ABSTRACT
Glycosaminoglycans (GAGs) are valuable bioactive polysaccharides with promising biomedical and pharmaceutical applications. In this study, we analyzed GAGs using HPLC-MS/MS from the bone (B), muscle (M), skin (S), and viscera (V) of Scophthalmus maximus (SM), Paralichthysi (P), Limanda ferruginea (LF), Cleisthenes herzensteini (G), Platichthys bicoloratus (PB), Pleuronichthys cornutus (PC), and Cleisthenes herzensteini (CH). Unsaturated disaccharide products were obtained by enzymatic hydrolysis of the GAGs and subjected to compositional analysis of chondroitin sulfate (CS), heparin sulfate (HS), and hyaluronic acid (HA), including the sulfation degree of CS and HS, as well as the content of each GAG. The contents of GAGs in the tissues and the sulfation degree differed significantly among the fish. The bone of S. maximus contained more than 12 µg of CS per mg of dry tissue. Although the fish typically contained high levels of CSA (CS-4S), some fish bone tissue exhibited elevated levels of CSC (CS-6S). The HS content was found to range from 10-150 ug/g, primarily distributed in viscera, with a predominant non-sulfated structure (HS-0S). The structure of HA is well-defined without sulfation modification. These analytical results are independent of biological classification. We provide a high-throughput rapid detection method for tissue samples using HPLC-MS/MS to rapidly screen ideal sources of GAG. On this basis, four kinds of CS were prepared and purified from flounder bone, and their molecular weight was determined to be 23-28 kDa by HPGPC-MALLS, and the disaccharide component unit was dominated by CS-6S, which is a potential substitute for CSC derived from shark cartilage.
Subject(s)
Chondroitin Sulfates , Flounder , Glycosaminoglycans , Tandem Mass Spectrometry , Animals , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/isolation & purification , Glycosaminoglycans/isolation & purification , Glycosaminoglycans/chemistry , Chromatography, High Pressure Liquid , Bone and Bones/chemistry , Skin/chemistry , Skin/metabolism , Hyaluronic Acid/chemistry , Hyaluronic Acid/isolation & purification , Muscles/chemistryABSTRACT
Thromboembolic diseases pose a serious risk to human health worldwide. Fucosylated chondroitin sulfate (FCS) is reported to have good anticoagulant activity with a low bleeding risk. Molecular weight plays a significant role in the anticoagulant activity of FCS, and FCS smaller than octasaccharide in size has no anticoagulant activity. Therefore, identifying the best candidate for developing novel anticoagulant FCS drugs is crucial. Herein, native FCS was isolated from sea cucumber Cucumaria frondosa (FCScf) and depolymerized into a series of lower molecular weights (FCScfs). A comprehensive assessment of the in vitro anticoagulant activity and in vivo bleeding risk of FCScfs with different molecule weights demonstrated that 10 kDa FCScf (FCScf-10 K) had a greater intrinsic anticoagulant activity than low molecular weight heparin (LMWH) without any bleeding risk. Using molecular modeling combined with experimental validation, we revealed that FCScf-10 K can specifically inhibit the formation of the Xase complex by binding the negatively charged sulfate group of FCScf-10 K to the positively charged side chain of arginine residues on the specific surface of factor IXa. Thus, these data demonstrate that the intermediate molecular weight FCScf-10 K is a promising candidate for the development of novel anticoagulant drugs.
Subject(s)
Anticoagulants , Chondroitin Sulfates , Factor IXa , Molecular Weight , Animals , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/pharmacology , Chondroitin Sulfates/isolation & purification , Anticoagulants/pharmacology , Anticoagulants/chemistry , Anticoagulants/isolation & purification , Factor IXa/metabolism , Factor IXa/antagonists & inhibitors , Factor IXa/chemistry , Cucumaria/chemistry , Sea Cucumbers/chemistry , Blood Coagulation/drug effects , Humans , Models, MolecularABSTRACT
In this study, glycosaminoglycans (GAGs) were extracted from corb (Sciaena umbra) heads and thoroughly examined for their structure. Through cellulose acetate electrophoresis, the GAGs were identified as chondroitin sulfate (CS), with a recovery yield of 10.35 %. The CS exhibited notable characteristics including a high sulfate content (12.4 %) and an average molecular weight of 38.32 kDa. Further analysis via 1H NMR spectroscopy and SAX-HPLC revealed that the CS primarily consisted of alternating units predominantly composed of monosulfated disaccharides at positions 6 and 4 of GalNAc (52.6 % and 38.8 %, respectively). The ratio of sulfate groups between positions 4 and 6 of GalNAc (4/6 ratio) was approximately 0.74, resulting in an overall charge density of 0.98. Thermal properties of the CS were assessed using techniques such as differential scanning calorimetry and thermogravimetric analysis. Notably, the CS demonstrated concentration-dependent prolongation of activated partial thromboplastin time (aPTT) and thrombin time (TT) while showing no effect on platelet function. At 200 µg/mL, aPTT and TT coagulation times were 1.4 and 3.7 times faster than the control, respectively. These findings suggest that CS derived from corb heads holds promise as an anticoagulant agent for therapy, although further clinical investigations are necessary to validate its efficacy.
Subject(s)
Anticoagulants , Chondroitin Sulfates , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/pharmacology , Chondroitin Sulfates/isolation & purification , Anticoagulants/chemistry , Anticoagulants/pharmacology , Anticoagulants/isolation & purification , Animals , Humans , Blood Coagulation/drug effectsABSTRACT
In the present study, a selenium-chondroitin sulfate (SeCS) was synthesized by the sodium selenite (Na2SeO3) and ascorbic acid (Vc) redox reaction using chondroitin sulfate derived from shark cartilage as a template, and characterized by SEM, SEM-EDS, FTIR and XRD. Meanwhile, its stability was investigated at different conditions of pH and temperatures. Besides, its antioxidant activity was further determined by the DPPH and ABTS assays. The results showed the SeCS with the smallest particle size of 131.3 ± 4.4 nm and selenium content of 33.18% was obtained under the optimal condition (CS concentration of 0.1 mg/mL, mass ratio of Na2SeO3 to Vc of 1:8, the reaction time of 3 h, and the reaction temperature of 25 °C). SEM image showed the SeCS was an individual and spherical nanostructure and its structure was evidenced by FTIR and XRD. Meanwhile, SeCS remained stable at an alkaline pH and possessed good storage stability at 4 °C for 28 days. The results on scavenging free radical levels showed that SeCS exhibited significantly higher antioxidant activity than SeNPs and CS, indicating that SeCS had a potential antioxidant effect.
Subject(s)
Antioxidants/chemistry , Cartilage/chemistry , Chondroitin Sulfates/chemistry , Nanoparticles/chemistry , Selenium/chemistry , Sharks , Animals , Benzothiazoles/chemistry , Biphenyl Compounds/chemistry , Chondroitin Sulfates/isolation & purification , Drug Stability , Hydrogen-Ion Concentration , Particle Size , Picrates/chemistry , Sulfonic Acids/chemistry , TemperatureABSTRACT
Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.
Subject(s)
Electrophoresis, Polyacrylamide Gel , Glycosaminoglycans/isolation & purification , Silver Staining , Animals , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/isolation & purification , Desiccation , Glycosaminoglycans/chemistry , Heparitin Sulfate/chemistry , Heparitin Sulfate/isolation & purification , Ion Exchange , Lung/metabolism , Mice , SolutionsABSTRACT
Sea cucumbers were nutritional food and traditional Chinese medicine. In this study, fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus (fCS-Sc), a potential anticoagulant agent and immunological adjuvant, was investigated for its immune activation effects on RAW 264.7 macrophage for the first time. The results indicated that fCS-Sc could significantly promote the proliferation, the pinocytic activity of RAW 264.7 cells, and the production of NO, TNF-α, IL-1ß, and IL-6. The fluorescence labeling assay indicated that fCS-Sc could bind to the macrophage. Moreover, the specific pattern recognition receptor inhibition assays showed that toll-like receptor 4 (TLR4) and TLR2 were involved in the recognition of fCS-Sc. Western blot assays indicated that fCS-Sc could induce degradation of cytoplasm IκB-α, and promotion of NF-κB p65 subunit translocation to nucleus, leading to a functional improvement of macrophage through NF-κB pathway. The results suggested that fCS-Sc might served as a promising candidate of immunomodulator.
Subject(s)
Chondroitin Sulfates/pharmacology , Macrophages/drug effects , Macrophages/immunology , Stichopus/chemistry , Animals , Cell Proliferation/drug effects , Chondroitin Sulfates/isolation & purification , Cytokines/immunology , Immunomodulation , Mice , NF-kappa B p50 Subunit/immunology , Pinocytosis/drug effects , RAW 264.7 CellsABSTRACT
Fucosylated chondroitin sulfates (FCSs) PC and HH were isolated from the sea cucumbers Paracaudina chilensis and Holothuria hilla, respectively. The purification of the polysaccharides was carried out by anion-exchange chromatography on a DEAE-Sephacel column. The structural characterization of the polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of nondestructive NMR spectroscopic methods. Both polysaccharides were shown to contain a chondroitin core [â3)-ß-d-GalNAc (N-acethyl galactosamine)-(1â4)-ß-d-GlcA (glucuronic acid)-(1â]n, bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in their pattern of sulfation: PC contained Fuc2S4S and Fuc4S in a ratio of 2:1, whereas HH included Fuc2S4S, Fuc3S4S, and Fuc4S in a ratio of 1.5:1:1. Moreover, some GalNAc residues in HH were found to contain an unusual disaccharide branch Fuc4S-(1â2)-Fuc3S4S-(1â at O-6. Sulfated GalNAc4S6S and GalNAc4S units were found in a ratio of 3:2 in PC and 2:1 in HH. Both polysaccharides demonstrated significant anticoagulant activity in a clotting time assay, which is connected with the ability of these FCSs to potentiate the inhibition of thrombin and factor Xa in the presence of anti-thrombin III (ATIII) and with the direct inhibition of thrombin in the absence of any cofactors.
Subject(s)
Anticoagulants/pharmacology , Blood Coagulation/drug effects , Chondroitin Sulfates/pharmacology , Holothuria/metabolism , Animals , Anticoagulants/isolation & purification , Antithrombin III/metabolism , Antithrombins/isolation & purification , Antithrombins/pharmacology , Chondroitin Sulfates/isolation & purification , Factor Xa/metabolism , Factor Xa Inhibitors/isolation & purification , Factor Xa Inhibitors/pharmacology , Molecular Structure , Structure-Activity Relationship , Thrombin/antagonists & inhibitors , Thrombin/metabolismABSTRACT
A capillary electrophoresis (CE) method combined with online and offline enrichment for improving the detection sensitivity of chondroitin sulfate (CS) is established. The online enrichment method is based on the field-amplified sample stacking and large volume electrokinetic injection, and offline enrichment is based on the association between cetyltrimethylammonium chloride and CS. Experimental parameters affecting CE method such as the type and pH of background electrolyte, the injection mode and time and the steps of offline enrichment were optimized. Under optimum conditions, the calibration plot between CS concentration and peak area was linear in the range of 1 ~ 100 µg/mL. The enrichment factor was 130 times and the limit of detection was 50 ng/mL. The average recovery was 103.5% and the relative standard deviation of peak area was <2.0%. The method was successfully applied to the quantitative analysis of CS in drugs.
Subject(s)
Chondroitin Sulfates/analysis , Electrophoresis, Capillary/methods , Cetrimonium/chemistry , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/isolation & purification , Limit of Detection , Linear Models , Reproducibility of Results , Sodium Chloride/chemistryABSTRACT
The present work deals with the extraction and purification of chondroitin sulfate/dermatan sulfate from skin (CSG) and bone (CBG) of corb (Sciaena umbra). Electrophoresis of these polymers in barium acetate buffer on cellulose acetate revealed two fractions similar to dermatan sulfate and chondroitin sulfate. The in vivo anticoagulant activity of both chondroitin sulfate/dermatan sulfate (CS/DS) were evaluated, at 25 and 75 mg kg-1 of body weight (b.w), using activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests. Results showed that aPTT of CSG and CBG at 75 mg kg-1 of b.w were prolonged by 1.59 and 1.48-fold respectively, compared with the control. Further, toxicity studies on liver performed by the catalytic activity of transaminases in plasma, oxidative stress markers and hepatic morphological changes demonstrated that CSG and CBG at both doses are not toxics. In summary, the higher activity and lower toxicity of both CS/DS, especially at 25 mg kg-1 of b.w, recommended these compounds as a better drug candidate.
Subject(s)
Anticoagulants/pharmacology , Chondroitin Sulfates/pharmacology , Dermatan Sulfate/pharmacology , Fishes/metabolism , Animals , Anticoagulants/isolation & purification , Anticoagulants/toxicity , Blood Coagulation Tests , Bone and Bones/chemistry , Calorimetry, Differential Scanning , Chondroitin Sulfates/isolation & purification , Chondroitin Sulfates/toxicity , Dermatan Sulfate/isolation & purification , Dermatan Sulfate/toxicity , Drug Evaluation, Preclinical , Electrophoresis, Cellulose Acetate , Female , Glycosaminoglycans/isolation & purification , Liver/drug effects , Liver Function Tests , Microscopy, Electron, Scanning , Oxidative Stress/drug effects , Rats, Wistar , Skin/chemistry , X-Ray DiffractionABSTRACT
A fucosylated glycosaminoglycan (AmFG) was extracted from the sea cucumber Acaudina molpadioides. And a series of oligosaccharides were purified from the size-homogeneous fractions, which were prepared from the ß-eliminative depolymerized AmFG. According to "bottom-up" strategy, the precise structure of AmFG was elucidated by analyzing the structures of these purified oligosaccharides, combining with NMR analysis of its free-radical depolymerized product. It contained a CS-E-like backbone, and each GlcUA was branched with a mono- or di-sulfated fucose (Fuc) at O-3. Intriguingly, besides two types of monosaccharide branches, Fuc2S4S (60 %) and Fuc4S (25 %), that were common in FG, AmFG also contained an unusual disaccharide branch GalNAc-α1,2-Fuc3S4S (15 %); this is the first report of such a structure in a glycosaminoglycan. Biological assays indicated that native AmFG and its oligosaccharides had potent anticoagulant and intrinsic tenase (iXase) inhibitory activities in a chain length-dependent manner. For these oligosaccharides, octasaccharide was the minimum structural fragment for potent anti-iXase activity, and the disaccharide branch might enhance this activity.
Subject(s)
Anticoagulants/chemistry , Anticoagulants/pharmacology , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/pharmacology , Disaccharides/chemistry , Fucose/chemistry , Neoplasm Proteins/antagonists & inhibitors , Sea Cucumbers/chemistry , Animals , Chondroitin Sulfates/isolation & purification , Cysteine Endopeptidases , Molecular Structure , Monosaccharides/chemistry , Structure-Activity Relationship , Sulfates/chemistryABSTRACT
Thrombin triggers cellular responses that are crucial for development and progression of cancer, such as proliferation, migration, oncogene expression and angiogenesis. Thus, biomolecules capable of inhibiting this protease have become targets in cancer research. The present work describes the in vitro antitumor properties of a chondroitin sulfate with anti-thrombin activity, isolated from the Litopenaeus vannamei shrimp (sCS). Although the compound was unable to induce cytotoxicity or cell death and/or cell cycle changes after 24 h incubation, it showed a long-term antiproliferative effect, reducing the tumor colony formation of melanoma cells by 75% at 100 µg/mL concentration and inhibiting the anchorage-independent colony formation. sCS reduced 66% of melanoma cell migration in the wound healing assay and 70% in the transwell assay. The compound also decreased melanin and TNF-α content of melanoma cells by 52% and 75% respectively. Anti-angiogenic experiments showed that sCS promoted 100% reduction of tubular structure formation at 100 µg/mL. These results are in accordance with the sCS-mediated in vitro expression of genes related to melanoma development (Cx-43, MAPK, RhoA, PAFR, NFKB1 and VEGFA). These findings bring a new insight to CS molecules in cancer biology that can contribute to ongoing studies for new approaches in designing anti-tumor therapy.
Subject(s)
Angiogenesis Inhibitors , Antineoplastic Agents , Chondroitin Sulfates , Melanoma, Experimental/drug therapy , Penaeidae/chemistry , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/isolation & purification , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/isolation & purification , Chondroitin Sulfates/pharmacology , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , RabbitsABSTRACT
Hyaluronic acid (HA) and chondroitin sulfate (CS) are valuable bioactive polysaccharides that have been highly used in biomedical and pharmaceutical applications. Extensive research was done to ensure their efficient extraction from marine and terrestrial by-products at a high yield and purity, using specific techniques to isolate and purify them. In general, the cartilage is the most common source for CS, while the vitreous humor is main used source of HA. The developed methods were based in general on tissue hydrolysis, removal of proteins and purification of the target biopolymers. They differ in the extraction conditions, enzymes and/or solvents used and the purification technique. This leads to specific purity, molecular weight and sulfation pattern of the isolated HA and CS. This review focuses on the analysis and comparison of different extraction and purification methods developed to isolate these valuable biopolymers from marine and terrestrial animal by-products.
Subject(s)
Biological Products/isolation & purification , Cartilage/chemistry , Chondroitin Sulfates/isolation & purification , Hyaluronic Acid/isolation & purification , Animals , Aquatic Organisms/chemistryABSTRACT
This study was designed to isolate (isolation yield of 36.51 %) and characterise chondroitin sulphate (CS) from skate (Raja porosa) cartilage. Gel permeation chromatography demonstrated that the Raja porosa chondroitin sulphate (RPCS) obtained was a relatively uniform polysaccharide with a molecular weight of 40,752 Da and a purity of 94.0 %. Fourier-transform infrared, nuclear magnetic resonance spectroscopy and strong anion-exchange high-performance liquid chromatography indicated that the content of the predominant GlcA-GalNAc6S unit in RPCS was 65.84 %, which was higher than that in shark CS (53.93 %). Furthermore, RPCS displayed more effective free radical scavenging effects than did shark CS, indicating the potential of RPCS to promote oxidative stress resistance and to act as an antioxidant agent. Skate cartilage could be exploited as a sufficient and sustainable source for the preparation of CS with higher 6S-sulphation, which in turn could be scaled up for use in the pharmaceutical industry.
Subject(s)
Antioxidants/chemistry , Cartilage/chemistry , Chondroitin Sulfates , Skates, Fish , Animals , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/isolation & purificationABSTRACT
Sea cucumber body wall contains several naturally occurring bioactive components that possess health-promoting properties. Isostichopus badionotus from Yucatan, Mexico is heavily fished, but little is known about its bioactive constituents. We previously established that I. badionotus meal had potent anti-inflammatory properties in vivo. We have now screened some of its constituents for anti-inflammatory activity in vitro. Glycosaminoglycan and soluble protein preparations reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory responses in HaCaT cells while an ethanol extract had a limited effect. The primary glycosaminoglycan (fucosylated chondroitin sulfate; FCS) was purified and tested for anti-inflammatory activity in vivo. FCS modulated the expression of critical genes, including NF-ĸB, TNFα, iNOS, and COX-2, and attenuated inflammation and tissue damage caused by TPA in a mouse ear inflammation model. It also mitigated colonic colitis caused in mice by dextran sodium sulfate. FCS from I. badionotus of the Yucatan Peninsula thus had strong anti-inflammatory properties in vivo.
Subject(s)
Anti-Inflammatory Agents , Chondroitin Sulfates/isolation & purification , Chondroitin Sulfates/pharmacology , Glycosaminoglycans/isolation & purification , Glycosaminoglycans/pharmacology , Otitis/drug therapy , Sea Cucumbers/chemistry , Tissue Extracts/isolation & purification , Tissue Extracts/pharmacology , Animals , Chondroitin Sulfates/therapeutic use , Colitis/chemically induced , Colitis/drug therapy , Dextran Sulfate/adverse effects , Disease Models, Animal , HaCaT Cells , Humans , In Vitro Techniques , Mexico , Mice , Otitis/chemically induced , Tetradecanoylphorbol Acetate/adverse effectsABSTRACT
Chondroitin sulfate (CS), mainly present in the cartilage and bone of animals, is known as a potential food-derived bioactive that has several biological functions, such as anti-arthritic and anti-inflammatory activity. Sturgeon (Acipenser), an important fishery resource in China, contains an abundance of CS in their cartilage. In our previous study, we have extracted and purified CS from sturgeon cartilage. Herein, we further investigate the health benefits of sturgeon-derived chondroitin sulfate (SCS), especially for colorectal cancer treatment. The in vitro study indicated that SCS could inhibit the proliferation of the human colon cancer cell line HCT-116 in a dose-dependent manner, which was associated with cell cycle arrest. In addition, SCS also led to extensive cellular apoptosis in colon cancer cell HCT-116 cells. Meanwhile, an in vivo study showed that SCS treatment significantly inhibited the tumor development of xenograft HCT-116 in mice via proliferation suppression and apoptosis induction. Further, a mechanistic study demonstrated that the apoptosis induction was mainly due to the activation of the Bcl-2 family-associated mitochondrial pathway. Overall, our results provided a basis for SCS as a promising agent against colon cancer.
Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Chondroitin Sulfates/pharmacology , Colonic Neoplasms/pathology , Fishes/metabolism , Animals , Apoptosis/genetics , Cartilage/metabolism , China , Chondroitin Sulfates/isolation & purification , Chondroitin Sulfates/therapeutic use , Colonic Neoplasms/drug therapy , Dose-Response Relationship, Drug , HCT116 Cells , Humans , Male , Mice, Inbred BALB C , Mice, Nude , Mitochondria/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/geneticsABSTRACT
Osteoarthritis is the most prevalent rheumatic disease. During disease progression, differences have been described in the prevalence of chondroitin sulfate (CS) isomers. Marine derived-CS present a higher proportion of the 6S isomer, offering therapeutic potential. Accordingly, we evaluated the effect of exogenous supplementation of CS, derived from the small spotted catshark (Scyliorhinus canicula), blue shark (Prionace glauca), thornback skate (Raja clavata) and bovine CS (reference), on the proliferation of osteochondral cell lines (MG-63 and T/C-28a2) and the chondrogenic differentiation of mesenchymal stromal cells (MSCs). MG-G3 proliferation was comparable between R. clavata (CS-6 intermediate ratio) and bovine CS (CS-4 enrichment), for concentrations below 0.5 mg/mL, defined as a toxicity threshold. T/C-28a2 proliferation was significantly improved by intermediate ratios of CS-6 and -4 isomers (S. canicula and R. clavata). A dose-dependent response was observed for S. canicula (200 µg/mL vs 50 and 10 µg/mL) and bovine CS (200 and 100 µg/mL vs 10 µg/mL). CS sulfation patterns discretely affected MSCs chondrogenesis; even though S. canicula and R. clavata CS up-regulated chondrogenic markers expression (aggrecan and collagen type II) these were not statistically significant. We demonstrate that intermediate values of CS-4 and -6 isomers improve cell proliferation and offer potential for chondrogenic promotion, although more studies are needed to elucidate its mechanism of action.
Subject(s)
Cell Proliferation/drug effects , Chondrocytes/drug effects , Chondrogenesis/drug effects , Chondroitin Sulfates/pharmacology , Aged , Aged, 80 and over , Animals , Cattle , Cell Differentiation/drug effects , Cell Line , Chondrocytes/metabolism , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/isolation & purification , Female , Humans , Isomerism , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Osteoblasts/drug effects , Osteoblasts/metabolism , Sharks , Skates, FishABSTRACT
Rationale: Placental-like chondroitin sulfate A (pl-CSA) is known to be exclusively synthesized in multiple cancer tissues and associated with disease severity. Here, we aimed to assess whether pl-CSA is released into bio-fluids and can serve as a cancer biomarker. Methods: A novel ELISA was developed to analyse pl-CSA content in bio-fluids using pl-CSA binding protein and an anti-pl-CSA antibody. Immunohistochemical staining of tissue chips was used as the gold standard control. Results: The developed ELISA method was specific and sensitive (1.22 µg/ml). The pl-CSA content was significantly higher in lysates and supernatants of cancer cell lines than in those of normal cell lines, in plasma from mouse cancer models than in that from control mice, and in plasma from patients with oesophageal, cervical, ovarian, or lung cancer than in that from healthy controls. Similar to the tissue chip analysis, which showed a significant difference in pl-CSA positivity between cancer tissues and normal adjacent tissues, the plasma pl-CSA analysis had 100% sensitivity and specificity for differentiating oesophageal and lung cancer patients from healthy controls. Importantly, in oesophageal and lung cancer patients, the pl-CSA content was significantly higher in late-stage disease than in early-stage disease, and it dramatically decreased after surgical resection of the tumour. Conclusion: These data indicate a direct link between plasma pl-CSA content and tumour presence, indicating that plasma pl-CSA may be a non-invasive biomarker with clinical applicability for the screening and surveillance of patients with multiple types of solid tumours.
Subject(s)
Chondroitin Sulfates/isolation & purification , Enzyme-Linked Immunosorbent Assay , Neoplasms/genetics , Animals , Antibodies, Anti-Idiotypic/genetics , Antibodies, Anti-Idiotypic/immunology , Chondroitin Sulfates/genetics , Chondroitin Sulfates/immunology , Female , Humans , Male , Mice , Middle Aged , Neoplasms/immunology , Neoplasms/pathology , Placenta/metabolism , Pregnancy , Protein Binding/immunologyABSTRACT
In the present study, we describe the development of a fast, 2-step salt gradient for analysis of chondroitin sulfate disaccharides. Using salt gradients, which is somewhat unusual in HILIC-based separations, provides relatively fast chromatography with excellent sensitivity (15 min cycle time, 10-20 fmol/µL detection, 30-50 fmol/µL quantitation limit), and good linearity. The efficiency of the new method is demonstrated by measuring human tissue slices of healthy, cirrhotic, and cancerous liver samples. Preliminary results show major differences among the quantity and sulfation pattern of the various sample types.
Subject(s)
Chondroitin Sulfates/isolation & purification , Chromatography, High Pressure Liquid/methods , Disaccharides/isolation & purification , Sodium Chloride/chemistry , Chondroitin Sulfates/chemistry , Disaccharides/chemistry , Humans , Liver/chemistryABSTRACT
Fucosylated chondroitin sulfate (FCS) HeSp was isolated from the Patagonian sea cucumber Hemioedema spectabilis. Ion-exchange chromatography was applied for purification of the FCS from the crude extract of sulfated polysaccharides. Analysis of monosaccharide and sulfate content of HeSp revealed the molar ratio of GlcA:GalNAc:Fuc:SO3Na as 1.15:1:1.1:3.9. Molecular weight of HeSp (44.1 kDa) was determined by GPC. According to the NMR spectral data, the main fragment of HeSp was the trisaccharide â3)-ß-d-GalNAc-(1â4)-ß-d-GlcA(3-O-α-l-Fuc)-(1â, where GalNAc units were sulfated either at O-4, at O-6 or both at O-4 and O-6. The fucosyl branches attached to O-3 of GlcA showed also different patterns of sulfation: Fucp2S4S, Fucp4S and Fucp3S4S were found in a ratio of 3.8:1.5:1. Besides, small amounts of the disaccharide fragment â3)-ß-d-GalNAc-(1â4)-ß-d-GlcA3S-(1â were observed in a structure of HeSp. The polysaccharide was found to block cancer cells adhesion to platelet-coated surface and to inhibit tubulogenesis, thus demonstrating the potential antitumor activity.
Subject(s)
Antineoplastic Agents/pharmacology , Chondroitin Sulfates/pharmacology , Polysaccharides/pharmacology , Sea Cucumbers/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Cell Adhesion/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/isolation & purification , Drug Screening Assays, Antitumor , HeLa Cells , Humans , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Species Specificity , Tumor Cells, CulturedABSTRACT
Glycans analysis is challenging due to their immense structural diversity. Isotachophoresis was investigated as separation method for the purification of isobaric sulfated disaccharides prior to their characterization by Mass Spectrometry (MS) and tunable IR multiple photon dissociation (IRMPD). This proof of feasibility study was applied to the separation and characterization of chondroitin sulfate (CS) disaccharides. ITP separation conditions were optimized. Separation starts using a 2.5 mM chloride ions and 10 mM glycine at pH 3.2 solution as leading electrolyte and a terminating electrolyte composed of formic acid 2.5 mM and glycine 10 mM at pH 3.5. The CS disaccharides sample were prepared in the terminating electrolyte. The length of injection was also investigated in order to create longer plateau-like bands of pure solutes. This strategy was helpful for collecting fraction at such microseparation scale. Indeed, capillary ITP affords the injection of few tens of nanoliter of sample. Fractionation of the CS disaccharides mixture in isolated ITP bands and collection of solutes were successfully done using a HPC coated fused silica capillary of 1m-length and 75 µm of internal diameter. Collected fractions in a final of volume 10 µL were analyzed by CZE, tandem MS and IRMPD spectroscopy. The purity of each fraction is higher than 90% and is well-adapted to IRMPD characterization.