ABSTRACT
Steviol glycosides, the active sweet components of stevia plant, have been recently found to possess a number of therapeutic properties, including some recorded anticancer ones against various cancer cell types (breast, ovarian, cervical, pancreatic, and colon cancer). Our aim was to investigate this anticancer potential on the two most commonly used breast cancer cell lines which differ in the phenotype and estrogen receptor (ER) status: the low metastatic, ERα+ MCF-7 and the highly metastatic, ERα-/ERß+ MDA-MB-231. Specifically, glycosides' effect was studied on cancer cells': (a) viability, (b) functionality (proliferation, migration, and adhesion), and (c) gene expression (mRNA level) of crucial molecules implicated in cancer's pathophysiology. Results showed that steviol glycosides induced cell death in both cell lines, in the first 24 hr, which was in line with the antiapoptotic BCL2 decrease. However, cells that managed to survive showcased diametrically opposite behavior. The low metastatic ERα+ MCF-7 cells acquired an aggressive phenotype, depicted by the upregulation of all receptors and co-receptors (ESR, PGR, AR, GPER1, EGFR, IGF1R, CD44, SDC2, and SDC4), as well as VIM and MMP14. On the contrary, the highly metastatic ERα-/ERß+ MDA-MB-231 cells became less aggressive as pointed out by the respective downregulation of EGFR, IGF1R, CD44, and SDC2. Changes observed in gene expression were compatible with altered cell functions. Glycosides increased MCF-7 cells migration and adhesion, but reduced MDA-MB-231 cells migratory and metastatic potential. In conclusion, the above data clearly demonstrate that steviol glycosides have different effects on breast cancer cells according to their ER status, suggesting that steviol glycosides might be examined for their potential anticancer activity against breast cancer, especially triple negative breast cancer (TNBC).
Subject(s)
Estrogen Receptor alpha , Neoplasms , Diterpenes, Kaurane , ErbB Receptors , Estrogen Receptor beta/physiology , Glucosides , Glycosides/pharmacology , Matrix Metalloproteinase 14 , Proto-Oncogene Proteins c-bcl-2 , RNA, Messenger , Receptors, EstrogenABSTRACT
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/ß. 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.
Subject(s)
Biomarkers, Tumor/metabolism , Lumican/metabolism , Melanoma/pathology , Podosomes/pathology , Skin Neoplasms/pathology , Animals , Cell Cycle , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Shape , Cortactin/metabolism , Cyclin D1/metabolism , Extracellular Matrix/metabolism , Focal Adhesions/metabolism , Humans , Hyaluronic Acid/metabolism , Lung Neoplasms/secondary , Melanoma/metabolism , Melanoma/ultrastructure , Mice, Inbred C57BL , Neoplasm Metastasis , Phosphorylation , Signal Transduction , Skin Neoplasms/metabolism , Skin Neoplasms/ultrastructure , Snail Family Transcription Factors/metabolism , Vinculin/metabolismABSTRACT
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.
Subject(s)
Extracellular Matrix/metabolism , Animals , Extracellular Matrix/chemistry , HumansABSTRACT
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 ERß-positive MDA-MB-231, cells with the respective ERß-suppressed (shERßMDA-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, αVß3, and αVß5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of α2 and ß1 integrin subunits both in MDA-MB-231 and in shERßMDA-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.
Subject(s)
Breast Neoplasms/metabolism , Cell Shape/drug effects , Cortactin/metabolism , Integrins/metabolism , Lumican/pharmacology , Signal Transduction/drug effects , Breast Neoplasms/pathology , Breast Neoplasms/ultrastructure , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Focal Adhesion Kinase 1/metabolism , Humans , Hyaluronan Receptors/metabolism , Hyaluronan Synthases/metabolism , MCF-7 Cells , Microscopy, Electron, Scanning , Phosphorylation/drug effectsABSTRACT
A great hallmark of breast cancer is the absence or presence of estrogen receptors ERα and ERß, 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 ERß 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.
Subject(s)
Biomarkers, Tumor , Breast Neoplasms/etiology , Breast Neoplasms/metabolism , Epithelial-Mesenchymal Transition , Podosomes/metabolism , Animals , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Epithelial-Mesenchymal Transition/genetics , Extracellular Matrix/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Lumican/genetics , Lumican/metabolism , Molecular Targeted Therapy , Signal TransductionABSTRACT
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.
Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Hyaluronic Acid/metabolism , Animals , Biomarkers, Tumor/genetics , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Female , Humans , Hyaluronic Acid/geneticsABSTRACT
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.
Subject(s)
Electrophoresis/methods , Enzyme Assays/methods , Hyaluronoglucosaminidase/analysis , Biotinylation , Enzyme-Linked Immunosorbent Assay/methods , Humans , Hyaluronic Acid/metabolism , Hyaluronoglucosaminidase/blood , Hyaluronoglucosaminidase/metabolism , Hyaluronoglucosaminidase/urine , Saliva/chemistry , Staining and Labeling/methodsABSTRACT
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 ß-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.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Hyaluronan Receptors/metabolism , Hyaluronan Synthases/metabolism , Hyaluronic Acid/metabolism , Hymecromone/pharmacology , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Down-Regulation , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Receptors, Estrogen/metabolismABSTRACT
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 ERß in highly aggressive MDA-MB-231 (ERß+) 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/ß type. These data support the anticancer activity of lumican and open a new area for the pharmacological targeting of the invasive breast cancer.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Extracellular Matrix/metabolism , Lumican/pharmacology , Receptors, Estrogen/metabolism , Cell Line, Tumor , Cellular Reprogramming/genetics , Female , Gene Expression Profiling , Gene Knockout Techniques , Humans , MCF-7 Cells , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , RNA, Small Interfering/geneticsABSTRACT
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.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Gene Expression Regulation, Neoplastic , Heparin/chemistry , Heparitin Sulfate/therapeutic use , Animals , Antineoplastic Agents/chemistry , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Communication/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cytokines/genetics , Cytokines/metabolism , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Female , Heparin/therapeutic use , Heparitin Sulfate/chemistry , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Protein Binding/drug effects , Signal Transduction , Stromal Cells/drug effects , Stromal Cells/metabolism , Stromal Cells/pathologyABSTRACT
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.
Subject(s)
Agriculture , Food Industry , Nanotechnology , Neoplasms/therapy , Toxicology , HumansABSTRACT
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.
Subject(s)
ADAM Proteins/genetics , ADAM Proteins/metabolism , Colorectal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Aged , Aged, 80 and over , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Cell Line, Tumor , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Drug Discovery , Female , Humans , Male , Middle Aged , Neoplasm MetastasisABSTRACT
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.
Subject(s)
Cartilage/metabolism , Glycosaminoglycans/metabolism , Joint Diseases/metabolism , Proteoglycans/metabolism , Cartilage/pathology , Golgi Apparatus/metabolism , Golgi Apparatus/pathology , Humans , Hydrolases/metabolism , Joint Diseases/pathology , Lysosomes/metabolism , Lysosomes/pathologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Pericardium , Tissue Engineering/methods , Tissue Scaffolds , Animals , Biocompatible Materials , Biomechanical Phenomena , Bioprosthesis , Cattle , Cell Adhesion , Cell Culture Techniques , Cell Movement , Elastic Modulus , Endothelial Cells/cytology , Humans , Materials Testing , Pericardium/chemistry , Pericardium/cytology , Tissue Scaffolds/chemistryABSTRACT
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.
Subject(s)
Colorectal Neoplasms/enzymology , Glucuronosyltransferase/metabolism , N-Acetylgalactosaminyltransferases/metabolism , Aged , Aged, 80 and over , Blotting, Western , Chondroitin Sulfates/metabolism , Colorectal Neoplasms/genetics , Dermatan Sulfate/metabolism , Female , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Glucuronosyltransferase/genetics , Humans , Male , Middle Aged , N-Acetylgalactosaminyltransferases/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Carcinoma, Squamous Cell/genetics , Chondroitin/metabolism , Dermatan Sulfate/metabolism , Enzymes/genetics , Epigenesis, Genetic , Gene Expression Regulation, Enzymologic , Laryngeal Neoplasms/genetics , Adult , Aged , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/metabolism , Case-Control Studies , Chondroitin/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dermatan Sulfate/biosynthesis , Enzymes/metabolism , Epigenesis, Genetic/physiology , Gene Expression Profiling , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Humans , Laryngeal Neoplasms/enzymology , Laryngeal Neoplasms/metabolism , Middle Aged , N-Acetylgalactosaminyltransferases/genetics , N-Acetylgalactosaminyltransferases/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Sulfotransferases/genetics , Sulfotransferases/metabolismABSTRACT
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.
Subject(s)
Biomarkers, Tumor/metabolism , Cell Adhesion Molecules/metabolism , Colon/enzymology , Colorectal Neoplasms/enzymology , Hyaluronoglucosaminidase/metabolism , Rectum/enzymology , Aged , Aged, 80 and over , Blotting, Western , Cell Adhesion Molecules/genetics , Colon/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Disease Progression , Female , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Humans , Hyaluronoglucosaminidase/genetics , Male , Middle Aged , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , Rectum/pathology , Reverse Transcriptase Polymerase Chain Reaction , Survival RateABSTRACT
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.
Subject(s)
Colorectal Neoplasms/chemistry , Disaccharides/analysis , Glycosaminoglycans/analysis , Aged , Aged, 80 and over , Chondroitin Sulfates/analysis , Colorectal Neoplasms/pathology , Dermatan Sulfate/analysis , Disaccharides/chemistry , Female , Glycosaminoglycans/chemistry , Humans , Male , Middle Aged , Neoplasm Staging , Sulfotransferases/analysisABSTRACT
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.