ABSTRACT
The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by ß1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.
Subject(s)
Antibodies, Monoclonal , Polyamines , Cricetinae , Animals , CHO Cells , Cricetulus , Tunicamycin , Putrescine/metabolism , Eflornithine/pharmacology , RNA, Messenger/metabolism , Glycoproteins , Polysaccharides , Immunoglobulin G , Spermine/metabolismABSTRACT
We developed a simple and sensitive analytical HPLC method for the determination of acetylated hyaluronic acid (AcHA) in moisturizing and milk lotions. AcHA with different molecular weights was separated as a single peak using a C4 column and detected through post-column derivatization using 2-cyanoacetamide. The limits of detection and quantification were 60 and 200 ng, respectively. We found that AcHA in water was successfully extracted into a strong anion exchange (SAX) spin column with a recovery rate of AcHA was 63.8 ± 1.8%. Although the supernatant from acetone precipitation of lotions could pass through the spin column, the recovery rate (%) and accuracy of AcHA were affected by the viscous properties of cosmetics and acidic and acetone-soluble ingredients. Upon conducting analytical methods in this study, the concentration of AcHA in nine lotions was found to have ranged from 7.50 to 83.3 µg/mL. These values are comparable to the concentration range of AcHA in emulsions that have been previously evaluated for their superior effects. We believe that the analytical and extraction method is useful for the qualitative analysis of AcHA in moisturizing and milk lotions.
Subject(s)
Hyaluronic Acid , Milk , Animals , Chromatography, High Pressure Liquid/methods , Acetone , EmulsionsABSTRACT
The König reaction is commonly used for the detection of cyanide and its derivatives, including thiocyanate and selenocyanate. We found that this reaction can be used to quantify glutathione fluorometrically, and applied it to the simultaneous determination of reduced and oxidized glutathiones (GSH and GSSG) using a conventional LC system with isocratic elution. The limits of detection were 6.04 nM and 9.84 nM for GSH and GSSG, respectively, and the limits of quantification were 18.3 nM and 29.8 nM, respectively. We also determined GSH and GSSG levels in PC12 cells exposed to paraquat, an oxidative stressor, and observed a decrease in GSH/GSSG ratio, as expected. Total GSH levels quantified by this method and by the conventional colorimetric method with 5,5'-dithiobis(2-nitrobenzoic acid) were comparable. Our new application of the König reaction offers a reliable and useful method to simultaneously quantify intracellular GSH and GSSG.
Subject(s)
Glutathione , Paraquat , Rats , Animals , Glutathione Disulfide/metabolism , Oxidation-ReductionABSTRACT
The structural and functional relationships of glycosaminoglycans (GAGs) derived from marine organisms have been investigated, suggesting that marine invertebrates, particularly Bivalvia, are abundant sources of highly sulfated or branched GAGs. In this study, we identified a novel fucosylated heparan sulfate (Fuc-HS) from the midgut gland of the Japanese scallop, Patinopecten yessoensis. Scallop HS showed resistance to GAG-degrading enzymes, including chondroitinases and heparinases, and susceptibility to heparinases increased when scallop HS was treated with mild acid hydrolysis, which removes the fucosyl group. Moreover, 1H NMR detected significant signals near 1.2-1.3 ppm corresponding to the H-6 methyl proton of fucose residues and small H-3 (3.59 ppm) or H-2 (3.39 ppm) signals of glucuronate (GlcA) were detected, suggesting that the fucose moiety is attached to the C-3 position of GlcA in scallop HS. GC-MS detected peaks corresponding to 1, 3, 5-tri-O-acetyl-2, 4-di-O-methyl-L-fucitol and 1, 4, 5-tri-O-acetyl-2, 3-di-O-methyl-L-fucitol, suggesting that the fucose moiety is 3-O- or 4-O-sulfated. Furthermore, scallop HS showed anti-coagulant and neurite outgrowth-promoting (NOP) activities. These results suggest that the midgut gland of scallops is a valuable source of Fuc-HS with biological activities.
Subject(s)
Chondroitin Sulfates , Pectinidae , Animals , Chondroitin Sulfates/chemistry , Fucose/chemistry , Glycosaminoglycans/chemistry , Heparitin Sulfate , Glucuronic Acid , GlucuronatesABSTRACT
Cartilage in the head of sturgeon or salmon has been gaining attention as a rich source of functional chondroitin sulfate (CS) or proteoglycans. Although the cartilage was found in the heads of other bony fishes, the structure of CS and its core protein, especially aggrecan, was not fully investigated. In this study, comprehensive analysis of CS and aggrecan in the head cartilage of 10 bony fishes including sturgeon and salmon was performed. The 4-O-sulfation to 6-O-sulfation ratio (4S/6S ratio; S: sulfate residue) of CS in Perciformes was â§1.0, while the 4S/6S ratios of CS from sturgeons and salmon were less than 0.5. Dot blotting and proteomic analysis revealed that aggrecan was a major core protein in head cartilage of all bony fishes. These results suggest that the head cartilage of bony fishes is a promising source for the preparation of CS or proteoglycans as a health food ingredient.
Subject(s)
Chondroitin Sulfates , Proteoglycans , Aggrecans/analysis , Animals , Cartilage/metabolism , Chondroitin Sulfates/chemistry , Fishes/metabolism , Proteoglycans/chemistry , Proteomics , Salmon/metabolismABSTRACT
Chondroitin sulfate (CS) and its isomeric variant, dermatan sulfate (DS), are complex glycosaminoglycans (GAGs) which are ubiquitous components of the extracellular matrix in various tissues including the brain. CS and/or DS are known to bind to a variety of growth factors and regulate many cellular events such as proliferation and differentiation. Although the biological activities of CS and/or DS towards neural stem/progenitor cells (NSPCs) have been well investigated, the CS and/or DS of hematopoietic stem cells (HSCs) have not been fully characterized. Here, we analyzed GAGs on mononuclear cells of rat umbilical cord blood cells (UCB-MNCs). CS was detected in vascular intima and media of rat umbilical cord at embryonic day 19 (E19) by immunohistochemistry. The stem-cell-enriched-UCBCs (SCE-UCBCs), which were expanded from rat UCB-MNCs, expressed CS. CS chains are composed of repeating disaccharide units, which are classified into several types such as O-, A-, B-, C-, D-, and E-unit according to the number and positions of sulfation. A disaccharide composition analysis revealed that CS and/or DS were abundant in rat UCB-MNCs as well as in their expanded SCE-UCBCs, while the amount of heparan sulfate (HS) was less. The degree of sulfation of CS/DS was relatively low and the major component in UCB-MNCs and SCE-UCBCs was the A-unit. A colony-forming cell assay revealed that the percentage of colony-forming cells decreased in culture with CS degradation enzyme. The CS and/or DS of UCBCs may be involved in biological activities such as stem cell proliferation and/or differentiation.
Subject(s)
Cell Differentiation/drug effects , Cell Proliferation/drug effects , Chondroitin Sulfates/pharmacology , Fetal Blood/metabolism , Stem Cells/metabolism , Animals , Cell Culture Techniques , Cells, Cultured , Chondroitin Sulfates/chemistry , Disaccharides/chemistry , Disaccharides/pharmacology , Female , Fetal Blood/cytology , Rats , Stem Cells/cytologyABSTRACT
The simultaneous detection of cyanide (CN), thiocyanate (SCN), and selenocyanate (SeCN) by a HPLC-fluorescence detector (FLD) with the post-column König reaction was recently reported. SCN and SeCN are also detectable by HPLC-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) because sulfur and selenium can be detected, respectively, without any pre- or post-treatment. ICP-MS has high sensitivity for selenium and sulfur detection and is robust to sample matrices. In this study, we compared HPLC-FLD with the post-column König reaction and HPLC-ICP-MS in terms of SCN and SeCN detection sensitivity and linearity. The limit of detection (LOD) for SCN indicated that HPLC-FLD with the post-column König reaction was 354 times more sensitive than HPLC-ICP-MS. Likewise, the LOD for SeCN indicated that HPLC-FLD was 51 times more sensitive than HPLC-ICP-MS. These results demonstrated that HPLC-FLD was a more suitable technique for SeCN and SCN detection than HPLC-ICP-MS. We previously reported that SeCN was generated in selenite-exposed mammalian cells to detoxify excess selenite. HPLC-FLD with the post-column König reaction enabled good separation and detection for quantifying SCN and SeCN in mammalian cell lines exposed to selenite. The intracellular SCN and SeCN concentrations determined by this technique suggested differences in the metabolic capacity for selenite to form SeCN among the cell lines. In addition, since the amount of intracellular SCN and SeCN were significantly decreased by pretreatment of myeloperoxidase (MPO) inhibitors, SCN and SeCN were resulted from the interaction of sulfur and selenium with endogenous CN, respectively, generated with MPO.
Subject(s)
Chromatography, High Pressure Liquid/methods , Cyanates/analysis , Mass Spectrometry/methods , Selenium Compounds/analysis , Spectrometry, Fluorescence/methods , Thiocyanates/analysis , Cyanates/metabolism , Hep G2 Cells , Humans , Limit of Detection , Linear Models , Selenium Compounds/metabolism , Thiocyanates/metabolismABSTRACT
Honeybee larvae have been recognized as nutrient-rich food in many countries. Although glycogen, a storage form of glucose in animals, is synthesized in honeybee larvae, there is no information on the structure of glycan and its biological activity. In this study, we successfully extracted glycogen from honeybee larvae using hot water extraction and investigated the structure and biological activity of glycan. It was found that the molecular weight of glycogen from honeybee larvae is higher than that of glycogen from bovine liver and oysters. In addition, treatment of RAW264.7 cells with glycogen from honeybee larvae resulted in a much higher production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 than treatment with glycogen from either bovine liver or oysters. These results suggest that the high molecular weight glycogen from honeybee larvae is a functional food ingredient with immunomodulatory activity.
Subject(s)
Bees/chemistry , Glycogen/pharmacology , Immunologic Factors/pharmacology , Interleukin-6/metabolism , Larva/chemistry , Tumor Necrosis Factor-alpha/metabolism , Animals , Cattle , Functional Food , Glycogen/analysis , Immunologic Factors/analysis , Liver/chemistry , Macrophages/metabolism , Mice , Molecular Weight , Ostreidae/chemistry , RAW 264.7 CellsABSTRACT
Physical mixtures of cationic polymers and heparin have been developed to overcome the limitations of unfractionated heparin. In this study, we found that heparin associates with natural polyamines in water, resulting in the generation of a poly-ion complex (PIC). PIC formation (or stability) was influenced by the concentration and ratio of heparin and polyamines, molecular weight of heparin, nature of polyamines, and pH conditions. Interestingly, the PIC obtained when heparin and tetrakis (3-aminopropyl) ammonium (Taa) were mixed exhibited stability and was sticky in nature. PIC formation was due to an electrostatic interaction between heparin and Taa. Heparin-Taa PIC was administered subcutaneously to mice, and the time to maximum heparin concentration within the therapeutic range of heparin was markedly increased compared to that after a single dose of heparin. These results suggest that the quaternary ammonium structure of Taa is critical for the preparation of a stable PIC, thereby allowing the sustained release of heparin into the blood.
ABSTRACT
We have found recently that dendritic spine extension is inhibited through acrolein conjugation with α- and ß-tubulin proteins during brain infarction. In this current study, we looked for other acrolein-conjugated proteins in the 100,000g precipitate fraction, to clarify how cytoskeleton structure is modified by acrolein. Acrolein-conjugated proteins were sought from acrolein-treated mouse FM3A and Neuro2a cells and from tissues isolated from mouse brain infarction. It was found that vimentin was conjugated with acrolein, and the conjugated amino acid residue was Cys328, which is the only Cys residue in vimentin. It was also found that Cys207, 257, 285, and Lys118 in actin, another cytoskeleton protein, were conjugated with acrolein. The structure and localization of vimentin and actin filaments were changed greatly in infarct brain in photochemically induced thrombosis model mice and in acrolein-treated Neuro2a cells. In addition, degradation of cytoskeleton proteins was accelerated in the order vimentin > tubulin > actin in mouse brain infarction. These findings indicate that a dysfunction of the cytoskeleton by acrolein is strongly involved in the tissue damage during brain infarction, together with the apoptosis caused by glyceraldehyde-3-phosphate dehydrogenase and protein degradation by matrix metalloproteinase-9.
Subject(s)
Acrolein/metabolism , Actins/metabolism , Brain Infarction/physiopathology , Cytoskeleton/metabolism , Vimentin/metabolism , Animals , Humans , Male , MiceABSTRACT
Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC-MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.
Subject(s)
Acrolein/pharmacology , Brain Ischemia/pathology , Endothelium, Vascular/pathology , Glucuronidase/metabolism , Glycocalyx/pathology , Ischemic Stroke/pathology , Amino Acid Sequence , Animals , Brain Ischemia/etiology , Brain Ischemia/metabolism , Chondroitin Sulfates/metabolism , Disease Models, Animal , Endothelium, Vascular/metabolism , Glucuronidase/chemistry , Glucuronidase/genetics , Glycocalyx/metabolism , Heparitin Sulfate/metabolism , Humans , Hyaluronoglucosaminidase/metabolism , Ischemic Stroke/etiology , Ischemic Stroke/metabolism , Male , Mice , Mice, Inbred C57BL , Photochemistry , Protein ConformationABSTRACT
Mast cells have secretory granules containing chemical mediators such as histamine and play important roles in the immune system. Polyamines are essential factors for cellular processes such as gene expression and translation. It has been reported that secretory granules contain both histamine and polyamines, which have similar chemical structures and are produced from the metabolism of cationic amino acids. We investigated the effect of polyamine depletion on mast cells using bone marrow-derived mast cells (BMMCs). Polyamine depletion was induced using α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. DFMO treatment resulted in a significant reduction of cell number and abnormal secretory granules in BMMCs. Moreover, the cells showed a 2.3-fold increase in intracellular histamine and up-regulation of histidine decarboxylase (HDC) at the transcriptional level during BMMC differentiation. Levels of the transcription factor kruppel-like factor 4 (KLF4) greatly decreased upon DFMO treatment; however, Klf4 mRNA was expressed at levels similar to controls. We determined the translational regulation of KLF4 using reporter genes encoding Klf4-luc2 fusion mRNA, for transfecting NIH3T3 cells, and performed in vitro translation. We found that the efficiency of KLF4 synthesis in response to DFMO treatment was enhanced by the existence of a GC-rich 5'-untranslated region (5'-UTR) on Klf4 mRNA, regardless of the recognition of the initiation codon. Taken together, these results indicate that the enhancement of histamine synthesis by DFMO depends on the up-regulation of Hdc expression, achieved by removal of transcriptional suppression of KLF4, during differentiation.
Subject(s)
Histamine/biosynthesis , Kruppel-Like Transcription Factors/metabolism , Mast Cells/metabolism , Animals , Bone Marrow/metabolism , Bone Marrow Cells/metabolism , Cell Differentiation/physiology , Eflornithine/pharmacology , Female , Histamine/metabolism , Histidine Decarboxylase/genetics , Kruppel-Like Factor 4 , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Ornithine Decarboxylase/metabolism , Polyamines/metabolism , Secretory Vesicles/metabolism , Spermidine/metabolismABSTRACT
We developed a simple and sensitive HPLC method for the determination of selenocyanate (SeCN-). The König reaction, which is generally used for the determination of cyanide and thiocyanate, was applied for the post-column detection, and using barbituric acid as a fluorogenic reagent made it possible to detect SeCN- with high sensitivity. The limits of detection (LOD) and quantification (LOQ) were 73.5 fmol and 245.1 fmol, respectively. Subsequently, the amounts of SeCN- in human blood and in cultured cell samples were analyzed, and no SeCN- was detected in human whole blood. Interestingly, we have found that some of the spiked SeCN- decomposed to cyanide in human whole blood. Ascorbic acid suppressed the decomposition of SeCN- to cyanide by reducing the ferric ion, which is typically involved in SeCN- decomposition. Then, SeCN- was detected in cultured HEK293 cells exposed to selenite. The established HPLC method with fluorescence detection of SeCN- is useful for investigating small amounts of SeCN- in biological samples.
Subject(s)
Cyanates/blood , Fluorescence , Selenium Compounds/blood , Cells, Cultured , Chromatography, High Pressure Liquid/instrumentation , HEK293 Cells , HumansABSTRACT
We have recently found that conjugation of acrolein with a 50 kDa protein(s) is strongly associated with tissue damage during brain infarction. In the current study, the identity and function of the 50 kDa protein(s) conjugated with acrolein during brain infarction were investigated. The 50 kDa protein(s) conjugated with acrolein were identified as α- and ß-tubulins. Ten cysteine residues in α- and ß-tubulins (Cys25, 295, 347 and 376 in α-tubulin and Cys12, 129, 211, 239, 303 and 354 in ß-tubulin) were mainly conjugated with acrolein. Since two cysteine residues of α-tubulin (Cys347 and 376) and four cysteine residues of ß-tubulin (Cys12, 129, 239 and 354) were located at the interaction site of α- and ß-tubulins, association between α- and ß-tubulins to form microtubules was strongly inhibited by conjugation with acrolein. Accordingly, dendritic spine extension consisting of microtubules was greatly inhibited in acrolein-treated Neuro2a cells. The results strongly suggest that acrolein contributes to the functional losses in brain signaling through its conjugation with α- and ß-tubulins.
Subject(s)
Acrolein/toxicity , Dendritic Spines/metabolism , Dendritic Spines/pathology , Neuroblastoma/metabolism , Stroke/metabolism , Stroke/pathology , Tubulin/metabolism , Acrolein/metabolism , Amino Acid Sequence , Animals , Cell Line, Tumor , Dendritic Spines/drug effects , Male , Mice , Mice, Inbred C57BL , Microtubules/drug effects , Microtubules/metabolism , Neuroblastoma/pathology , Protein Conformation , Stroke/chemically induced , Tubulin/chemistryABSTRACT
Macrophage mannose receptor (MMR/CD206) is a promising target for the detection and identification of sentinel lymph node (SLN). MMR-targeting probes have been developed using mannosylated dextran, however, impairment of efficient targeting of SLN was often caused because of retention of injection site in which macrophages and dendritic cells exist. In this study, we prepared new MMR-targeting probes from yeast mannan (85 kDa), and its bioditribution was investigated. In-vivo evaluation showed that 11.9% of injected dose of 99mTc-labeled mannan-S-cysteines (99mTc-MSCs) was accumulated in popliteal lymph node (the SLN in this model), however, significant level of radioactivity (approximately 80%) was remained in injection site. Interestingly, 99mTc-labeled low molecular weight mannan-S-cysteine mannan (99mTc-LSC) prepared from 50 and 25 kDa mannan showed a decreased specific accumulation of 99mTc-LSC in the popliteal lymph node, while the radioactivity at the injection site remained unchanged. These results suggest that the molecular size, or nature/shape of the sugar chain is important for the specific accumulation of 99mTc-MSC in popliteal lymph node.
Subject(s)
Cysteine/pharmacokinetics , Lymph Nodes/metabolism , Mannans/pharmacokinetics , Animals , Cysteine/chemistry , Mannans/chemistry , Mice , Molecular Weight , Single Photon Emission Computed Tomography Computed Tomography , Technetium , Tissue DistributionABSTRACT
Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions -202 to -117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5'-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.
Subject(s)
5' Untranslated Regions/genetics , G-Quadruplexes , Gene Expression/drug effects , N-Acetylgalactosaminyltransferases/genetics , Polyamines/pharmacology , RNA Folding/drug effects , A549 Cells , Animals , CHO Cells , Caco-2 Cells , Cell Line, Tumor , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/metabolism , Cricetinae , Cricetulus , Glucuronosyltransferase , HCT116 Cells , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , MCF-7 Cells , Mice , Multifunctional Enzymes , N-Acetylgalactosaminyltransferases/metabolism , NIH 3T3 Cells , Polyamines/metabolism , RNA Folding/genetics , RNA InterferenceABSTRACT
This report describes the development of a target-protein-oriented natural-products-isolation (TPO-NAPI) method for Hedgehog inhibitors and the direct GLI1 inhibitor, 5'- O-methyl-3-hydroxyflemingin A (3), which inhibited hedgehog (Hh) signal transduction and diminished characteristics of cancer stem cells. Eight natural products (including three newly described products) that directly bind to GLI1 were rapidly obtained via the TPO-NAPI method developed using GLI1 protein-immobilized beads. 5'- O-Methyl-3-hydroxyflemingin A (3) inhibited Hh signaling (IC50 7.3 µM), leading to decreasing production of the Hh target proteins BCL2, PTCH1, and BMI1. 5'- O-Methyl-3-hydroxyflemingin A (3) was cytotoxic to Hh-related cancer cells. CD experiments revealed that 5'- O-methyl-3-hydroxyflemingin A (3) directly bound GLI1 ( Kd = 7.7 µM). Moreover, 5'- O-methyl-3-hydroxyflemingin A (3) diminished cancer stem cell characters of Huh7 such as sphere formation and production of the cancer stem cell marker EpCAM. These results suggest that Hh inhibitors can efficiently suppress the activity of cancer stem cells.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Chalcones/pharmacology , Hedgehog Proteins/antagonists & inhibitors , Neoplastic Stem Cells/drug effects , Zinc Finger Protein GLI1/antagonists & inhibitors , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Cell Line, Tumor , Chalcones/chemistry , Chalcones/isolation & purification , Fabaceae/chemistry , Hedgehog Proteins/metabolism , Humans , Molecular Docking Simulation , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Signal Transduction/drug effects , Zinc Finger Protein GLI1/metabolismABSTRACT
BACKGROUND AND PURPOSE: We recently found that acrolein (CH2=CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. METHODS: Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N-acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. RESULTS: NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S-transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S-transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S-transferases. CONCLUSIONS: The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S-transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.
Subject(s)
Acetylcysteine/therapeutic use , Brain Infarction/drug therapy , Brain/drug effects , Neuroprotective Agents/therapeutic use , Oxidative Stress/drug effects , Acetylcysteine/pharmacology , Acrolein/metabolism , Animals , Brain/metabolism , Brain Infarction/metabolism , Cell Line, Tumor , Disease Models, Animal , Glutathione/metabolism , Mice , Reactive Oxygen Species/metabolismABSTRACT
We have previously reported that the keratan sulfate (KS) disaccharide was branched to the C-3 position of glucuronate in chondroitin sulfate (CS)-E derived from the Mactra chinensis. We carried out the comprehensive disaccharide analysis of GAGs from 10 shellfish, Ruditapes philippinarum, Scapharca broughtonii, Mizuhopecten yessoensis, Turbo cornutus, Crassostrea nippona, Corbicula japonica, Mytilus galloprovincialis, Neptunea intersculpta, Pseudocardium sachalinense and Crassostrea gigas, to better understand the glycan structures in marine organisms. The contents of CS, heparan sulfate and hyaluronic acid and their compositions depend on the species of shellfish. Interestingly, a peak corresponding to a pentasaccharide containing KS disaccharide was observed when GAGs from T. cornutus was treated with chondroitinase (Chase) ACII but not Chase ABC. In addition, unidentified peaks were also observed when CS derived from R. philippinarum, S. broughtonii were treated with Chase ACII. These results suggest the presence of additional unidentified structure of CS in these shellfish.
Subject(s)
Glycosaminoglycans/chemistry , Shellfish , Animals , Chondroitin Sulfates/chemistry , Disaccharides/chemistry , Heparitin Sulfate/chemistry , Hyaluronic Acid/chemistry , Oligosaccharides/chemistryABSTRACT
Glycomics represents one of the last frontiers and most challenging in omic analysis. Glycosylation occurs in the endoplasmic reticulum and the Golgi organelle and its control is neither well-understood nor predictable based on proteomic or genomic analysis. One of the most structurally complex classes of glycoconjugates is the proteoglycans (PGs) and their glycosaminoglycan (GAG) side chains. Previously, our laboratory solved the structure of the chondroitin sulfate chain of the bikunin PG. The current study examines the much more complex structure of the dermatan sulfate GAG chain of decorin PG. By utilizing sophisticated separation methods followed by compositional analysis, domain mapping, and tandem mass spectrometry coupled with analysis by a modified genetic algorithm approach, the structural motif for the decorin dermatan sulfate chain was determined. This represents the second example of a GAG with a prominent structural motif, suggesting that the structural variability of this class of glycoconjugates is somewhat simpler than had been expected.