Biomedical subjects

A D Theocharis

Publications and source records attributed to A D Theocharis.

16 recordsLinked to original sources

PubMed2006

The increased accumulation of structurally modified versican and decorin is related with the progression of laryngeal cancer.

Versican and decorin, two proteoglycans (PGs) with contradictory roles in the pathophysiology of cancer, comprise important stromal components in many tumor types and play a crucial role in the progression of cancer. In this study, we provide direct evidence for a significant and stage-related accumulation of versican and decorin in the tumor-associated stroma of laryngeal squamous cell carcinoma (LSCC) in comparison to normal larynx. Both PGs were found to be co-localized within the peritumorous stroma. In addition, the accumulated versican and decorin were markedly modified on both protein core and glycosaminoglycan (GAG) levels. Decorin, which was present under both glycanated and non-glycanated forms, perceptibly increased with the progression of LSCC, compared to the normal larynx. Tumor-associated glycanated decorin was found to contain significant amounts of dermatan sulfate (DS) sequences. Versican was also found to undergo stage-related structural modifications since a marked heterogeneity of protein cores was observed, being intense in late stage of laryngeal cancer. The increased accumulation of both versican and decorin was associated with a significant stage-related increase of the molar ratio of Delta di-mono4S to Delta di-mono6S up to approximately threefold in LSCC compared to the normal ones. The modified chemical structure of both PGs could be associated with the degree of aggressiveness of laryngeal squamous cell carcinomas.

Carcinoma, Squamous Cell↗

PubMed2006

Immunization with specific polysaccharide antigen reduces alterations in corneal proteoglycans during experimental slime-producing Staphylococcus epidermidis keratitis.

PURPOSE: Staphylococcus epidermidis is a leading cause of bacterial keratitis associated with corneal damage. Corneal integrity is closely associated with matrix macromolecules, such as proteoglycans (PGs) and collagen. The aim of this study was to examine whether active immunization (AI) using a major immunogenic polysaccharide determinant of slime (20-kDa PS) as antigen, and passive immunization (PI) after administration of specific antibodies toward 20-kDa PS affect the distribution of PGs as well as corneal lesions in an experimental model of slime-producing S. epidermidis keratitis. METHODS: For AI, seven rabbits were immunized with 20-kDa PS, whereas for PI, seven rabbits received specific antibodies against 20-kDa PS. Lesions were graded clinically for a 21-day period. Levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by ELISA. The distribution of certain extracellular matrix PGs during corneal healing was analyzed immunohistochemically. RESULTS: Levels of specific anti-20-kDa PS antibodies in serum and aqueous humor obtained after either AI or PI were significantly higher as compared with those in the respective nonimmunized control groups (p<0.001). Clinical grading showed that both AI and PI rabbits had a significantly less corneal damage as compared with infected nontreated rabbits. Immunohistochemical analyses for PGs exhibited significant differences to the wounded regions as compared with noninfected corneal tissue. Accumulation of keratan sulfate PGs and decorin was observed in the corneal stroma of infected rabbits and of heparan sulfate PGs around the new-formed vessels. This phenomenon was significantly reduced in immunized animals in accordance with macroscopically decreased corneal damage observed in these animals. CONCLUSIONS: Results of this study suggest a key role of 20-kDa PS and its antibodies as prophylactic and therapeutic agents in keratitis caused by slime-producing S. epidermidis.

Animals↗

PubMed2006

Transforming growth factor-beta as a key molecule triggering the expression of versican isoforms v0 and v1, hyaluronan synthase-2 and synthesis of hyaluronan in malignant osteosarcoma cells.

Versican, a large sized chondroitin-sulphate proteoglycan (PG), and its binding partner, hyaluronan (HA), are extracellular matrix (ECM) components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in cell migration and proliferation of cancer cells and, on the other hand, disruption of HA synthesis by inhibiting hyaluronan synthase-2 (HAS2) expression in osteosarcoma cells by suppressing cell proliferation, invasiveness and motility. Considering that growth factors, such as TGF-beta, bFGF and PDGF-BB, are important regulators for the expression of the ECM macromolecules, in this study we examined the effect of these growth factors on the expression of the various versican isoforms, HA synthases as well as HA synthesis by MG-63 osteosarcoma cells and normal human osteoblastic periodontal ligament cells (hPDL). Real-time PCR and metabolic labelling followed by fine HPLC analysis coupled to radiochemical detection were the methods utilized. It was found that, contrary to normal hPDL cells, osteosarcoma MG-63 cells do not constitutively express the versican isoforms V0 and V1. Exogenous addition of TGF-beta2 stimulated the versican transcript levels mainly by forcing osteosarcoma cells to express V1 and V0 isoforms. PDGF-BB and bFGF had only minor effects in these cells. In hPDL cells a strong stimulation of the V3 transcript by all growth factors was observed. TGF-beta2 was also the major stimulator of HAS2 isoform expression as well as hyaluronan synthesis in osteosarcoma cells, while PDGF-BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. The obtained results show for the first time that TGF-beta2 triggers the malignant phenotype pattern of versican and hyaluronan expression in human osteosarcoma cells and indicate that this growth factor may account for the metastatic potential of these cells.

Becaplermin↗

PubMed2006

Versican but not decorin accumulation is related to metastatic potential and neovascularization in testicular germ cell tumours.

AIMS: To investigate the expression of versican and decorin in patients with testicular germ cell tumours (GCTs) and to correlate this with the clinicopathological findings. Matrix proteoglycans versican and decorin are frequently overexpressed in various malignancies and are involved in the progression of cancer. METHODS AND RESULTS: Overexpression of versican and decorin was detected in GCTs by immunoblotting. Immunohistochemical staining for proteoglycans was performed on 71 cases of paraffin-embedded tissues. In most of the cases increased decorin and versican stromal staining was demonstrated. In both seminomas and non-seminomatous germ cell tumours (NSGCTs) strong staining of decorin was not found to be related to any of the clinicopathological variables. Accumulation of versican was found to be associated with vascular and lymphatic invasion, nodal metastasis and disease stage in seminomas and NSGCTs and, in addition, with tumour size and distant metastasis only in NSGCTs. Additionally, only the deposition of versican was linearly correlated with the number of microvessels in the tumour stroma in GCTs. CONCLUSIONS: Ectopic versican and decorin expression is a frequent feature in GCTs. Versican but not decorin accumulation in GCTs is related to metastatic potential and neovascularization and might be a useful marker for testicular malignancy.

Adolescent↗

PubMed2005

Signal transduction by IL-2 and its receptors as target in treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic and destructive arthropathy with systemic features, the etiopathogenesis of which remains unclear. It is characterized by relapsing and remitting inflammation and hyperplasia of synovial cells. Proinflammatory cytokines, such as interleukin-2 (IL-2), play an important role in maintaining cartilage damage and severe destruction of the joints due to an uncontrolled activation of cellular immunity. An imbalance between proinflammatory and anti-inflammatory mediators is likely to contribute to the chronicity of the disease. Therefore, insight into the activation state of T-cells in different stages of the disease may be important to understand pathogenetic mechanisms underlying RA and could be a lead for the design of future therapeutic strategies. Because of the central role of the IL-2/IL-2 receptor (IL-2R) system in mediation of the immune system, monitoring and manipulation of this system has important diagnostic and therapeutic implications. New approaches in RA therapy with anticytokine agents, which block cytokines and their receptors, are now used as antirheumatic drugs in clinical practice.

Animals↗

PubMed2005

Genistein suppresses the invasive potential of human breast cancer cells through transcriptional regulation of metalloproteinases and their tissue inhibitors.

Progression of breast cancer implicates the degradation of extracellular matrix (ECM) by metallo-proteinases (MMPs), a process with important consequences on the growth and invasiveness of cancer cells in adjacent and distant sites. The isoflavone, genistein--a natural inhibitor of protein tyrosine kinase pathway--inhibits the growth of a wide range of cancer cells in vitro. The aim of this study was to investigate: i) the expression of mRNAs encoded for MMPs and their endogenous inhibitors (TIMPs) associated with pathogenesis and metastatic potential of breast cancer cells; and ii) the effect of genistein on the transcription of MMPs and TIMPs and the invasive potential of breast cancer cells. Gene expression at transcriptional level was examined in cell cultures of two epithelial breast cancer cell lines, the high invasive (ER-negative) MDA-MB-231 and the low invasive (ER-positive) MCF-7, as well as the normal mammary cells (MCF-12A) following RNA isolation and reversed transcriptase polymerase chain reaction (RT-PCR). The inhibitory effect of genistein on functional invasiveness was examined by a cell invasion assay. Cell cycle distribution showed that genistein arrested breast cancer MDA-MB-231, MCF-7 and BT-20 cells in the G2/M phase. Both normal and breast cancer cell lines express the genes of MMP-2, -9, MT1-, MT2-, MT3-MMP and TIMP-1, -2 and -3. MCF-7 express notably less MMPs than MDA-MB-231 cell line. The addition of genistein resulted in down-regulation of the transcription of all MMP genes in MDA-MB-231 and most of MMPs in MCF-7 cells. The inhibitory effect of genistein on MMPs was functionally confirmed, since it significantly reduced the invasion properties of cancer cells in vitro. The obtained results indicate that genistein may be of great value in prevention of breast cancer cell metastasis, since it represents both a transcriptional modulator of genes involved in this pathogenetic process and a suppressor of breast cancer cell invasiveness.

Antineoplastic Agents↗

PubMed2003

Versican undergoes specific alterations in the fine molecular structure and organization in human aneurysmal abdominal aortas.

Versican is the major matrix proteoglycan in aortic wall and participates in various biological functions of the tissue. In the present study the molecular characteristics of versican isolated from normal human aorta as well as those of versican expressed in aneurysmal aortic tissue were examined. Versican was isolated by combined anion-exchange and gel permeation chromatography and was further characterized by high-performance liquid chromatography, polyacrylamide gel electrophoresis and immunoblotting. In both tissues versican is exclusively substituted with chondroitin sulfate chains, in contrast to other human tissues where both chondroitin and dermatan sulfate chains are attached onto versican core proteins. Except for the significant decrease in the concentration of versican in the aneurysmal tissue, this PG undergoes specific alterations in the aneurysmal tissue. The molecular size of versican isolated from diseased tissue is decreased with a simultaneous increase in the ratio of glycosaminoglycan to protein in this tissue. The latter reflect the extensive fragmentation of versican in the diseased tissue and most probably the generation of shorter peptides enriched to glycosaminoglycan chains. Although the size of chondroitin sulfate chains is identical in both versican preparations, a significant increase in the percentage of 6-sulfated disaccharides is observed in chondroitin sulfate chains of versican in aneurysmal aortas, which is accompanied by decrease in 4-sulfated and non-sulfated units.

Aged↗

PubMed2001

Characterization of glycosaminoglycans from human normal and scoliotic nasal cartilage with particular reference to dermatan sulfate.

The composition and the distribution of glycosaminoglycans (GAGs) present in normal human nasal cartilage (HNNC), were examined and compared with those in human scoliotic nasal cartilage (HSNC). In both tissues, hyaluronan (HA), keratan sulfate (KS) and the galactosaminoglycans (GalAGs)--chondroitin sulfate (CS) and dermatan sulfate (DS)--were identified. The overall GAG content in HSNC was approx. 30% higher than the HNNC. Particularly, a 114% increase in HA, and 46% and 86% in KS and DS, respectively, was recorded. CS was the main type of GAG in both tissues with no significant compositional difference. GalAG chains in HSNC exhibited an altered disaccharide composition which was associated with significant increases of non-sulfated and 6-sulfated disaccharides. DS, which was identified and quantitated for the first time in HNNC and HSNC, contained low amounts of iduronic acid (IdoA), 18% and 28% respectively. In contrast to other tissues, where IdoA residues are organized in long IdoA rich repeats, the IdoA residues of DS in human nasal cartilage seemed to be randomly distributed along the chain. DS chains in HSNC were of larger average molecular size than those from HNNC. These results clearly indicate the GAG content and pattern in both HNNC and HSNC and demonstrate that scoliosis of nasal septum cartilage is related to quantitative and structural modifications at the GAG level.

Adult↗

PubMed2001

Human abdominal aortic aneurysm is characterized by decreased versican concentration and specific downregulation of versican isoform V(0).

Abdominal aortic aneurysm (AAA) is a common disease of human aorta with increased incidence. It is a complication to atherosclerosis and it is closely associated with alterations in extracellular macromolecules. In this study, the levels of mRNA for versican--the major extracellular arterial proteoglycan (PG)--present in AAA and normal aortas were evaluated by reverse-transcriptase polymerase chain reaction. The concentration of versican was also examined in corresponding tissue samples. Versican was almost completely extracted with 4 M guanidine hydrochloride in the presence of Triton X-100, isolated by chromatography on DEAE-Sephacel and characterized using treatment with specific chondro-/dermato-lyases and agarose gel electrophoresis. Versican localization in tissue as well as the variation and distribution of smooth muscle cells (SMCs) and macrophages were also investigated immunohistochemically. The mRNAs coding for versican isoforms V(0) and V(1) were identified in both tissues, whereas V(2) was absent. The expression of V(0) was decreased 40% in aneurysmal vessel wall, whereas that for V(1) remained constant. This change was simultaneous with a significant decrease in versican concentration by 89%. In normal aortas, most versican was seen in the intima, whereas in AAA, this layer is characterized by advanced atherosclerotic lesion, rich in lipids and macrophages but poor in versican. The decreased transcription and the still lower amount of versican in the AAA may correlate to (i) a decrease in density of SMCs, these cells being the major source of versican in aorta, and (ii) the presence of macrophages, which may induce versican degradation and modulate versican synthesis. It is proposed that the decreased synthesis and increased degradation of versican, particularly of isoform V(0), and the resulting low concentration in the intima are crucial factors contributing to the altered viscoelastic and compressive properties and thereby to the deformity and dilatation of aorta.

Adult↗

PubMed2001

Identification, quantification and fine structural characterization of glycosaminoglycans from uterine leiomyoma and normal myometrium.

The type, amount and fine chemical composition of glycosaminoglycans (GAGs) present both in human normal myometrium and uterine leiomyoma have been studied. GAGs were fractionated by ion-exchange chromatography on DEAE-Sephacel, isolated by gel-permeation chromatography on Sepharose CL-6B and characterized using electrophoresis in cellulose acetate membranes, specific enzymic treatments and analysis by high-performance capillary electrophoresis (HPCE). No statistical intrabatch differences in total GAG content in both tissues were identified, whereas significant interbatch differences between normal myometrium and uterine leiomyoma were recorded. Hyaluronan (HA), chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate (HS) and keratan sulphate (KS) were identified in both tissues. Statistically significant (P </= 0.001) differences in uterine leiomyoma in the content of HA (-27.7%), DS (+158.5%), KS (+116.4%) and HS (-52.5%), as compared to normal myometrium, were determined. Despite the differences in the amount of GAGs, no significant differences in the fine chemical composition of CS and DS were identified. It is worth noting that DS of both tissues is constructed by glucuronic acid-containing long repeats separated by a few iduronic acid residues. The results of this study suggest that the development of uterine leiomyoma is closely related to quantitative changes at the GAG level. The increase of DS and KS may well be related to increased neosynthesis of fibrotic connective tissue, whereas the decrease of HA and the unaltered amounts of CS may also indicate benign tumors of low growth rate.

Chromatography, Gel↗

PubMed2001

Keratan sulphate in cerebrum, cerebellum and brainstem of sheep brain.

Keratan sulphate was identified in sheep brain. We describe here the isolation and partial characterization of keratan sulphate from cerebrum, cerebellum and brainstem of young sheep brains. The galactosaminoglycan was isolated by using ion-exchange chromatography and gel filtration after exhaustive digestion with papain of the delipidated tissues, followed by alkaline borohydride degradation and chondroitinase ABC and heparinases I, II and III treatment. The material isolated by ion-exchange chromatography from each tissue was eluted as single but polydispersed peak from Sephadex G-75, with average molecular masses 8.4, 7.9 and 8.8 kDa for cerebrum, cerebellum and brainstem, respectively. Keratanase I and II totally degraded keratan sulphate from cerebrum and brainstem, but only partially that from cerebellum. The content of keratan sulphate was found to be about 215, 173 and 144 microg/g dry delipidated tissue for cerebrum, brainstem and cerebellum, respectively.

Animals↗

PubMed2000

Pancreatic carcinoma is characterized by elevated content of hyaluronan and chondroitin sulfate with altered disaccharide composition.

The amount and the types of glycosaminoglycans (GAGs) present in human pancreatic carcinoma were examined and compared with those in normal pancreas. Human pancreatic carcinoma contained increased levels (4-fold) of total GAGs. Particularly, this carcinoma is characterized by a 12-fold increase of hyaluronan (HA) and a 22-fold increase in chondroitin sulfate (CS) content. CS in pancreatic carcinoma exhibited an altered disaccharide composition which is associated with marked increase of non-sulfated and 6-sulfated disaccharides. Dermatan sulfate (DS) was also increased (1.5-fold) in carcinoma, whereas heparan sulfate (HS), the major GAG of normal pancreas, becomes the minor GAG in pancreatic carcinoma without significant changes in the content and in molecular size. In all cases, the galactosaminoglycans (GalGAGs, i.e. CS and DS) derived from pancreatic carcinomas were of lower molecular size compared to those from normal pancreas. The results in this study indicate, for the first time, that human pancreatic carcinoma is characterized by highly increased amounts of HA and of a structurally altered CS.

Carbohydrate Sequence↗

PubMed1999

Human abdominal aortic aneurysm is closely associated with compositional and specific structural modifications at the glycosaminoglycan level.

Human abdominal aortic aneurysm (AAA) is a commonly occuring disease of blood vessels and is related to alterations in extracellular matrix molecules. In this study we report on the type and fine structural characterization of glycosaminoglycans (GAGs) present in AAA as compared with those present in normal abdominal aorta. Hyaluronan (HA), the galactosaminoglycans-chondroitin sulfate (CS) and dermatan sulfate (DS) with average molecular size (Mr) of 35-kDa-as well as heparan sulfate (HS) with Mr of 40-kDa were identified in both tissues. No significant intrabatch differences in total GAG content were identified in normal and aneurysmal aortas. Comparing, however, tissue composition and structure of GAGs between AAAs and normal aortas, significant differences (P < or = 0.001) were found. The overall GAG content in AAAs was approx. 60% lower than the normal ones. A 90% decrease in HS content, and 65 and 73% in CS and HA, respectively, were also recorded. In contrast, only a slight decrease in the amount of DS was noted (8%). Structural alterations in disaccharide composition of GAGs correspond mainly to significant decreases (P < or = 0.001) of HS-derived N-sulfated disaccharides, CS-derived 6-sulfated disaccharide and DS-derived disulfated disaccharides. These results demonstrate that the development of AAA is related to dramatic quantitative and structural modifications at the GAG level and this may well be attributed to the destruction of arterial wall architecture and further significant functional inadequacies of the tissue.

Aged↗

PubMed1999

Isolation and analysis of a novel acidic polysaccharide from the case of squid pen.

A new non-sulphated acidic polysaccharide with an average molecular mass of 55 kDa was isolated from squid pen case after papain digestion and beta-elimination. This polysaccharide contains mainly L-iduronic acid, D-glucuronic acid, D-galactosamine, D-glucosamine and significant amounts of neutral sugars as glucose, galactose and fucose. The polysaccharide was not degraded to the relative disaccharides by chondroitinases ABC, AC and B, hyaluronidase and keratanase or by treatment with heparinases, suggesting a structure different from those of known glycosaminoglycans. The polysaccharide cannot form self aggregates.

Animals↗

PubMedDate not supplied

STI571 as a potent inhibitor of growth and invasiveness of human epithelial breast cancer cells.

STI571, a specific tyrosine kinase inhibitor, exhibits a substantial therapeutic activity in patients with chronic myeloid leukaemia and gastrointestinal stromal tumors. In this study we examined the activity of STI571 on the growth and invasiveness of three human epithelial breast cancer cell lines of low (MCF-7) and high (ZR-75-1 and MDA-MB-231) invasive potential. Growth of all cell lines in serum-containing medium was significantly inhibited by STI571 in a dose-dependent manner, with an average IC50 of approximately 5-6 microM. Flow cytometric analysis revealed that this effect is characterized by an accumulation of all breast cancer cell types tested in the G2/M-phase of the cell cycle with a concomitant decrease of the percentage of cells in the S-phase. Interestingly, no increase in apoptosis was observed, indicating that the effect of this kinase inhibitor is cytostatic rather than cytotoxic. In addition, STI571 exerts a significant inhibition effect on the invasion of the highly invasive breast cancer cell lines ZR-75-1 and MDA-MB-231. These results encourage further preclinical investigations on the mechanisms underlying the inhibitory effects of STI571, which may be of great value in breast cancer treatment.

Adenocarcinoma↗

PubMedDate not supplied

Expression of MMPs and TIMPs genes in human breast cancer epithelial cells depends on cell culture conditions and is associated with their invasive potential.

Growth and invasiveness of breast cancer cells in adjacent and distant sites is associated with the expression of metalloproteinases (MMPs), which are capable of degrading almost all extracellular matrix macromolecules of supporting stroma. In order to identify markers useful for monitoring breast cancer pathogenesis and metastatic potential, we examined the expression of mRNAs encoded for MMPs and their endogenous inhibitors (TIMPs) in a panel of four epithelial breast cancer cell lines of high (MDA-MB-231 and ZR-75-1) and low (MCF-7 and BT-20) metastatic potential, and their expression was compared with that of normal mammary cells (MCF-12A). Expression patterns were evaluated using cell cultures in serum-containing and serum-free media. Gene expression studies were performed following cell cultures, RNA isolation, reversed transcription and polymerase chain reaction. Both normal and breast cancer cells express MMPs and TIMPs at various levels, depending on cell type and culture conditions. Comparison of their mRNA levels from serum-containing media showed that MMP-9, MT2-MMP and TIMP-1 are highly expressed in all cancer cells as compared to normal ones, whereas MMP-1 and -7 are overexpressed only in breast cancer cells of high invasion potential. In serum-free cultures, the highly metastatic cells retain the overexpression profile for MMP-1 and -7. Furthermore, MT2-MMP and TIMP-1 were constitutively expressed and they can also be correlated with cancer cells, whereas constitutive expression of MMP-9 was similar in normal and cancer cells. The results of this study indicate that the expression of MMPs is dependent on the culture conditions, i.e. the growth factors present in serum-containing media. Furthermore, data suggest that, independently of cell culture conditions, the expression of MT2-MMP may be associated with malignant transformation of mammary cells and the overexpression of MMP-1 and -7 with the highly metastatic potential of epithelial breast cancer cells.

Breast Neoplasms↗