Publications by authors named "Demitrios Vynios"

30 Publications

  • Page 1 of 1

Lumican Inhibits In Vivo Melanoma Metastasis by Altering Matrix-Effectors and Invadopodia Markers.

Cells 2021 Apr 8;10(4). Epub 2021 Apr 8.

CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire, 51100 Reims, France.

It was reported that lumican inhibits the activity of metalloproteinase MMP-14 and melanoma cell migration in vitro and in vivo. Moreover, Snail triggers epithelial-to-mesenchymal transition and the metastatic potential of cancer cells. Therefore, the aim of this study was to examine the effect of lumican on Mock and Snail overexpressing melanoma B16F1 cells in vivo. Lung metastasis was analyzed after intravenous injections of Mock-B16F1 and Snail-B16F1 cells in Lum and Lum mice. At day 14, mice were sacrificed, and lungs were collected. The number of lung metastatic nodules was significantly higher in mice injected with Snail-B16F1 cells as compared to mice injected with Mock-B16F1 cells confirming the pro-metastatic effect of Snail. This effect was stronger in Lum mice as compared to Lum, suggesting that endogenous lumican of wild-type mice significantly inhibits metastasis to lungs. Scanning electron and confocal microscopy investigations demonstrated that lumican inhibits the development of elongated cancer cell phenotypes which are known to develop invadopodia releasing MMPs. Moreover, lumican was shown to affect the expression of cyclin D1, cortactin, vinculin, hyaluronan synthase 2, heparanase, MMP-14 and the phosphorylation of FAK, AKT, p130 Cas and GSK3α/β. Altogether, these data demonstrated that lumican significantly inhibits lung metastasis in vivo, as well as cell invasion in vitro, suggesting that a lumican-based strategy targeting Snail-induced metastasis could be useful for melanoma treatment.
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http://dx.doi.org/10.3390/cells10040841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068222PMC
April 2021

A guide to the composition and functions of the extracellular matrix.

FEBS J 2021 Feb 19. Epub 2021 Feb 19.

Department of Biomedical Sciences, University of Padova, Italy.

Extracellular matrix (ECM) is a dynamic 3-dimensional network of macromolecules that provides structural support for the cells and tissues. Accumulated knowledge clearly demonstrated over the last decade that ECM plays key regulatory roles since it orchestrates cell signaling, functions, properties and morphology. Extracellularly secreted as well as cell-bound factors are among the major members of the ECM family. Proteins/glycoproteins, such as collagens, elastin, laminins and tenascins, proteoglycans and glycosaminoglycans, hyaluronan, and their cell receptors such as CD44 and integrins, responsible for cell adhesion, comprise a well-organized functional network with significant roles in health and disease. On the other hand, enzymes such as matrix metalloproteinases and specific glycosidases including heparanase and hyaluronidases contribute to matrix remodeling and affect human health. Several cell processes and functions, among them cell proliferation and survival, migration, differentiation, autophagy, angiogenesis, and immunity regulation are affected by certain matrix components. Structural alterations have been also well associated with disease progression. This guide on the composition and functions of the ECM gives a broad overview of the matrisome, the major ECM macromolecules, and their interaction networks within the ECM and with the cell surface, summarizes their main structural features and their roles in tissue organization and cell functions, and emphasizes the importance of specific ECM constituents in disease development and progression as well as the advances in molecular targeting of ECM to design new therapeutic strategies.
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http://dx.doi.org/10.1111/febs.15776DOI Listing
February 2021

Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling.

FEBS J 2020 11 31;287(22):4862-4880. Epub 2020 Mar 31.

Laboratoire de Biochimie Médicale et Biologie Moléculaire, Université de Reims Champagne-Ardenne, Reims, France.

The small leucine-rich proteoglycan lumican regulates estrogen receptors (ERs)-associated functional properties of breast cancer cells, expression of matrix macromolecules, and epithelial-to-mesenchymal transition. However, it is not known whether the ER-dependent lumican effects on breast cancer cells are related to the expression of integrins and their intracellular signaling pathways. Here, we analyzed the effects of lumican in three breast cancer cell lines: the highly metastatic ERβ-positive MDA-MB-231, cells with the respective ERβ-suppressed (shERβMDA-MB-231), and lowly invasive ERα-positive MCF-7/c breast cancer cells. Scanning electron microscopy, confocal microscopy, real-time PCR, western blot, and cell adhesion assays were performed. Lumican effects on breast cancer cell morphology were also investigated in 3-dimensional collagen cultures. Lumican treatment induced cell-cell contacts and cell grouping and inhibited microvesicles and microvilli formation. The expression of the cell surface adhesion receptor CD44, its isoform and variants, hyaluronan (HA), and HA synthases was also investigated. Lumican inhibited the expression of CD44 and HA synthases, and its effect on cell adhesion revealed a major role of α1, α2, α3, αVβ3, and αVβ5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of α2 and β1 integrin subunits both in MDA-MB-231 and in shERβMDA-MB-231 as compared to MCF-7/c cells. Downstream signaling pathways for integrins, such as FAK, ERK 1/2 MAPK 42/44, and Akt, were found to be downregulated by lumican. Our data shed light to the molecular mechanisms responsible for the anticancer activity of lumican in invasive breast cancer.
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http://dx.doi.org/10.1111/febs.15289DOI Listing
November 2020

Epithelial-to-mesenchymal transition and invadopodia markers in breast cancer: Lumican a key regulator.

Semin Cancer Biol 2020 05 8;62:125-133. Epub 2019 Aug 8.

CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire, Reims, France; Université de Reims Champagne Ardenne, Laboratoire de Biochimie Médicale et Biologie Moléculaire, Reims, France. Electronic address:

A great hallmark of breast cancer is the absence or presence of estrogen receptors ERα and ERβ, with a dominant role in cell proliferation, differentiation and cancer progression. Both receptors are related with Epithelial-to-Mesenchymal Transition (EMT) since there is a relation between ERs and extracellular matrix (ECM) macromolecules expression, and therefore, cell-cell and cell-ECM interactions. The endocrine resistance of ERα endows epithelial cells with increased aggressiveness and induces cell proliferation, resulting into a mesenchymal phenotype and an EMT status. ERα signaling may affect the transcriptional factors which govern EMT. Knockdown or silencing of ERα and ERβ in MCF-7 and MDA-MB-231 breast cancer cells respectively, provoked pivotal changes in phenotype, cellular functions, mRNA and protein levels of EMT markers, and consequently the EMT status. Mesenchymal cells owe their migratory and invasive properties to invadopodia, while in epithelial cells, lamellipodia and filopodia are mostly observed. Invadopodia, are actin-rich protrusions of plasma membrane, promoting proteolytic degradation of ECM and tumor invasion. Cortactin and MMP-14 govern the formation and principal functions of invadopodia. In vitro experiments proved that lumican inhibits cortactin and MMP-14 expression, alters the formation of lamellipodia and transforms mesenchymal cells into epithelial-like. Conclusively, lumican may inhibit or even reverse the several metastatic features that EMT endows in breast cancer cells. Therefore, a lumican-based anti-cancer therapy which will pharmacologically target and inhibit EMT might be interesting to be developed.
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http://dx.doi.org/10.1016/j.semcancer.2019.08.003DOI Listing
May 2020

A guide to hyaluronan and related enzymes in breast cancer: biological significance and diagnostic value.

FEBS J 2019 08 13;286(15):3057-3074. Epub 2019 May 13.

Biochemistry, Biochemical Analysis & Matrix Pathobiochemistry Research Group, Department of Chemistry, University of Patras, Greece.

Hyaluronan (HA) is a unique nonsulfated glycosaminoglycan that contributes to breast cancer cells growth and functional properties, including cell migration, invasion, adhesion, as well as tumor-associated angiogenesis in different stages of breast cancer progression and especially metastasis. Latest data show that the levels of HA and/or low molecular mass HA in blood serum and plasma of breast cancer patients may be a useful biomarker for breast cancer prognosis, differential diagnosis, and patients' treatment monitoring. Therefore, the qualitative and quantitative determination of HA in biological samples is an emerging area of research. This review gathers, categorizes, and sums up all the currently used methodologies to analyze HA and HA-related enzymes. The advantages, disadvantages, limitations in use, and the information they provide, are critically considered and discussed. Moreover, emphasis is given to the significance of HA determination in breast cancer, as well as of its related enzymes, for diagnosis and prognosis of this type of cancer.
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http://dx.doi.org/10.1111/febs.14860DOI Listing
August 2019

Analyzing Hyaluronidases in Biological Fluids.

Methods Mol Biol 2019 ;1952:127-142

Biochemistry, Biochemical Analysis and Matrix Pathobiochemistry Research Group, Department of Chemistry, University of Patras, Patras, Greece.

Hyaluronidases are a group of enzymes responsible for the degradation of hyaluronan. They seem to be associated with a plethora of pathological conditions, as it has been showcased by numerous studies over the past years. The emerging role of hyaluronidases in various pathological states, especially cancer, is of a great interest. Thus, they are considered as important research targets.In this chapter the popular assay for hyaluronidase analysis in biological fluids is presented and discussed in detail. The assay is divided into two steps; the first is zymography that aims mainly to detect different hyaluronidase enzymes in a biological sample, and the second is the direct quantitative measurement of enzymatic activity by a microtiter plate assay. Both steps are characterized by high sensitivity, simplicity, and limited time consumption.
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http://dx.doi.org/10.1007/978-1-4939-9133-4_12DOI Listing
July 2019

Tumor-suppressive functions of 4-MU on breast cancer cells of different ER status: Regulation of hyaluronan/HAS2/CD44 and specific matrix effectors.

Matrix Biol 2019 05 16;78-79:118-138. Epub 2018 Apr 16.

Biochemistry, Biochemical Analysis & Matrix Pathobiology Res. Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26110 Patras, Greece. Electronic address:

The malignant phenotype of various cancers is linked to enhanced expression of hyaluronan, a pro-angiogenic glycosaminoglycan whose expression is suppressed by 4-methylumbelliferone (4-MU), a non-toxic oral agent used as a dietary supplement to improve health and combat prostate cancer. In this study, we investigated the role of 4-MU in mammary carcinoma cells with distinct malignant phenotypes and estrogen receptor (ER) status, a major prognostic factor in the clinical management of breast cancers. We focused on two breast cancer cell lines, the low metastatic and ERα+ MCF-7 cells, and the highly-aggressive and ERα- MDA-MB-231 cells. Treatment with 4-MU caused a dose-dependent decrease of hyaluronan accumulation in the extracellular matrix as well as within the breast cancer cells, most prevalent in cells lacking ERα. This decrease in hyaluronan was accompanied by suppression of Hyaluronan Synthase 2 (HAS2), the major enzyme responsible for the synthesis of hyaluronan, and by induction of hyaluronidases (HYALs) -1 and -2. Moreover, 4-MU induced intense phenotypic changes and substantial loss of CD44, a major hyaluronan receptor, from cell protrusions. Importantly, 4-MU evoked differential effects depending on the absence or presence of ERα. Only the ERα+ cells showed signs of apoptosis, as determined by cleaved PARP-1, and anoikis as shown by concurrent loss of E-cadherin and β-catenin. Interestingly, 4-MU significantly reduced migration, adhesion and invasion of ERα- breast cancer cells, and concurrently reduced the expression and activity of several matrix degrading enzymes and pro-inflammatory molecules with tumor-promoting functions. Collectively, our findings suggest that 4-MU could represent a novel therapeutic for specific breast cancer subtypes with regard to their ER status via suppression of hyaluronan synthesis and regulation of HAS2, CD44, matrix-degrading enzymes and inflammatory mediators.
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http://dx.doi.org/10.1016/j.matbio.2018.04.007DOI Listing
May 2019

Lumican effectively regulates the estrogen receptors-associated functional properties of breast cancer cells, expression of matrix effectors and epithelial-to-mesenchymal transition.

Sci Rep 2017 03 23;7:45138. Epub 2017 Mar 23.

Université de Reims Champagne Ardenne, Laboratoire de Biochimie Médicale et Biologie Moléculaire, Reims, France.

Lumican is a small leucine-rich proteoglycan that has been shown to contribute in several physiological processes, but also to exert anticancer activity. On the other hand, it has been recently shown that knockdown of the estrogen receptor α (ERα) in low invasive MCF-7 (ERα+) breast cancer cells and the suppression of ERβ in highly aggressive MDA-MB-231 (ERβ+) cells significantly alter the functional properties of breast cancer cells and the gene expression profile of matrix macromolecules related to cancer progression and cell morphology. In this report, we evaluated the effects of lumican in respect to the ERs-associated breast cancer cell behaviour, before and after suppression of ERs, using scanning electron and confocal microscopies, qPCR and functional assays. Our data pinpointed that lumican significantly attenuated cell functional properties, including proliferation, migration and invasion. Furthermore, it modified cell morphology, inducing cell-cell junctions, evoked EMT/MET reprogramming and suppressed the expression of major matrix effectors (matrix metalloproteinases and EGFR) implicated in breast cancer progression. The effects of lumican were found to be related to the type of breast cancer cells and the ERα/β type. These data support the anticancer activity of lumican and open a new area for the pharmacological targeting of the invasive breast cancer.
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http://dx.doi.org/10.1038/srep45138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362815PMC
March 2017

The role of heparins and nano-heparins as therapeutic tool in breast cancer.

Glycoconj J 2017 06 24;34(3):299-307. Epub 2016 Oct 24.

Biochemistry, Biochemical Analysis and Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26110, Patras, Greece.

Glycosaminoglycans are integral part of the dynamic extracellular matrix (ECM) network that control crucial biochemical and biomechanical signals required for tissue morphogenesis, differentiation, homeostasis and cancer development. Breast cancer cells communicate with stromal ones to modulate ECM mainly through release of soluble effectors during cancer progression. The intracellular cross-talk between cell surface receptors and estrogen receptors is important for the regulation of breast cancer cell properties and production of ECM molecules. In turn, reorganized ECM-cell surface interface modulates signaling cascades, which regulate almost all aspects of breast cell behavior. Heparan sulfate chains present on cell surface and matrix proteoglycans are involved in regulation of breast cancer functions since they are capable of binding numerous matrix molecules, growth factors and inflammatory mediators thus modulating their signaling. In addition to its anticoagulant activity, there is accumulating evidence highlighting various anticancer activities of heparin and nano-heparin derivatives in numerous types of cancer. Importantly, heparin derivatives significantly reduce breast cancer cell proliferation and metastasis in vitro and in vivo models as well as regulates the expression profile of major ECM macromolecules, providing strong evidence for therapeutic targeting. Nano-formulations of the glycosaminoglycan heparin are possibly novel tools for targeting tumor microenvironment. In this review, the role of heparan sulfate/heparin and its nano-formulations in breast cancer biology are presented and discussed in terms of future pharmacological targeting.
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http://dx.doi.org/10.1007/s10719-016-9742-7DOI Listing
June 2017

Emerging aspects of nanotoxicology in health and disease: From agriculture and food sector to cancer therapeutics.

Food Chem Toxicol 2016 May 8;91:42-57. Epub 2016 Mar 8.

Center of Toxicology Science & Research, Medical School, University of Crete, Heraklion, Crete, Greece; Scientific Educational Center of Nanotechnology, Far Eastern Federal University, Engineering School, Vladivostok, Russia. Electronic address:

Nanotechnology is an evolving scientific field that has allowed the manufacturing of materials with novel physicochemical and biological properties, offering a wide spectrum of potential applications. Properties of nanoparticles that contribute to their usefulness include their markedly increased surface area in relation to mass, surface reactivity and insolubility, ability to agglomerate or change size in different media and enhanced endurance over conventional-scale substance. Here, we review nanoparticle classification and their emerging applications in several fields; from active food packaging to drug delivery and cancer research. Nanotechnology has exciting therapeutic applications, including novel drug delivery for the treatment of cancer. Additionally, we discuss that exposure to nanostructures incorporated to polymer composites, may result in potential human health risks. Therefore, the knowledge of processes, including absorption, distribution, metabolism and excretion, as well as careful toxicological assessment is critical in order to determine the effects of nanomaterials in humans and other biological systems. Expanding the knowledge of nanoparticle toxicity will facilitate designing of safer nanocomposites and their application in a beneficial manner.
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http://dx.doi.org/10.1016/j.fct.2016.03.003DOI Listing
May 2016

ADAMTS expression in colorectal cancer.

PLoS One 2015 18;10(3):e0121209. Epub 2015 Mar 18.

Biochemistry, Biochemical Analysis and Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece.

ADAMTSs are a family of secreted proteinases that share the metalloproteinase domain with matrix metalloproteinases (MMPs). By acting on a large panel of extracellular substrates, they control several cell functions such as fusion, adhesion, proliferation and migration. Through their thrombospondin motifs they also possess anti-angiogenic properties. We investigated whether ADAMTSs participate in colorectal cancer progression and invasion. Their expression was investigated at both mRNA and protein levels. Using RT-PCR, the expression of ADAMTS-1, -4, -5 and ADAMTS-20 was estimated in colorectal tumors of different cancer stage and anatomic site and 3 cell lines of different aggressiveness. An overexpression of ADAMTS-4 and -5 was observed, especially in tissue samples, whereas ADAMTS-1 and -20 were found to be down-regulated. Western blot analysis further supported the RT-PCR findings, revealing in addition the degradation of ADAMTS-1 and -20 in cancer. In situ expression and localization of ADAMTS-1, -4, -5 and -20 was also investigated by immunohistochemical analysis. Our data suggest a positive correlation between ADAMTS-4 and -5 expression and cancer progression, in contrast with the anti-angiogenic members of the family, ADAMTS-1 and -20, which were found to be down-regulated. Our findings support the notion that overexpression of ADAMTS-4 and ADAMTS-5 in colorectal cancer might be a possible invasive mechanism of cancer cells in order to degrade proteoglycans of ECM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121209PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364768PMC
February 2016

Metabolism of cartilage proteoglycans in health and disease.

Biomed Res Int 2014 3;2014:452315. Epub 2014 Jul 3.

Biochemistry, Biochemical Analysis and Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26500 Patras, Greece.

Cartilage proteoglycans are extracellular macromolecules with complex structure, composed of a core protein onto which a variable number of glycosaminoglycan chains are attached. Their biosynthesis at the glycosaminoglycan level involves a great number of sugar transferases well-orchestrated in Golgi apparatus. Similarly, their degradation, either extracellular or intracellular in lysosomes, involves a large number of hydrolases. A deficiency or malfunction of any of the enzymes participating in cartilage proteoglycan metabolism may lead to severe disease state. This review summarizes the findings regarding this topic.
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http://dx.doi.org/10.1155/2014/452315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106107PMC
April 2015

Syndecans as modulators and potential pharmacological targets in cancer progression.

Front Oncol 2014 3;4. Epub 2014 Feb 3.

Biochemistry, Biochemical Analysis and Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras , Patras , Greece.

Extracellular matrix (ECM) components form a dynamic network of key importance for cell function and properties. Key macromolecules in this interplay are syndecans (SDCs), a family of transmembrane heparan sulfate proteoglycans (HSPGs). Specifically, heparan sulfate (HS) chains with their different sulfation pattern have the ability to interact with growth factors and their receptors in tumor microenvironment, promoting the activation of different signaling cascades that regulate tumor cell behavior. The affinity of HS chains with ligands is altered during malignant conditions because of the modification of chain sequence/sulfation pattern. Furthermore, matrix degradation enzymes derived from the tumor itself or the tumor microenvironment, like heparanase and matrix metalloproteinases, ADAM as well as ADAMTS are involved in the cleavage of SDCs ectodomain at the HS and protein core level, respectively. Such released soluble SDCs "shed SDCs" in the ECM interact in an autocrine or paracrine manner with the tumor or/and stromal cells. Shed SDCs, upon binding to several matrix effectors, such as growth factors, chemokines, and cytokines, have the ability to act as competitive inhibitors for membrane proteoglycans, and modulate the inflammatory microenvironment of cancer cells. It is notable that SDCs and their soluble counterparts may affect either the behavior of cancer cells and/or their microenvironment during cancer progression. The importance of these molecules has been highlighted since HSPGs have been proposed as prognostic markers of solid tumors and hematopoietic malignancies. Going a step further down the line, the multi-actions of SDCs in many levels make them appealing as potential pharmacological targets, either by targeting directly the tumor or indirectly the adjacent stroma.
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http://dx.doi.org/10.3389/fonc.2014.00004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910246PMC
June 2014

Biomechanical and structural changes following the decellularization of bovine pericardial tissues for use as a tissue engineering scaffold.

J Mater Sci Mater Med 2012 Jun 28;23(6):1387-96. Epub 2012 Mar 28.

Department of Mechanical Engineering & Aer/tics, University of Patras, Patras, Greece.

To achieve natural scaffolds for tissue engineering applications we decellularized bovine pericardial (BP) tissues according to two different protocols: a novel treatment based on Triton(®) X-100 (12 h, 4 °C) (BP1) and a trypsin/EDTA treatment (37 °C, 48 h) (BP2). Results were compared with commercially available acellular xenogeneic biomaterials, Veritas(®) and Collamed(®). Biomechanical characteristics, high (E(h)) and low (E(l)) modulus of elasticity, of the fresh untreated tissue varied with the anatomical direction (apex to base (T) to transverse (L)) (mean ± SDEV): (41.63 ± 14.65-48.12 ± 10.19 MPa and 0.27 ± 0.05-0.30 ± 0.12 MPa respectively). BP1 had no mechanical effect (44.65 ± 19.73-52.67 ± 7.59 MPa and 0.37 ± 0.14-0.37 ± 0.11 MPa, respectively) but BP2 resulted in significant decrease in E(h) and E(l) (20.96 ± 8.17-36.82 ± 3.23 MPa and 0.20 ± 0.06-0.23 ± 0.06 MPa). Hysteresis ratio (h) varied (19-26 % of the loading energy) independently of anatomical direction. Glycosaminoglycans content was unaffected by BP1, while 22 % of chondroitin/dermatan sulphate and 60 % of hyaluronan were removed after BP2 treatment. Endothelial cell adhesion was achieved after 24 h and 3 days cell culture.
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http://dx.doi.org/10.1007/s10856-012-4620-8DOI Listing
June 2012

Chondroitin synthases I, II, III and chondroitin sulfate glucuronyltransferase expression in colorectal cancer.

Mol Med Rep 2011 Mar-Apr;4(2):363-8. Epub 2011 Jan 25.

Department of Chemistry, Laboratory of Biochemistry, Section of Organic Chemistry and Natural Products, University of Patras, 26110 Patras, Greece.

Glycosaminoglycans undergo significant structural alterations in cancer, namely in terms of their sulfation pattern and hydrodynamic size. Numerous studies have focused on this issue, and have demonstrated that glycosaminoglycans play a crucial role in cancer growth and invasion. However, the majority of the enzymes involved in glycosaminoglycan alterations have yet to be examined in detail. The present study focused on the expression of chondroitin-synthesizing enzymes in colorectal cancer. Specimens from healthy controls and cancer patients were subjected to RT-PCR analysis after RNA isolation, and to Western blotting after sequential extraction. The results indicated that chondroitin polymerizing factor and glucuronyltransferase gradually increased with cancer stage, and were expressed at much higher levels in adenomas compared to adjacent normal tissue. The opposite profile was obtained for chondroitin synthase I. Chondroitin synthase III was present at low levels in all the samples examined; however, its expression was higher in the samples from the cancer patients than in those from the healthy controls. It can therefore be concluded that, among the various factors regulating the structure of glycosaminoglycans in cancer, the differential expression of chondroitin-synthesizing enzymes is of the most significance.
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http://dx.doi.org/10.3892/mmr.2011.431DOI Listing
July 2011

The chondroitin/dermatan sulfate synthesizing and modifying enzymes in laryngeal cancer: expressional and epigenetic studies.

Head Neck Oncol 2010 Oct 7;2:27. Epub 2010 Oct 7.

1Department of Chemistry, Laboratory of Biochemistry, Section of Organic Chemistry and Natural Products, Karatheodori str, University of Patras, Patras, 26500, Greece.

Background: Significant biochemical changes are observed in glycosaminoglycans in squamous cell laryngeal carcinoma. The most characteristics are in chondroitin/dermatan sulfate fine structure and proportion, which might be due to differential expression of the enzymes involved in their biosynthesis. The aim of the present work was the investigation in expressional and epigenetic level of the enzymes involved in chondroitin/dermatan sulfate biosynthesis in laryngeal cancer.

Methods: Tissues subjected to total RNA and DNA isolation, and protein extraction. The techniques used in this study were RT-PCR analysis, western blotting and methylation specific PCR.

Results: We identified that many enzymes were expressed in the cancerous specimens intensively. Dermatan sulfate epimerase was expressed exclusively in the cancerous parts and in minor amounts in healthy tissues; in the macroscopically normal samples it was not detected. Furthermore, chondroitin synthase I and chondroitin polymerizing factor were strongly expressed in the cancerous parts compared to the corresponding normal tissues. Sulfotransferases, like chondroitin 6 sulfotransferase 3, were highly expressed mainly in healthy specimens.

Conclusions: The study of the various chondroitin/dermatan synthesizing enzymes revealed that they were differentially expressed in cancer, in human laryngeal cartilage, leading to specific chondroitin/dermatan structures which contributed to proteoglycan formation with specific features. The expression of the examined enzymes correlated with the glycosaminoglycan profile observed in previous studies.
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http://dx.doi.org/10.1186/1758-3284-2-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958872PMC
October 2010

Involvement of hyaluronidases in colorectal cancer.

BMC Cancer 2010 Sep 17;10:499. Epub 2010 Sep 17.

Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece.

Background: Hyaluronidases belong to a class of enzymes that degrade, predominantly, hyaluronan. These enzymes are known to be involved in physiological and pathological processes, such as tumor growth, infiltration and angiogenesis, but their exact role in tumor promotion or suppression is not clear yet. Advanced colorectal cancer is associated with elevated amounts of hyaluronan of varying size. The aim of the present study was therefore to illuminate the importance of hyaluronidases in colon carcinoma progression.

Methods: The patients' samples (macroscopically normal and cancerous) were subjected to sequential extraction with PBS, 4 M GdnHCl and 4 M GdnHCl --1% Triton X-100. The presence of the various hyaluronidases in the extracts was examined by zymography and western blotting. Their expression was also examined by RT-PCR.

Results: Among hyaluronidases examined, Hyal-1, -2, -3 and PH-20 were detected. Their activity was higher in cancerous samples. Hyal-1 and Hyal-2 were overexpressed in cancerous samples, especially in advanced stages of cancer. Both isoforms were mainly extracted with PBS. Hyal-3 was observed only in the third extract of advanced stages of cancer. PH-20 was abundant in all three extracts of all stages of cancer. The expression of only Hyal-1 and PH-20 was verified by RT-PCR.

Conclusion: A high association of hyaluronidases in colorectal cancer was observed. Each hyaluronidase presented different tissue distribution, which indicated the implication of certain isoforms in certain cancer stages. The results provided new evidence on the mechanisms involved in the progression of colorectal cancer.
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http://dx.doi.org/10.1186/1471-2407-10-499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2949809PMC
September 2010

Alterations of glycosaminoglycan disaccharide content and composition in colorectal cancer: structural and expressional studies.

Oncol Rep 2009 Aug;22(2):369-75

Department of Chemistry, University of Patras, 26110 Patras, Greece.

The glycosaminoglycans are implicated in many processes important in the growth and progression of malignant tumors. In the present study glycosaminoglycans were purified from healthy, macroscopically normal and cancerous specimens of different anatomic sites and different stages of cancer and analyzed by FACE after chondroitinases and sulfatases digestion. The cancerous samples contained increased levels of 6-sulfated unsaturated disaccharides compared to macroscopically normal and healthy samples, the increase being stage-related. The differences in sulfation were found to be related to the anatomic site and the stage of cancer. RT-PCR analysis of 4-sulfotransferase mRNA revealed its presence in decreasing amounts as the stage of the cancer increased. Furthermore, the percent content of hyaluronan disaccharides was elevated in macroscopically normal samples compared to the cancerous, and in addition, it was much more elevated than that of healthy samples. Haluronan levels increase with stage in cancerous tissues. Therefore, it could be concluded that the glycosaminoglycans in colorectal cancer are biosynthetically directed to contribute in different ways depending on the cancer stage and anatomical site.
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August 2009

Keratan sulfate-containing proteoglycans in sheep brain with particular reference to phosphacan and synaptic vesicle proteoglycan isoforms.

Biomed Chromatogr 2009 May;23(5):455-63

Department of Chemistry, Laboratory of Biochemistry, Section of Organic Chemistry, Biochemistry and Natural Products, University of Patras, Patras, Greece.

Proteoglycans (PGs) are widely expressed in all areas of the brain. In this study, the keratan sulfate-containing PGs (KS-PGs) from cerebrum (CB), cerebellum (CL) and brainstem (BS) of young sheep brain were isolated, purified and characterized. The amount of KS-PGs in CL was significantly lower than that in CB and BS. KS-PGs were characterized by increased extent of glycosylation and heterogeneity of KS chains in CL. Western blot analyses demonstrated the presence of the KS-PGs phosphacan, SV2A and SV2B isoforms of synaptic vesicle proteoglycan in all three areas of the young sheep brain. Phosphacan predominated in BS and CB, showing significant molecular heterogeneity. SV2A and SV2B were found in two forms of high and low molecular sizes according to their extent of glycosylation in sheep brain. SV2A predominated in CL, where forms with very high molecular sizes were detected. Immunohistochemical examination revealed that SV2A was localized in the extracellular matrix of both gray and white matter. In contrast, phosphacan and SV2B were mainly localized in the white matter in all brain regions. The results of the present study demonstrated that KS-PGs are present in the three areas of the sheep brain, showing significant variations in their content, structure and localization among the distinct areas. These differences may be important for the physiology of the brain.
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http://dx.doi.org/10.1002/bmc.1127DOI Listing
May 2009

Citrullination of linear and cyclic altered peptide ligands from myelin basic protein (MBP(87-99)) epitope elicits a Th1 polarized response by T cells isolated from multiple sclerosis patients: implications in triggering disease.

J Med Chem 2008 Dec;51(24):7834-42

Department of Chemistry, University of Patras, Patras 26500, Greece.

Derangement of cellular immunity is central in the pathophysiology of multiple sclerosis (MS) and is often manifested by abnormal cytokine production. We investigated cytokine secretion in peripheral blood mononuclear cells (PBMC) of 18 MS patients and 15 controls and correlated cytokine polarization with the nature of antigenic stimulus. We synthesized two novel citrullinated peptides, linear [Cit(91), Ala(96), Cit(97)]MBP(87-99) and cyclo(87-99)[Cit(91), Ala(96), Cit(97)]MBP(87-99) that resulted from citrullination of 91,97 Arg residues in antagonists, linear [Arg(91), Ala(96)]MBP(87-99) and cyclo(87-99)[Arg(91), Ala(96)]MBP(87-99) peptides. PBMC from MS patients and controls were cultured with citrullinated peptides, and both peptides caused a Th1 polarization in all MS patients studied. In contrast, culture with noncitrullinated MBP peptides resulted in heterogeneous cytokine secretion that differed between individual patients. Thus, citrullination of self-antigens may potentially trigger disease in susceptible individuals. This finding may open new avenues in drug design of new substances that inhibit citrullination and arrest epitope spreading and worsening of MS.
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http://dx.doi.org/10.1021/jm800891nDOI Listing
December 2008

Biochemical changes of extracellular proteoglycans in squamous cell laryngeal carcinoma.

Connect Tissue Res 2008 ;49(3):239-43

Department of Chemistry, Laboratory of Biochemistry, University of Patras, Patras, Greece.

Larynx is a complicated organ with peculiar properties, having a noticeable impact in vocal and respiratory physiology. In squamous cell laryngeal carcinoma, the extracellular matrix components underwent significant modifications concerning their fine chemical structure. Degradation of aggrecan is observed, whereas versican and decorin amounts are increased. The expression of aggrecan is almost totally ceased in later cancer stages, whereas decorin is expressed in normal and cancerous samples. But its expression is increased in cancer, being related to cancer stage. However, the expression of versican seems to be characteristic of the tumor, since none or traces expression is observed in normal samples. Chondroitin/dermatan sulfate is the major glycosaminoglycan, but its sulfation shows a shift from C6 position of galactosamine in normal samples to C4 in malignancy. Dermatan sulfate represents minor amounts in normal samples but increases in proportion up to one-fourth of total sulfated glycosaminoglycans in malignancy. In addition, an increase in the amounts of hyaluronan is also observed in malignant samples. Accumulated data demonstrate that tumor progression is closely related to the alteration of the expression and biochemical composition of specific extracellular constituents that describes the mild aggressive phenotype of squamous cell laryngeal carcinoma.
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http://dx.doi.org/10.1080/03008200802147662DOI Listing
September 2008

The structural and compositional changes of glycosaminoglycans are closely associated with tissue type in human laryngeal cancer.

Biochimie 2007 Dec 17;89(12):1573-80. Epub 2007 Jul 17.

Laboratory of Biochemistry, Department of Chemistry, School of Natural Sciences, University of Patras, 265 00 Patras, Greece.

Hyaluronan and sulfated glycosaminoglycans, as intrinsic components of proteoglycans, are playing important roles in cancer biology. In the present study, we investigated in detail the glycosaminoglycans on both fine chemical and structural levels in laryngeal cartilaginous and non-cartilaginous tissues at different stages of laryngeal cancer. The results indicated that in cartilaginous tissues the amounts of chondroitin sulfate, keratan sulfate, dermatan sulfate and hyaluronan presented a dramatic decrease in contrast to the non-cartilaginous tissues, which showed a significant increase of these glycosaminoglycans compared to their normal counterparts. On fine chemical structure, the molar ratios of 4-sulfated to 6-sulfated and non-sulfated to sulfated disaccharides from both cartilaginous and non-cartilaginous cancerous tissues showed a significant increase. On molecular-size level, in laryngeal cancer, the chromatographic behaviour of the sulfated glycosaminoglycan chains from both tissue-types revealed their lower M(r) with a more polydisperse and heterogeneous distribution compared to the normal ones. In addition, in both tissues, a significant decrease of high molecular-size hyaluronan was observed. Of particular interest was the great increase of hyaluronan of low molecular mass in the laryngeal non-cartilaginous tissues, which ranged from 330 to 890 kDa. The kind and the extent of these alterations, which presented an intense stage-related behaviour, depended on the tissue origin and could be associated with the malignant phenotype of human laryngeal cancer.
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http://dx.doi.org/10.1016/j.biochi.2007.07.006DOI Listing
December 2007

Cartilage aggrecan undergoes significant compositional and structural alterations during laryngeal cancer.

Biochim Biophys Acta 2006 Jul 9;1760(7):1046-53. Epub 2006 Mar 9.

Laboratory of Biochemistry, Section of Organic Chemistry, Biochemistry and Natural Products, Department of Chemistry, University of Patras, 26500 Patras, Greece.

Aggrecan is a key component of cartilage and is responsible for the integrity and function of the tissue. In this study, the content of aggrecan and its structural modifications in adjacent to cancer apparently normal cartilages (AANCs) from various stages of laryngeal squamous cell carcinoma (LSCC) were investigated. Our data demonstrated a stage-related loss of aggregable aggrecan in AANCs, compared to the healthy laryngeal cartilage (HLC), which was excessive in advanced stages of disease. On aggregable aggrecan level, AANCs were characterized by significant compositional and structural modifications, the extent of which was closely related with the stage of LSCC. Four concrete subpopulations of aggregable molecules with particular physicochemical characteristics were identified with a strong tendency to prevail subpopulations of molecules of lower hydrodynamic sizes with increasing LSCC stage. These findings demonstrated that the cleavage of aggregable aggrecan occurred in concrete peptide bonds within the CS-1 and CS-2 attachment domains. These significant alterations were closely associated with the process of cartilage destruction, indicating the crucial role of aggrecan during LSCC.
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http://dx.doi.org/10.1016/j.bbagen.2006.02.007DOI Listing
July 2006

Preparation of cross-linked cellulases and their application for the enzymatic production of glucose from municipal paper wastes.

Prep Biochem Biotechnol 2006 ;36(2):111-25

Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece.

Hydrolysis of cellulosic wastes has been applied for environmental purposes and glucose production. An enzymatic process is proposed for such treatment of municipal cellulosic wastes, and the optimum conditions are described. It was found that different conditions should be applied for the treatment of soft or hard paper wastes, the most characteristic being pretreatment of wastes and temperature of the treatment process. Optimization of enzyme characteristics was also examined after stabilization of the enzymes by cross-linking. Endocellulase was better stabilized after cross-linking with EDAC whereas, exocellulase was better with glutaraldehyde. The application of cross-linked enzyme in the waste paper treatment process resulted in about a 25% increase of glucose liberation.
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http://dx.doi.org/10.1080/10826060500533901DOI Listing
April 2006

The extractability of extracellular matrix components as a marker of cartilage remodeling in laryngeal squamous cell carcinoma.

Biochim Biophys Acta 2005 Jan 1;1721(1-3):81-8. Epub 2004 Nov 1.

Laboratory of Biochemistry, Section of Organic Chemistry, Biochemistry and Natural Products, Department of Chemistry, University of Patras, 265 00 Patras, Greece.

Sequential extraction was applied to investigate the proteoglycan (PG) organization in healthy laryngeal cartilage (HLC) and laryngeal cartilage squamous cell carcinoma (LCSCC). Highly stable aggrecan aggregates, extracted from both HLC and LCSCC with strong dissociative reagents, i.e., 4 M guanidine HCl (GdnHCl), represented 53% and 7%, respectively, of total extracted macromolecules. Less stable complexes/aggregates, extracted with mild dissociative reagents (1 and 2 M GdnHCl), represented 40% and 61% of total extracted PGs from healthy and cancerous cartilage, respectively. Interestingly, a relative high proportion (32%) of uronic acid (UA)-containing macromolecules were removed from the cancerous cartilage using associative extracting solutions (PBS and 0.5 M GdnHCl), which obviously represented molecules freely extractable from the tissue. In contrast, the corresponding proportion in HLC was impressively low (about 7%). The major proportion of these molecules was chondroitin sulfate-containing PGs (CSPGs), which identified mainly as aggrecan. Differential digestion of the sequential extracts with chondroitinase ABC and chondroitinase AC II demonstrated the presence of dermatan sulfate-containing PGs (DSPGs) in both HLC and LCSCC, being mainly present in the 1 M GdnHCl extract, and identified as decorin. All cancerous extracts were found to be rich in 4-sulfated disaccharides, mostly participating in DS structures. In conclusion, the applied procedure permitted the elucidation of the changes in the cartilage status, regarding the stability and identity of its proteoglycan aggregates/complexes, in both HLC and LCSCC.
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http://dx.doi.org/10.1016/j.bbagen.2004.10.004DOI Listing
January 2005

Changes of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the serum of patients with autoimmune diseases: association with age and disease activity.

Clin Chem Lab Med 2004 ;42(8):880-8

Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece.

Matrix metalloproteinases participate in the degradation of the extracellular matrix proteins and are regulated mainly by their respective tissue inhibitors. In a variety of inflammatory connective tissue diseases, variations in the tissue content of both metalloproteinases and tissue inhibitors have been reported. The purpose of this study was to determine the serum levels of metalloproteinases and tissue inhibitors in patients with autoimmune diseases and compare with those of healthy individuals of similar age. The metalloproteinase content was analyzed by zymography and it was found that the serum levels of metalloproteinase-2 and metalloproteinase-9 of all autoimmune disease samples were decreased, in all diseases examined and independently of clinical activity, while those of active metalloproteinase-9 were significantly elevated. Both tissue inhibitors were quantitated by direct enzyme-linked immunosorbent assay and were also found decreased in autoimmune disease samples, confirming the balance that should exist in the secretion of metalloproteinases and tissue inhibitors. These results suggested that the increased active form of metalloproteinase-9, together with the decreased concentration of tissue inhibitor-2, could be used for diagnostic purposes and for the follow-up of patients with autoimmune diseases.
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http://dx.doi.org/10.1515/CCLM.2004.143DOI Listing
February 2005

Matrix proteoglycans are markedly affected in advanced laryngeal squamous cell carcinoma.

Biochim Biophys Acta 2004 Jun;1689(2):152-61

Laboratory of Biochemistry, Section of Organic Chemistry, Biochemistry and Natural Products, Department of Chemistry, University of Patras, 26500 Patras, Greece.

Proteoglycans (PGs) are implicated in the growth and progression of malignant tumors. In this study, we examined the concentration and localization of PGs in advanced (stage IV) laryngeal squamous cell carcinoma (LSCC) and compared with human normal larynx (HNL). LSCC and HNL sections were examined immunohistochemically with a panel of antibodies, and tissues extracts were analyzed by biochemical methods including immunoblotting and high performance liquid chromatography (HPLC). The results demonstrated significant destruction of cartilage in LSCC, which was followed by marked decrease of aggrecan and link protein. In contrast to the loss of aggrecan in LSCC, accumulation of versican and decorin was observed in the tumor-associated stroma. Biochemical analyses indicated that aggrecan, versican, decorin and biglycan comprise the vast majority of total PGs in both healthy and cancerous tissue. In LSCC the absolute amounts of KS/CS/DS-containing PGs were dramatically decreased about 18-fold in comparison to HNL. This decrease is due to the loss of aggrecan. Disaccharide analysis of CS/DSPGs from LSCC showed a significant reduction of 6-sulfated Delta-disaccharides (Deltadi-6S) with a parallel increase of 4-sulfated Delta-disaccharides (Deltadi-4S) as compared to HNL. The obtained data clearly demonstrate that tumor progression is closely related to specific alteration of matrix PGs in LSCC. The altered composition of PGs in cartilage, as well as in tumor-associated stroma, is crucial for the biological behaviour of cancer cells in the diseased tissue.
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http://dx.doi.org/10.1016/j.bbadis.2004.03.006DOI Listing
June 2004

Proteoglycans in human laryngeal cartilage. Identification of proteoglycan types in successive cartilage extracts with particular reference to aggregating proteoglycans.

Biochimie 2004 Mar;86(3):221-9

Organic Chemistry, Biochemistry and Natural Products Division, Department of Chemistry, University of Patras, Patras 26500, Greece.

The content, composition and structure of proteoglycans (PGs) in adult human laryngeal cartilage (HLC) were investigated. PGs were extracted from the tissue by using two different extraction protocols. In the first protocol, PGs were extracted under dissociative conditions, 4 M guanidine HCl (GdnHCl), and in the second protocol, sequentially, with phosphate buffered saline (PBS) and solutions of increasing GdnHCl concentration (0.5, 1, 2 and 4 M). Chemical and immunological analyses of dissociate extracts (first protocol) revealed the presence of four, at least, different types of PGs. Aggrecan was the major PG, versican, decorin and biglycan being in small amounts. Galactosaminoglycan-containing PGs (GalAGPGs) represented the vast majority of total PGs present in extracts of HLC. Differential digestion with chondroitinase ABC and AC II showed that the GalAGPGs from HLC contained a significant proportion of dermatan sulphate (DS). In addition, disaccharide analysis showed that 6-sulphated disaccharides predominated in chondroitin sulphate (CS) chains. The sequential extraction (second protocol) indicated that PBS extract contained very little amount of PGs. The 0.5, 1 and 2 M GdnHCl extracts contained 6.3%, 24.5% and 15.2% of total extracted PGs, respectively. Four molar GdnHCl extracted the larger proportion, about 53%, of total PGs. This extract contained almost only proteoglycan aggregate components i.e., G1 bearing aggrecan, hyaluronan and link protein. The characterization of the aggrecan showed that it constituted a polydisperse population of monomers with an average molecular mass of 720 kDa. The glycosaminoglycans (GAGs) present were chondroitin sulphate with a M(r) of 15 kDa, and keratan sulphate (KS) with a M(r) of 10 kDa, in proportions 84% and 16%, respectively.
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http://dx.doi.org/10.1016/j.biochi.2004.01.005DOI Listing
March 2004

Immunological studies of sheep brain keratan sulphate proteoglycans.

Biochimie 2002 Dec;84(12):1225-8

Department of Chemistry, University of Patras, Patras, 26500, Greece.

Recently, we reported the isolation and partial characterization of keratan sulphate (KS) from sheep brain. In this study, a panel of monoclonal antibodies (Mab) recognizing epitopes within KS chains and core proteins of KS-containing proteoglycans were used to detect, by immunoblotting, antigenically related molecules extracted from cerebrum, cerebellum and brainstem, respectively. Although the intensity of labelling varied with each of the antibodies, the brain KSPGs were recognized by all the monoclonals used, confirming the presence of KS side chains, which react with the Mabs: 5-D-4, EFG-11, EFG-4, I22, as also the presence of KSPGs related to phosphacan-KS (3H1 proteoglycan). Extracts of all the three brain areas could bind both anti-KS and anti-core protein Mabs, as also anti-HNK-1 monoclonal antibody. Binding was sensitive to keratanases degradation in the cerebrum and brainstem except cerebellum where the presence of a large molecular size hybrid CS/KSPG bearing KS chains partially resistant to keratanases was identified. This population reacts only with 5-D-4, EFG-11 and EFG-4 antibodies. Furthermore, the presence of HNK-1 epitope in CSPGs was detected in the cerebellum and brainstem. In contrast, in the cerebrum the coexistence of HNK-1 epitope and KS in KSPGs was identified. These data suggest that the KSs of sheep brain are part of proteoglycans containing protein and KS antigenic sites related to those of corneal and cartilage KSPG, as also of the brain proteoglycan phosphacan-KS.
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http://dx.doi.org/10.1016/s0300-9084(02)00023-8DOI Listing
December 2002

Altered content composition and structure of glycosaminoglycans and proteoglycans in gastric carcinoma.

Int J Biochem Cell Biol 2003 Mar;35(3):376-90

Laboratory of Biochemistry, Section of Organic Chemistry, Biochemistry and Natural Products, Department of Chemistry, University of Patras, 26110 Patras, Greece.

Glycosaminoglycans (GAGs) in proteoglycan (PG) forms or as free GAGs are implicated in the growth and progression of malignant tumors. These macromolecules were investigated in human gastric carcinoma (HGC) and compared with those in human normal gastric mucosa (HNG). We report that HGC contained about 2-fold increased amounts of GAGs in comparison to HNG. Specifically, HGC showed 3- and 2.5-fold net increase in chondroitin sulphate (CS) and hyaluronan (HA) contents, respectively. Dermatan sulphate (DS) was slightly increased, but the amount of heparan sulphate (HS) was decreased. Of particular, interest were the quite different sulphation profiles of CS and DS chains in HGC in which, non-sulphated and 6-sulphated disaccharide units were increased 10 and 4 times, respectively, in comparison to HNG. On PG level, three different populations were identified in both HNG and HGC, being HSPGs, versican (CS/DS chains) and decorin (CS/DS chains). In HGC, the amounts of versican and decorin were significantly increased about 3- and 8-fold, respectively. These PGs were also characterized by marked decrease in hydrodynamic size and GAG content per PG molecule. Analysis of Delta-disaccharide of versican and decorin from HGC showed an increase of 6-sulphated Delta-disaccharides (Delta di-6S) and non-sulphated Delta-disaccharides (Delta di-0S) with a parallel decrease of 4-sulphated Delta-disaccharides (Delta di-4S) as compared to HNG, which closely correlated with the increase of CS content. In addition, the accumulation of core proteins of versican and decorin in HGC was also associated with many post-translational modifications, referring to the number, size, degree and patterns of sulphation and epimerization of CS/DS chains. Studies on the modified metabolism of PGs/GAGs are under progress and will help in deeper understanding of the environment in which tumor cells proliferate and invade.
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http://dx.doi.org/10.1016/s1357-2725(02)00264-9DOI Listing
March 2003