genes and possibly enhance their malignancy as cancer cells under inflammatory, infectious and hypoxic conditions.

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.

L-NAME inhibits tumor cell progression and pulmonary metastasis of r/m HM-SFME-1 cells by decreasing NO from tumor cells and TNF-alpha from macrophages.

Highly metastatic ras/myc-transformed serum-free mouse embryo (r/m HM-SFME-1) cells were injected subcutaneously to mice and the effects of Nomega-nitro-L-arginine methyl ester (L-NAME) on the tumor progression and pulmonary metastasis were investigated. In addition, production of nitric oxide (NO), matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-alpha) in the tumor cells and in a mouse macrophage-like cell line, J774.1 cells, was analyzed. The increase in footpad thickness was significantly smaller in the mice which were fed the L-NAME containing water (4.24+/-0.39 mg/day/mouse). The number of the tumor cells metastasized to the lungs was smaller in the L-NAME treated mice, although statistical significance was not found. Co-treatment of r/m HM-SFME-1 cells with interferon-gamma (IFN-gamma; 100 U/ml) and lipopolysaccharide (LPS; 0.5 microg/ml) significantly enhanced NO production, and the presence of L-NAME at 1 mM significantly decreased this response. In r/m HM-SFME-1 cells, MMP-2 was undetectable and MMP-9 was also very little in the basal level, and both MMPs were unaffected by the IFN-gamma and/or LPS treatments, not to mention by the L-NAME treatment. In J774.1 cells, any treatment including LPS appeared to enhance MMP-9 production, however, this upregulation was not inhibited by the additional presence of L-NAME. Production of TNF-alpha by J774.1 cells was markedly enhanced with LPS treatment, and this enhancement was significantly reduced in the presence of L-NAME. These results indicate that the inhibitory effects of L-NAME on the tumor cell progression and pulmonary metastasis could be due to suppression of NO from tumor cells and TNF-alpha from macrophages (Mol Cell Biochem, 2007).

Interferon-gamma and lipopolysaccharide stimulation increases matrix metalloproteinase-9 expression and enhances invasion activity in ras/myc-transformed serum-free mouse embryo cells.

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma, 100 units/ml) and/or lipopolysaccharide (LPS, 0.5 microg/ml) for 24 h to investigate the effects of these ligands on the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Aminoguanidine (AG, 1mM; a nitric oxide synthase [NOS] inhibitor) was also added along with IFN-gamma and LPS to analyze a possible association of NO with invasiveness. Treatment of cells with IFN-gamma alone did not alter MMP-9 mRNA expression or pro-MMP-9 production, but LPS alone and IFN-gamma+LPS co-treatment enhanced them significantly. TIMP-1 mRNA expression remained unchanged with or without treatment and the mRNA expression of MMP-9 exceeded that of TIMP-1 in LPS- or IFN-gamma+LPS-treated cells. Co-treatment of cells with IFN-gamma and LPS up-regulated invasiveness and indicated a possible involvement of NO in the enhancement of invasiveness. These results suggest that ras/myc SFME cells respond to inflammatory and infectious conditions and that they may possibly modulate their characteristics as cancer cells due to their increase in MMP-9 expression and invasion activity.