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1.
J Microbiol Biotechnol ; 30(1): 31-37, 2020 Jan 28.
Article in English | MEDLINE | ID: mdl-31752054

ABSTRACT

We previously identified a new heparinase inhibitor fungal metabolite, named CRM646-A, which showed inhibition of heparinase and telomerase activities in an in vitro enzyme assay and antimetastatic activity in a cell-based assay. In this study, we elucidated the mechanism by which CRM646-A rapidly induced nucleus condensation, plasma membrane disruption and morphological changes by increasing intracellular Ca2+ levels. Furthermore, PD98059, a mitogen-activated protein kinase (MEK) inhibitor, inhibited CRM646-A-induced nucleus condensation through ERK1/2 activation in rat 3Y1 fibroblast cells. We identified CRM646-A as a Ca2+ ionophore-like agent with a distinctly different chemical structure from that of previously reported Ca2+ ionophores. These results indicate that CRM646-A has the potential to be used as a new and effective antimetastatic drug.


Subject(s)
Calcium/metabolism , Cell Nucleus/metabolism , Fibroblasts/drug effects , Hydroxybenzoates/pharmacology , Animals , Cell Line , Enzyme Activation , Enzyme Inhibitors/pharmacology , Fibroblasts/metabolism , Fungi/chemistry , Heparin Lyase/antagonists & inhibitors , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Rats , Signal Transduction
3.
Chem Commun (Camb) ; 50(31): 4056-8, 2014 Apr 21.
Article in English | MEDLINE | ID: mdl-24463939

ABSTRACT

Metalloglycomics - the effects of defined coordination compounds on oligosaccharides and their structure and function - opens new areas for bioinorganic chemistry and expands its systematic study to the third major class of biomolecules after DNA/RNA and proteins.


Subject(s)
Coordination Complexes/chemistry , Heparin Lyase/chemistry , Heparin/analogs & derivatives , Platinum/chemistry , Polysaccharides/chemistry , Proteoglycans/chemistry , Fondaparinux , Heparin/chemistry , Heparin Lyase/antagonists & inhibitors
4.
Anal Chem ; 83(20): 7815-22, 2011 Oct 15.
Article in English | MEDLINE | ID: mdl-21863856

ABSTRACT

Heparin and the low molecular weight heparins are extensively used as medicinal products to prevent and treat the formation of venous and arterial thrombi. In early 2008, administration of some heparin lots was associated with the advent of severe adverse effects, indicative of an anaphylactoid-like response. Application of orthogonal analytical tools enabled detection and identification of the contaminant as oversulfated chondroitin sulfate (OSCS) was reported in our earlier report. Herein, we investigate whether enzymatic depolymerization using the bacterially derived heparinases, given the structural understanding of their substrate specificity, can be used to identify the presence of OSCS in heparin. We also extend this analysis to examine the effect of other persulfonated glycosaminoglycans (GAGs) on the action of the heparinases. We find that all persulfonated GAGs examined were effective inhibitors of heparinase I, with IC(50) values ranging from approximately 0.5-2 µg/mL. Finally, using this biochemical understanding, we develop a rapid, simple assay to assess the purity of heparin using heparinase digestion followed by size-exclusion HPLC analysis to identify and quantify digestion products. In the context of the assay, we demonstrate that less than 0.1% (w/w) of OSCS (and other persulfonated polysaccharides) can routinely be detected in heparin.


Subject(s)
Chromatography, Gel , Chromatography, High Pressure Liquid , Glycosaminoglycans/chemistry , Heparin Lyase/antagonists & inhibitors , Heparin/analysis , Area Under Curve , Chondroitin Sulfates/chemistry , Drug Contamination/prevention & control , Heparin/metabolism , Heparin Lyase/metabolism , Kinetics , Protein Structure, Tertiary , Sulfonic Acids/chemistry
5.
Exp Dermatol ; 17(12): 1017-23, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18557927

ABSTRACT

Heparanase is a heparan sulphate endo-glycosidase which was previously detected in the outer root sheath of murine hair follicles. Heparanase overexpression was reported to improve mouse hair (re)growth. In this study, we investigated its involvement in human hair biology. Immunofluorescence detection was used to explore heparanase distribution in both anagen and catagen hair follicles. Heparanase functionality was assessed in in vitro cultured hair follicles, in the presence of a heparanase activity inhibitor. Our results showed that heparanase expression was (i) primarily located in the inner root sheath (IRS) of human hair follicle, and there (ii) restricted to anagen phase. Furthermore, inhibition of heparanase in in vitro cultured hair follicles induced a catagen-like process. Hair shaft retreat upward was accompanied by a decrease in Ki67-positive cells, the formation of an epithelial strand as evidenced by K14 keratin expression, and the loss of IRS as assessed by transglutaminase 1 and desmoglein labelling. IRS distribution of heparanase and the induction of catagen-like involution of hair follicles when a potent heparanase inhibitor is added suggest that heparanase is a key actor of IRS differentiation and hair homeostasis.


Subject(s)
Cell Differentiation/physiology , Hair Follicle/physiology , Heparin Lyase/metabolism , Homeostasis/physiology , Aged , Desmoglein 1/metabolism , Enzyme Inhibitors/pharmacology , Epidermis/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Fibronectins/metabolism , Hair Follicle/cytology , Hair Follicle/drug effects , Heparin Lyase/antagonists & inhibitors , Heparitin Sulfate/metabolism , Humans , Immunohistochemistry , Keratin-14/metabolism , Keratins, Hair-Specific/metabolism , Keratins, Type I/metabolism , Ki-67 Antigen/metabolism , Middle Aged , Tissue Culture Techniques , Transglutaminases/metabolism
6.
Anticancer Agents Med Chem ; 8(1): 64-76, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18220506

ABSTRACT

Malignant tumor cells invade normal tissues in the vicinity of cancer through devastating the extracelluar matrix and blood vessel wall of the tissues. An important step in this process is degradation of heparan sulfate proteoglycan, a carbohydrate-protein complex. Heparan sulfate proteoglycan is a major component of the extracellular matrix, and is essential for the self-assembly, insolubility and barrier properties of basement membranes. Heparanase is an endoglucuronidase that cleaves heparan sulfate and expression level of this enzyme correlates with metastatic potential of tumor cells. Treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin, a widely used anticoagulant, is structurally related to heparan sulfate and a natural substrate of heparanase. Long-term treatment of cancer patients having venous thromboembolism with low molecular weight heparin showed improved survival rate. Understanding the functional roles and the corresponding molecular mechanisms of heparin, heparan sulfate and heparanase in cancer development may pave the way for exploring remedies against tumor metastasis.


Subject(s)
Heparin Lyase/physiology , Heparin/physiology , Heparitin Sulfate/physiology , Neoplasms , Neovascularization, Pathologic , Animals , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Heparin/biosynthesis , Heparin Lyase/antagonists & inhibitors , Heparin Lyase/biosynthesis , Heparin, Low-Molecular-Weight/pharmacology , Heparin, Low-Molecular-Weight/therapeutic use , Heparitin Sulfate/biosynthesis , Humans , Neoplasm Metastasis , Neoplasms/enzymology , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/prevention & control , Neovascularization, Pathologic/enzymology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Neovascularization, Pathologic/prevention & control , Substrate Specificity , Thrombosis/blood , Thrombosis/prevention & control
7.
Semin Thromb Hemost ; 33(5): 557-68, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17629854

ABSTRACT

The heparan sulfate (HS) mimetic PI-88 is a promising inhibitor of tumor growth and metastasis expected to commence phase III clinical evaluation in 2007 as an adjuvant therapy for postresection hepatocellular carcinoma. Its anticancer properties are attributed to inhibition of angiogenesis via antagonism of the interactions of angiogenic growth factors and their receptors with HS. It is also a potent inhibitor of heparanase, an enzyme that plays a key role in both metastasis and angiogenesis. A series of PI-88 analogs have been prepared with enhanced chemical and biological properties. The new compounds consist of single, defined oligosaccharides with specific modifications designed to improve their pharmacokinetic properties. These analogs all inhibit heparanase and bind to the angiogenic fibroblast growth factor 1 (FGF-1), FGF-2, and vascular endothelial growth factor with similar affinity to PI-88. However, compared with PI-88, some of the newly designed compounds are more potent inhibitors of growth factor-induced endothelial cell proliferation and of endothelial tube formation on Matrigel. Representative compounds were also tested for antiangiogenic activity in vivo and were found to reduce significantly blood vessel formation. Moreover, the pharmacokinetic profile of several analogs was also improved, as evidenced primarily by lower clearance in comparison with PI-88. The current data support the development of HS mimetics as potent antiangiogenic anticancer agents.


Subject(s)
Biomimetic Materials/therapeutic use , Carcinoma, Hepatocellular/therapy , Heparitin Sulfate/therapeutic use , Neovascularization, Pathologic/drug therapy , Oligosaccharides/therapeutic use , Animals , Biomimetic Materials/pharmacokinetics , Biomimetic Materials/pharmacology , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Proliferation/drug effects , Chemotherapy, Adjuvant , Clinical Trials, Phase III as Topic , Endothelial Cells/metabolism , Endothelial Cells/pathology , Fibroblast Growth Factor 1/antagonists & inhibitors , Fibroblast Growth Factor 1/metabolism , Fibroblast Growth Factor 2/antagonists & inhibitors , Fibroblast Growth Factor 2/metabolism , Heparin Lyase/antagonists & inhibitors , Heparin Lyase/metabolism , Heparitin Sulfate/pharmacokinetics , Humans , Neoplasm Metastasis , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Oligosaccharides/pharmacokinetics , Oligosaccharides/pharmacology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism
8.
Semin Thromb Hemost ; 33(5): 547-56, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17629853

ABSTRACT

Heparin-like polysaccharides possess the capacity to inhibit cancer cell proliferation, angiogenesis, heparanase-mediated cancer cell invasion, and cancer cell adhesion to vascular endothelia via adhesion receptors, such as selectins. The clinical applicability of the antitumor effect of such polysaccharides, however, is compromised by their anticoagulant activity. We have compared the potential of chemically O-sulfated and N,O-sulfated bacterial polysaccharide (capsular polysaccharide from E. COLI K5 [K5PS]) species to inhibit metastasis of mouse B16-BL6 melanoma cells and human MDA-MB-231 breast cancer cells in two in vivo models. We demonstrate that in both settings, O-sulfated K5PS was a potent inhibitor of metastasis. Reducing the molecular weight of the polysaccharide, however, resulted in lower antimetastatic capacity. Furthermore, we show that O-sulfated K5PS efficiently inhibited the invasion of B16-BL6 cells through Matrigel and also inhibited the in vitro activity of heparanase. Moreover, treatment with O-sulfated K5PS lowered the ability of B16-BL6 cells to adhere to endothelial cells, intercellular adhesion molecule-1, and P-selectin, but not to E-selectin. Importantly, O-sulfated K5PSs were largely devoid of anticoagulant activity. These findings indicate that O-sulfated K5PS polysaccharide should be considered as a potential antimetastatic agent.


Subject(s)
Breast Neoplasms/enzymology , Melanoma/enzymology , Neoplasm Metastasis/prevention & control , Polysaccharides, Bacterial/pharmacology , Animals , Bacterial Capsules , Bone Neoplasms/prevention & control , Bone Neoplasms/secondary , Breast Neoplasms/pathology , Cell Adhesion/drug effects , Cell Line , E-Selectin/metabolism , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Female , Heparin Lyase/antagonists & inhibitors , Heparin Lyase/metabolism , Humans , Intercellular Adhesion Molecule-1/metabolism , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Melanoma/pathology , Mice , Neoplasm Metastasis/pathology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , P-Selectin/metabolism , Polysaccharides, Bacterial/therapeutic use
9.
Life Sci ; 79(17): 1661-5, 2006 Sep 20.
Article in English | MEDLINE | ID: mdl-16806278

ABSTRACT

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Heparin Lyase/analysis , Neoplasm Metastasis/drug therapy , Plant Extracts/pharmacology , Animals , Biological Assay , Cell Line, Tumor , Colorimetry/methods , Drug Screening Assays, Antitumor , Enzyme Inhibitors/analysis , Female , Flavobacterium/enzymology , Heparin/analysis , Heparin/metabolism , Heparin Lyase/antagonists & inhibitors , Intestinal Mucosa , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Melanoma/drug therapy , Melanoma/pathology , Melanoma/secondary , Mice , Mice, Inbred C57BL , Morus/chemistry , Neoplasm Metastasis/pathology , Specific Pathogen-Free Organisms
10.
J Org Chem ; 70(22): 8884-9, 2005 Oct 28.
Article in English | MEDLINE | ID: mdl-16238322

ABSTRACT

[reaction: see text] CRM646-A (1) and -B (2), two fungal glucuronides with a dimeric 2,4-dihydroxy-6-alkylbenzoic acid (orcinol p-depside) aglycone showing significant heparinase and telomerase inhibition activities, were synthesized for the first time. The successful approach involved construction of the phenol glucuronidic linkage, via coupling of the orsellinate derivative 27 with glucuronate bromide 7, before assembly of the phenolic ester linkage in the depside aglycone. Attempts via direct glycosylation of the depside aglycone derivatives were not successful.


Subject(s)
Heparin Lyase/antagonists & inhibitors , Hydroxybenzoates/chemical synthesis , Acremonium/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Heparin Lyase/metabolism , Hydroxybenzoates/chemistry , Hydroxybenzoates/pharmacology , Molecular Structure , Oxidation-Reduction
11.
Cardiovasc Res ; 63(2): 226-35, 2004 Aug 01.
Article in English | MEDLINE | ID: mdl-15249180

ABSTRACT

OBJECTIVES: Platelets contain numerous substances regulating angiogenic response. However, the regulatory role of platelets in blood vessel development remains to be elucidated. We investigated the comprehensive effect of platelets as a cellular system on angiogenesis. METHODS: The following approaches were applied: (a) in vitro-aortic ring assay and chemotaxis assay; (b) in vivo-injection of platelet-containing matrigel plug subcutaneously into a mouse followed by immunohistochemical analysis of angiogenic response. RESULTS: Platelets stimulated formation of blood vessels in vitro in the rat aortic ring model via VEGF and bFGF, while blocking of platelet factor-4 promoted this effect. Addition of platelets to the matrigel followed by its subcutaneous injection into a mouse resulted in an intensive migration of fibroblasts into the matrigel as well as formation of blood capillaries de novo. This platelet effect was mediated through bFGF, VEGF, and heparanase. Furthermore, platelet releasate was found to induce endothelial cell chemotaxis. This effect was mediated by a concerted action of intraplatelet bFGF, PDGF, VEGF, and heparanase. CONCLUSION: Platelets affect different stages of the angiogenic response with a trend to a pro-angiogenic net effect despite the presence of angiogenesis inhibitors such as platelet factor 4. While a concomitant effect of bFGF and VEGF seemed to be essential for the entire process of vessel formation (aortic ring and matrigel models), PDGF and heparanase were effective only at the migration stage.


Subject(s)
Blood Platelets/physiology , Endothelium, Vascular/pathology , Neovascularization, Pathologic , Animals , Antibodies, Blocking/pharmacology , Aorta , Capillaries/pathology , Cell Movement , Collagen , Drug Combinations , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/metabolism , Fibroblast Growth Factor 2/immunology , Fibroblast Growth Factor 2/metabolism , Fibroblasts/pathology , Heparin Lyase/antagonists & inhibitors , Heparin Lyase/metabolism , Humans , In Vitro Techniques , Laminin , Male , Mice , Mice, Inbred Strains , Platelet Factor 4/immunology , Platelet Factor 4/metabolism , Platelet-Derived Growth Factor/immunology , Platelet-Derived Growth Factor/metabolism , Proteoglycans , Rats , Rats, Inbred Strains , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism
12.
J Allergy Clin Immunol ; 113(4): 703-9, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15100677

ABSTRACT

BACKGROUND: Heparanase and eosinophils are involved in several diseases, including inflammation, cancer, and angiogenesis. OBJECTIVE: We sought to determine whether eosinophils produce active heparanase. METHODS: Human peripheral blood eosinophils were isolated by immunoselection and tested for heparanase protein (immunocytochemistry, Western blot), mRNA (RT-PCR) and activity (Na(2)[(35)S]O(4)-labeled extracellular matrix degradation) before and after activation. Heparanase intracellular localization (confocal laser microscopy) and ability to bind to eosinophil major basic protein (MBP) were also evaluated (immunoprecipitation). A model of allergic peritonitis resulting in eosinophilia was induced in TNF knockout and wild-type mice for in vivo studies. RESULTS: Eosinophils synthesized heparanase mRNA and contained heparanase in the active (50-kd) and latent (65-kd) forms. Heparanase partially co-localized with and was bound to MBP. No heparanase enzymatic activity was detected in eosinophils resting or activated with various agonists, including GM-CSF/C5a. Eosinophil lysates and MBP inhibited recombinant heparanase activity in a concentration-dependent manner (100%, 2 x 10(-7) mol/L). Eosinophil peroxidase and eosinophil cationic protein, but not myelin basic protein or compound 48/80, partially inhibited heparanase activity. Poly-l-arginine at very high concentrations caused an almost complete inhibition. In allergic peritonitis, heparanase activity in the peritoneal fluid inversely correlated with eosinophil number. CONCLUSIONS: MBP is the first identified natural heparanase-inhibiting protein. Its presence in the eosinophil granules might indicate a protective function of these cells in diseases associated with inflammation and cancer progression.


Subject(s)
Blood Proteins/physiology , Heparin Lyase/antagonists & inhibitors , Ribonucleases/physiology , Animals , Cells, Cultured , Cytoplasmic Granules/metabolism , Eosinophil Granule Proteins , Eosinophils/metabolism , Eosinophils/pathology , Eosinophils/physiology , Heparin Lyase/genetics , Heparin Lyase/metabolism , Humans , Hypersensitivity/complications , Leukocyte Count , Mice , Peritonitis/etiology , Peritonitis/pathology , RNA, Messenger/metabolism , Tissue Distribution
13.
J Med Chem ; 43(13): 2591-600, 2000 Jun 29.
Article in English | MEDLINE | ID: mdl-10891119

ABSTRACT

Poly(N-acryl amino acids) bearing side groups with a lipophilic character or having charged functional groups (i.e. -NH(2), -COOH, -SH, -OH, and phenols) were synthesized from the radical polymerization of N-acryl amino acid monomers. Monomers were prepared from the reaction of acryloyl chloride and amino acid esters in dry solvents. Polymers of a broad molecular weight ranging from 3 000 to 60 000 Da were obtained. The polymers were optically active, and their structures were confirmed by (1)H NMR and IR spectra and elemental analysis. Hydroxyl-containing polymers were sulfated in high conversion yields by SO(3)/pyridine complex. The newly synthesized linear homopolyanions were tested for heparin-like activities: (i) inhibition of heparanase enzyme, (ii) release of basic fibroblast growth factor (bFGF) from the extracellular matrix (ECM), and (iii) inhibition of smooth muscle cell (SMC) proliferation. Polymers based on tyrosine and leucine were highly active in all three tests (microgram level). Polymers based on phenylalanine, tert-leucine, and proline were active as heparanase inhibitors and FGF release, and polymers of trans-hydroxyproline, glycine, and serine were active only as heparanase inhibitors. The polymer of cis-hydroxyproline was inactive. It was found that a net anionic charge (i.e. carboxylic acid) is essential for biological activity. Thus, methyl ester derivatives of the active polymers, zwitterionic amino acid dependent groups (lysine, histidine), and decarboxylated amino acids (tyramine, ethanolamine) were inactive. The above active polymers did not exhibit anticoagulation activity which is considered the main limitation of heparin and heparinomimetics for clinical use. These synthetic poly(N-acryl amino acids) may have potential use in the inhibition of heparanase-mediated degradation of basement membranes associated with tumor metastasis, inflammation, and autoimmunity.


Subject(s)
Acrylic Resins/chemical synthesis , Amino Acids/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Acrylic Resins/chemistry , Acrylic Resins/pharmacology , Amino Acids/chemistry , Amino Acids/pharmacology , Animals , Anions , Anticoagulants/chemical synthesis , Anticoagulants/chemistry , Anticoagulants/pharmacology , Cattle , Cell Division/drug effects , Cells, Cultured , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Extracellular Matrix/metabolism , Fibroblast Growth Factor 2/metabolism , Heparin/chemistry , Heparin/pharmacology , Heparin Lyase/antagonists & inhibitors , Humans , Magnetic Resonance Spectroscopy , Molecular Weight , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Spectrophotometry, Infrared , Structure-Activity Relationship
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