Earticle

A carbohydrate microarray has been suggested as a powerful tool for robust, rapid, and diverse screening of carbohydrate-protein interaction. Here we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrate by reductive amination, in which reducing sugar groups were coupled with 4-(2-aminoethyl)aniline, which has di-amine groups at both ends. The modified carbohydrates were attached to amine-reactive NHS-activated glass surface by formation of stable amide bonds. This new method was applied for efficient construction of a carbohydrate microarray platform to analyze carbohydrate-protein interactions and assay glycosyltransferase activity. This array prepared using our method can be successfully used for screening the specificity of GBPs (glycan-binding proteins) such as lectins, growth factors, antibody, and microbial toxins, assaying new glycosyltransferase activity, and identification of carbohydrate-protein interactions

The a(1,6)-fucose attached to the core N-glycan (core fucose) of glycoproteins has been known to play essential roles in various pathophysiological events, including oncogenesis and metastasis. Aspergillus oryzae lectin (AOL) encoded by the fleA gene has been reported to bind to N-glycans containing this core fucose. In our previous study, the fleA gene was fused with genes of fluorescent proteins to generate fluorescent probes. Their binding capabilities to core fucose was confirmed by lectin blot and cytochemical staining. Here, we describe the internalization properties of the FleA-fluorescent fusion proteins. During cell imaging experiments, we surprisingly found that these proteins could be internalized into intracellular compartments, especially Golgi apparatus, when applied to live cells. In contrast, their fluorescences were observed only on the cell surface when applied to the fixed cells or the metabolically arrested cells at low temperature. These results suggested that FleA-fluorescent fusion proteins first bound to the core-fucosylated glycoprotein located on the cell surface and then could be internalized by the active cellular process. This internalization was shown to occur depending on a clathrin-mediated pathway by endocytosis inhibitor assay. Taken together, FleA-fluorescent fusion proteins can be employed as a vesicle for delivery of useful materials to the inside of cells as well as a valuable fluorescent probe for detection of core fucose.

Several classes of peptide:N-glycanse (PNGase) enzymes have been identified and used for release of N-glycan from glycoproteins. PNGase F, derived from Flavobacterium meningosepticum, has been most widely used while an almond PNGase A has been employed specifically for deglycosylation of plant- orinsect-derived glycoproteins. In the present study, we identified a new PNGase enzyme from Yarrowia lipolytica, of which amino acid sequence shows weak homology with PNGase A. This gene was cloned and successfully expressed in Pichia pastrois using the commercially available vector for secretory expression. The prepared enzyme, designated as PNGase Y, was shown to release various types of N-glycans including high-mannose and complex type glycans. Especially, it could release the glycans containing core α (1,3)-fucosefound in plant and insect as well as bi-phosphorylated glycans for lysosomal proteins, which are not good substrates for PNGase F. Moreover, PNGase Y was shown to release N-glycans efficiently from denatured glycoprotein as well as glycopeptide. It is a great advantage compared to PNGas A which requires an additional step of protease digestion due to the preference for glycopeptides as a substrate. Taken together, we suggest that PNGase Y will be a good tool to release various types of N-glycans with broad range of substrate specificity under favorable conditions without the need of glycopeptides preparation.

Tunicamycin is an antibiotic that blocks the synthesis of all N-linked glycoproteins. It causes endoplasmic reticulum (ER) stress and induce unfolded protein response (UPR). Recent evidence suggests tunicamycin-inducd ER stress is a possible cause of inflammation. In the present study, we have investigated the role of tunicamycin on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage. Tunicamycin decreaced inducible nitric oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), and pro-inflammatory cytokine such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpa (TNF-α) expression in response to LPS in RAW264.7 macrophage cells. However, other well-known ER stress inducers, A23187 and Thapsigargin increased LPS-induced COX-2 expression and had no effect on LPS-induced iNOS, TNF-α and IL-1β expression. These results suggest that the inhibitory effect of tuncimaycin on LPS-induced inflammation is independent of its effect on ER stress induction. We further demonstrated that tunicamycin inhibited LPS-induced NF-κB activation. in RAW264.7 macrophage cells.

Glucosamine (GlcN) derivative, GNK407 suppressed lipopolysaccharide (LPS)-induction of inflammatory genes such as inducible nitric oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), interleukin-1beta (IL-1 β) in mouse macrophage cell line (RAW 264.7). GNK407 inhibited LPS-induced activation of nuclear factor-kB (NF-kB). The inhibitory effect of GNK407 was mediate by inhibition of p65 DNA binding to NF-kB binding sequence of iNOS promoter in RAW 264.7 cells. Additionally, p65 is O-GlcNAcylated by LPS, and this modification suppressed by GNK407 in RAW 264.7 cells. Taken together, GNK407 inhibited LPS-induced inflammatory responses by suppressing NF-kB activation potentially through the inhibition LPS-induced upregulation of p65 O-GlcNAcylation. Our finding suggest that GNK407 may serve as novel anti-inflammatory agent.

It has been shown that glucosamine (GlcN) inhibits lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2) expression in macrophage. However, in this study, we demonstrated that GlcN enhanced the LPS-induced COX-2 expression in RAW 264.7 cells in low glucose cultured condition. It suggests that glucose concentration may be an important for LPS-induced COX-2 expression. We found that in low glucose condition, O-GlcNAc modification of Sp1 was increased by GlcN, although LPS did not further enhanced O-GlcNAc modification of Sp1. Further investigations regarding molecular mechanisms underlying the LPS-induced COX-2 expression and functional role of Sp1 transcription factor in the low glucose condition cultured RAW 264.7 cells.

GQ1b is a tetrasialo-ganglioside that has four sialic acid residues, and seemed to be involved in the learning and memory. However, the molecular mechanisms and functions of GQ1b is not well understood. Studies were initiated to determine the effect of GQ1b on the improvement of the learning and memory of rats by using the Y-maze and Morris water maze test. The spatial learning and memory of rats was improved by GQ1b treatment into the rat brain. The improvements in spatial memory as measured by these two maze tests are dependent on the hippocampal learning and memory function and related to the N-methyl-D-aspartate (NMDA) receptor signaling pathway. Thus, we showed the activation of NMDA receptor signaling pathway by GQ1b treatment in rat hippocampal cells and rat hippocampus. The activation of NMDA receptor signaling pathway can leads to an enhancement of Brain-derived neurotropic factor(BDNF) mRNA levels and stimulates released of the BDNF protein. BDNF is known to support the survival, growth and differentiation of neurons and mainly expressed in synapses of the hippocampus, cortex, and basal forebrain. We investigated that the expression and the secretion of BDNF were increased by GQ1b treatment in SH-SY5Y cells and rat primary neurons. In addition, GQ1b treatment prevented the effect of D-PDMP, ganglioside synthesis inhibitor, on decrease of BDNF expression in SH-SY5Y cells. To investigate whether NMDA receptor signaling pathways was involved in GQ1b-induced BDNF expression, rat primary neurons were treated with GQ1b and NMDA receptor antagonist D-AP5. Pretreatment with the D-AP5 significantly abolished BDNF expression following GQ1b treatment in primary cortical neurons. These data suggested that GQ1b mediates its effect on BDNF expression through the NMDA receptor activation in the brain. Therefore, ganglioside GQ1b might improve spatial learning and memory and regulate the expression and the secretion of BDNF via activation of NMDA receptors signaling pathway.

Autoantibodies, which are generated by immune system recognizing the presence of the abnormal tumor-associated antigens, are promising biomarkers for the early detection of tumors. Recently, we established hundreds of B cell hybridomas from H-ras12V transgenic mouse, a typical tumor model system, as a source of tumor-associated autoantibodies without using any extracellular antigens and have characterized specific target antigens against them. TAB-K94 monoclonal antibody, one of tumor-associated autoantibodies obtained from H-ras12V transgenic mouse, was investigated in this study and its target antigen was identified as cytokeratin 8/18 (CK8/18) complex, an intermediate filament (IF) protein complex of epithelial cells which is involved in cell motility and cancer progression. To establish a method for the detection of autoantibodies against CK8/18 complex in tumor patients sera, a specific mimotope against TAB-K94 autoantibody was screened from the cyclic random hepta-peptide phage library and, by enzyme-linked immunosorbent assay (ELISA) using it as a binder of anti-CK8/18 autoantibodies, we could distinguish breast cancer patients vs. normal subjects with 50.00 % sensitivity and 82.61 % specificity. These results imply that anti-CK8/18 autoantibody is induced in a certain group of breast cancer patients and detection of anti-CK8/18 autoantibody would be useful for the diagnosis of breast cancer.

Cancer is often difficult to achieve early diagnosis, which is, however, a decisive requisite for favorable outcome in cancer treatments. Cancer biomarkers have been sought for several purposes preferably in blood and pinpointing cancer cells-derived aberrant glycoproteins would be a well-grounded approach to cancer biomarker discovery. One of the glycosyltransferases responsible for aberrant glycosylation in cancer is N-acetylglucosaminyltransferase V (GnT-V), which catalyzes an addition of b1,6-N-acetylglucosamine (GlcNAc) to the core N-glycan, and many lines of evidence have demonstrated the role of N-acetylglucosaminyltransferase V (GnT-V) in cancer development. Tissue inhibitor of metalloproteinase-1 (TIMP-1) and protein tyrosine phosphatase kappa (PTPk) were suggested to be involved in cancer malignancy upon aberrantly glycosylation by GnT-V. In addition to GnT-V, several glycosyltransferase co-works to render the altered glycan structures in cancer cells including sialyl Lewis antigen and core-fucosylation, which prompted us to mine serological biomarker candidates in cancer sera. Immunodepleted on an immune-LC column, serological proteins were enriched by carbohydrate beads conjugated with lectins like L-PHA, DSA, E-selectin, AAL, Con-A. The fraction refractive to lectin enrichments was resolved on an SDS-PAGE gel, and fractionated by molecular mass. Both the captured glycoproteins and gel-separated proteins were tryptic-digested for sequence determination in an LTQ-FTICR mass spectrometer. Candidate proteins showing high sensitivity and specificity during the discovery phase were selected and the panel of biomarker candidates is currently under in-depth analyses for validation.

The development of cancer biomarker is of a great promise to conquer cancer since it leads to an early detection and provide an opportunity for better cancer treatment. Validation is a time-consuming step for biomarker developments requiring investigation of biomarker candidates using thousands or more of biosamples, which is why a sensitive, multiplexing validation method is necessary. To address this challenge, we are developing as antibody-based validation method, in which antibody with oxidized N-glycans was covalently linked to an identifiable DNA tag for use of the template in the transcription by T7 polymerase. The transcripts were designed for analysis on an oligonucleotide chip, where oligonucleotides with complementary sequences were embedded. The transcripts will be quantified and used for an indicator of the amounts of the antigen bound to the DNA-tagged antibody. This strategy will be multiplexed for an establishment of a novel validation method

Polyketides are a large class of natural products that include clinically important antibiotics, antitumor compounds, immunosuppressants, pigments and herbicides. Herboxidiene is a polyketide natural product that was found to control several important biannual weeds at relatively low application rates (<250 g/hectare) without damaging wheat. Early study indicated that the epoxide and the C-18 hydroxyl groups are important for the activity of herboxidiene. While Sakai et al., 2002 reported that herboxidiene showed antitumor activity against human tumor cell lines, Koguchi et al., 1997 reported that herboxidiene up-regulated the gene expression of low density lipoprotein receptors. The interesting biological profile of this natural product has prompted us to produce its glycosylated analogues so as to alter its activity. It is well known that bioactive natural products are often complexed with sugar moieties which are essential for activity of the compound. In this study, we carried out in vitro experiments to generate glucosyl-herboxidiene.

Streptomyces peucetius ATCC 27952 contains two similar genes (1068 nucleotides), dnmL and rmbA, which encode for glucose-1-phosphate (G-1-P) thymidylyltransferases (69% identity and 86% similarity). When the genes were expressed in E. coli under similar conditions, rmbA was expressed in soluble form, and it showed G-1-P thymidylyltransferase activity. But, dnmL was expressed as inclusion bodies. Thorough comparative analysis of DnmL and RmbA by sequence alignment, hydrophobicity scale and homology modeling inferred that the expression of RmbA in soluble form is contributed to the helical structure formed by the high hydrophobicity content in C-terminal sequence of RmbA. Similarly, the formation of inclusion bodies of DnmL was attributed to a loop conformation attained by its C-terminal sequence. To test the significance of these sequences, the C-terminal sequences of DnmL was substituted with that of RmbA. The fusion DnmL was expressed in soluble form and it had similar G-1-P thymidylyltransferase activity as compared to RmbA. These observations demonstrated that the hydrophobic effect plays a significant role in protein folding or structure, and the solubility of the expressed proteins depends on the protein conformations. In conclusion, this method of protein engineering will be a rational tool for enhancing solubility of proteins expressed in E. coli.

Polysaccharides from brown seaweeds are interesting due to their various biological activities. A water-soluble polysaccharide was isolated and purified from the sporophyll of Korean Undaria pinnatifida by dilute acid extraction, CaCl2 precipitation to remove alginates, and purified DEAE-cellulose chromatography. Structure of this polysaccharide was characterized by various methods such as elemental analysis, HPAEC-PAD, HPLC analysis, FT-IR, FT-Raman, and 1H and 13C NMR. The average molecular mass of this polysaccharide was estimated about 2.1×103 kDa by HPLC. The polymer showed lower electrophoretic mobility (RSH 0.127) than carrageenans (0.096-0.100) and chondroitin sulphate A (0.103), but higher than alginate (0.170). It is consisted of neutral sugar (52.34 % in mass) and sulfate esters (7.4 % in mass), no uronic acid was observed. From the HPAEC-PAD analysis, the monosaccaride composition was shown to be fucose, galactose, xylose, and mannose, with a molar ratio of 1, 0.8, 0.08, and 0.005, respectively. The major monosaccharide was fucose (50.9 mol%), suggesting that it is a fucoidan, a general name of seaweeds polysaccharides containing fucose residues. Other sugars, galactose (44.6 mol%), xylose (4.2 mol%), and mannose (0.3 mol%), were present as minor component. Very intense and broad FT-IR band at 1258-1259 cm-1 were attributed to S=O stretching of sulfate esters. The IR band centered at 840-842 cm-1 was attributed to COS bending of sulfate substituents. Elemental analysis (EA) of this polysaccharide resulted in C (23.03%), H (3.07%) and S (9.18 %). Taken together, these results clearly demonstrated the presence of sulfate groups in this polysaccharide. The degree of sulfation was determined to me 0.93 by DS (moles of SO3- per mole of sugar units) value, which shows about one sulfate group per sugar unit. Consequently the purified polysaccharide was defined as O-acetylated sulphated galactofucan.

Mulberry is well known to produce plenty of anthocyanins and other biologically active materials. In the present study, we investigated immune stimulating activity of a water-soluble polysaccharide purified from the Korean Mulberry fruits (Morus bombycis Koidz). The crushed raw materials were extracted with 75% ethanol at room temperature overnight and from this extract a polysaccharide was purified mainly by 75% ethanol precipitation and DEAE-cellulose chromatography. The chemical composition of the purified mulberry polysaccharide (tentatively named as JS-MP-1) was determined by High-Performance Anion Exchange Chromatography and Pulsed Amperometric Detection (HPAEC-PAD), elemental analysis, and FT-IR. Based on HPAEC-PAD analysis, major component monosaccharides of JS-MP-1 were shown to be galactose (37.6% in mole percentage), arabinose (36.3%) and rhamnose (18.4%), respectively. Elemental analysis resulted in C (34%), H (4.97%), N (0.96%) and S (0.07%). FT-IR analysis has shown -OH band at (ex, 840-842 cm-1 ), the typical pattern being seen in carbohydrates. Biochemical assays showed neutral sugars (35.25%), uronic acid (11.25%), sulfate (0.16%) and proteins (0.3%). Thus, taken collectively, the JS-MP-1 was shown to be a heteropolysaccharide, a variant of arabinogalactan containing some other minor sugars such as rhamnose and uronic acids but no detectable level of sulfate and protein. Enzyme Linked Immunosorbent Assay (ELISA) showed that the purified mulberry polysaccharide, JS-MP-1, remarkably stimulates the release of the proinflammatory cytokines, TNF-α and IL-6, in a dose-dependent manner, from the RAW 264.7 cells. RT-PCR analysis also showed a significant induction of the COX-2 gene expression in RAW264.7 murine macrophage by JS-MP-1. These results clearly demonstrated that the water-soluble polysaccharide isolated from the Mulberry fruits has immune stimulating activities.

Effects of the degree of deacetylation (DDA) and the molecular mass of chitosan oligosaccharides (CTS-OS), obtained from the enzymatic hydrolysis of high molecular weight chitosan (HMWC), on antitumor activity was explored. The DDA and molecular weights of CTS-OS were determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF MS) analysis. The CTS-OS were found to be a mixture of mainly dimers (18.8%), trimers (24.8%), tetramers (24.9%), pentamers (17.7%), hexamers (7.1%), heptamers (3.3%), and octamers (3.4%). The CTS-OS were further fractionated by gel-filtration chromatography into two major fractions: (1) COS, consisting of glucosamine (GlcN)n, n = 3–5 with DDA 100%; and (2) HOS, consisting of (GlcN)5 as the minimum residues and varying number of N-acetylglucosamine (GlcNAc)n, n = 1 –2 with DDA about 87.5% in random order. The cytotoxicities, expressed as the concentration needed for 50% cell death (CC50), of CTS-OS, COS, and HOS against PC3 (prostate cancer cell), A549 (lung cancer cell), and HepG2 (hepatoma cell), were determined to be 25 mg∙mL-1, 25 mg∙mL-1, and 50 mg∙ mL-1, respectively. The HMWC was approximately 50% less effective than both CTS-OS and COS. These results demonstrate that the molecular weight and DDA of chitosan oligosaccharides are important factors for suppressing cancer cell growth.

Stabilization, detoxification, and solubilization of small molecules based therapeutics and natural products can be altered by glycosylation. In vitro glycosylation of small molecules is difficult to scale up as well as costly to implement in industrial level. To overcome these hurdles, Escherichia coli has been engineered by heterologous overexpression of TDP-4-amino-4, 6-dideoxy-D-galactose biosynthetic gene clusters, and glycosyltransferase gene to produce a range of small molecule glycosides. By applying this metabolic engineering approach, flavonoids, the polyphenolic secondary plant metabolites- Quercetin and Kaempferol were glycosylated to produce Quercetin glycoside and Kaempferol glycoside by whole cell biotransformation. This strategy of in vivo glycosylation offers vast combinatorial biosynthesis potential to produce glycosylated natural products by simple fermentation. Key Words: glycosylation, heterologous overexpression, biotransformation, combinatorial biosynthesis potential.

The β-galactoside α 2,6-sialyltransferase (ST6Gal I) is an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins. It is highly expressed in tumors, which probably the basis for the increase of α (2, 6)-sialylation found in cancer cells. Previously we found that ionizing radiation (IR) increased the expression of ST6Gal I, cleaved it into catalytically active form as comparable to enzyme localized in golgi, and these soluble forms were secreted into the culture media. Induction of ST6Gal I significantly increasedthe adhesion and migration of colon cancer cells via sialylation of integrin β1 that is mostlyrelated to cell adhesion and migration. In this study, we further investigated the function of ST6Gal I underlying cleavage, solubilization and release from the cells, especially focusing on the metastasis. ST6Gal I was cleaved by Alzheimer's beta-site amyloid precursor protein-cleaving enzyme (BACE), which was activated by IR, and besides golgi-localized ST6Gal I, soluble form of ST6Gal I was released into the culture media. Although soluble form of ST6Gal I also had sialylation activity, these forms enhanced the migration and invasion of colon cancer cells which was less dependent of intergrin b1 sialylation as compared with golgi-anchored form. Especially, we found that ST6 Gal I repress the E-cadherin promoter activity via induction of Snail. These data suggests that the glycoconjugation with unknown function of soluble sialyltransferase may involve in the process of cancer progression and metastasis which may be in cooperation with golgi-form.

Microarray technologies have received considerable attention owing to the fact that they serve as effective tools for high-throughput analysis of boimolecular interactions and the identification of bioactive substances that bind to biomolecules. Most of the current methods used to construct microarrays rely on the immobilization of substances on properly derivatized surfaces. Among various functional groups used for this purpose, the N-hydroxysuccinimide (NHS) ester group has been largely employed since it can be readily reacted with amine or hydrazide functionalities in substances of interest. However, the NHS ester group is usually introduced onto the surface of a glass slide by employing inconvenient and time consuming multi-step processes. In recent studies, it was developed an efficient, single step method for derivatization of glass surfaces with NHS ester groups that takes advantage of an acid-mediated reaction of NHS ester functionalized dimethallysilanes with silanols on the glass surface. Conditions for the surface modification procedure that utilize TfOH rather than Sc(OTf)3 were found to be superior. Protein and RNA-binding experiments show that glass surfaces modified by employing this method are suitable for efficient immobilization of various substances that are appended by amine, hydrazide and alcohol functionalities. The newly developed acid-mediated, glass surface modification method should be generally applicable to the preparation of various functional group-modified surfaces.

The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. Gangliosides are sialic acid-containing glycosphingolipids involved in inflammation and oncogenesis. We have demonstrated that treatment with anti-EpCAM mAb and RAW264.7 cells significant inhibited the cell growth in SW620 cancer cells, but neither anti-EpCAM mAb nor RAW264.7 cells alone induced cytotoxicity. The relationship between ganglioside expression and the anti-cancer effects of anti-EpCAM mAb and RAW264.7 was investigated by high-performance thin-layer chromatography. The results demonstrated that GM1 play important roles in the ability of anti-EpCAM to inhibit cell growth in SW620 cells. Anti-EpCAM mAb treatment increased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins were unaltered. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. In light of these results, further clinical investigation should be conducted on anti-EpCAM mAb to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

The cell membrane glycoprotein KAI1 (CD82) is a candidate metastasis suppressor gene that has been indicated in the progression of solid tumors. KAI1 is significantly downregulated in various human malignancies. However, the expression and mechanism of regulation of KAI1 in metastasis has not yet been fully elucidated. Previously, it is suggested that N-glycosylation of KAI1 occurred at high level and lead to significant inhibition or promotion of cell motility depending on the extracellular matrix. In particular, little is known about the glycosylation of KAI1 and its functional role in cancer metastasis. Recently, we demonstrated that expression of ST6 Gal I, which was highly expressed in colon cancer, was responsible for integrin 1-mediated adhesion and migration in human colon cancer. Remarkably, KAI1 inhibits the tumor metastasis via interaction with tetraspanins, integrins and chemokines which are responsible for cell migration, adhesion and signalling. We found for the first time that KAI1 was sialylated by ST6 Gal I in human colon cancer cells and sialic acid modification was involved in stabilization of KAI1 by proteasome-dependent manner. Also, abolishment of Asn residues largely disrupts the migration-, invasion-, suppressive activities of KAI1. Importantly, when KAI1 was expressed in HCT116 human colon cancer cells with ST6 Gal I, inhibitory effects on migration and invasion was abrogated by altering of p130CAS-Crk coupling, a signaling step for integrin dependent migration. Moreover, sialylated KAI1 interacted with PKC-integrin and is unlikely to inhibit cell migration through its associated proteins. These new observations may indicated that KAI1 can be stably expressed by sialylation in the process of tumor malignancy and sialylation profoundly affects KAI1, leading to significant alterations of functions as metastasis suppressor.

Cyperus rotundus is a multipurpose plant, extensively used in traditional medicine around the world to treat stomach ailments, wounds, boils and blisters. A number of pharmacological and biological activities including anti-Candida, anti-inflammatory, antidiabetic, antidiarrhoeal, cytoprotective, antimutagenic, antimicrobial, antibacterial, antioxidant, cytotoxic and apoptotic, anti-pyretic and analgesic activities have been reported for this plant. In the present study, the water and 70% ethanol extracts showed no significant effect on NO production, and NF-κB SEAP reporter gene assay using LPS-stimulated RAW264.7 cells, murine macrophages. Similarly, the anti-inflammatory effects of seventeen essential oils isolated from C. rotundus were evaluated on LPS-stimulated RAW264.7 cells. From the tested compounds, α-cyperone, nootkatone, carophylline oxide, oleanolic acid, showed promising anti-inflammatory and NF-κB inhibition effect on LPS-stimulated RAW264.7 cells. Furthermore, the anti-inflammatory potential of four structurally divergent sesquiterpens (nootkatone, α-cyperone, valencene and β-selinene) were brought under molecular investigation in terms of LPS-stimulated RAW264.7 cells. Moreover, the nootkatoon, α-cyperone, were selected for detail study in order to evaluate various inflammatory mediators (iNOS and COX-2). The results showed that, nootkatone, α-cyperone, down-regulated the expressions of iNOS and COX-2 and correlated with the suppression of NF-κB signaling pathway. Additionally, electrophoretic mobility shift assay (EMSA) elucidated that nootkatone and α -cyperone dramatically suppressed LPS-induced NF-κB-DNA binding activity using 32P-labeled NF-κB probe. Hence, our data suggest that α-cyperone and nootkatone are potential therapeutic agents for inflammation associated diseases.

Natural products have played major roles in drug discovery including development of anti-cancer agents. Especially, saponins have been reported to have important anti-cancer properties. Platycodin D (PD) is a triterpenoid saponin isolated from the root of Platycodon grandiflorum (Campanulaceae). In the present study, the detailed signaling pathway involved in PD-induced apoptosis was investigated in human gastric adenocarcinoma AGS cells. We found that PD treatment resulted in a dose- and time-dependent decrease in the viability of AGS cells. The anti-proliferative effects of PD were accompanied by a G(1) phase cell cycle arrest and apoptosis both in doseand time-dependent manners. The characteristics of apoptosis were further determined by DAPI staining, DNA fragmentation assay, Annexin-V/PI double staining induced by exposure ratio of phosphatidylserine (PS) to the cell surface and sub-G(1) phase ratio. The apoptosis induced by PD was associated with the activation of initiator caspase-8 and -9, as well as the effector caspase-3. PD stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Moreover, PD induced an activation of the phosphorylation of c-Jun N-terminal kinase (JNK), but not p38 and ERK1/2. These results indicate that PD induces cellular apoptosis in AGS cell lines by the activation of caspase cascade and can be a potent anticancer agent against human gastric cancer.

This study investigates the anti-inflammatory effects of several components isolated from Rheum tanguticum Maxim. (Polygonaceae). Previously, we found that 70 % ethanol extracts of Rheum Rhizoma (Rheum tanguticum, R. undulatum, R. palmatum, R. officinale, Rumex crispus) had anti-inflammatory properties on LPS-stimulated RAW 264.7 macrophages and carrageenan-induced paw edema model. Thus, we studied five anthraquinone derivatives isolated from Rheum tanguticum, including chrysophanol, chrysophanol-8-O-Glc, 2-methoxy-4-hydroxyanthraquinone-5-O-Glc, emodin, and physcion. Chrysophanol, chrysophanol-8-O-Glc, physcion and emodin inhibited nitric oxide (NO) production at micromolar concentrations in LPS-induced RAW 264.7 macrophages. Among these anthraquinones, emodin was found to be a potent inhibitor with its IC50 (the half maximal inhibitory concentration) value of 57.23 μM. In addition, these four compounds dose-dependently reduced inducible nitricoxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression conducted by Western blotting analysis. Taken together, our data suggest that chrysophanol, chrysophanol-8-O-Glc, physcion and emodin from R. tanguticum showed anti-inflammatory activities through their inhibition of iNOS and COX-2 expression. Emodin was already reported to exhibit anti-inflammatory effects especially on acute pacreatitis animal model (1-3). Therefore, the further study is required to clarify anti-inflammatory mechanism of emodin in detail.

Leonurus japonica (syn L. heterophyllus Sweet) or Chinese motherwort is a herbaceous annual of pantropical distribution of the Lamiaceae family. Extracts of aerial parts are used in Chinese medicine for various purposes such as heart anti-arrhythmic, sedative, antimicrobial, anticoagulant, antioxidant and anti-tumoral properties, as well as booster of the immune response. The present study illustrated that, the water and 70% ethanol extracts of L. japonica exhibited no significant effect on NO and NF-κB SEAP reporter gene assay, using LPS-stimulated RAW264.7 cells. Several metabolites have been isolated from L. japonicus that substantiate the recorded activities. Five compounds were tested for anti-inflammatory effect. Leonurine, rutine, genkwanine, and stachydrine hydrochloride showed no promising effect on NO and NF-κB. On the other hand, the novel labdan diterpene derivative, 15, 16-epoxy-3-hydroxylabda-8, 13(16), 14-trien-7-one, exhibited potent inhibitory effect on the iNOS and COX-2 protein expression using LPS-stimulated RAW264.7 cells, macrophages. As a mechanism of anti-inflammatory action shown by 15,16-epoxy-3-hydroxylabda-8,13(16), 14-trien-7-one, suppression of nuclear factor (NF)-κB activation has been documented. Furthermore, the labdan compound exhibited significant inhibitory effect on LPS-medicated NF-κB-DNA binding and AP-1-DNA binding activitiesusing electrophoretic mobility shift assay (EMSA) 32P-labeled NF-κB and AP-1 probes. Taken together, labdan diterpene 15, 16-epoxy-3-hydroxylabda-8, 13(16), 14-trien-7-one, suppressed LPS-induced NF-κB activation, resulting down-regulation of iNOS and COX-2 protein expression, which was attributable to its inhibitory action of LPS-induced NO and PGE2 production. These results may contribute to the anti-inflammatory activity of 15, 16-epoxy-3-hydroxylabda-8, 13(16), 14-trien-7-one.