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Biomedical subjects

G N Tzanakakis

Publications and source records attributed to G N Tzanakakis.

At least 19 recordsLinked to original sources
PubMed2006

Chondroitin sulfate A chains enhance platelet derived growth factor-mediated signalling in fibrosarcoma cells.

Platelet derived growth factor is involved in the autocrine growth stimulation of malignant cells, the stimulation of angiogenesis and the recruitment and regulation of tumor fibroblasts. PDGF has been shown to physically interact with glycosaminoglycans which are abundant in the fibrosarcoma cell microenvironment. Aim of the present study was to examine the effects of glycosaminoglycans on the mitogenic function of platelet derived growth factor in two human fibrosarcoma cell lines (B6FS, HT1080). For this purpose exogenously added glycosaminoglycans, regulators of endogenous glycosaminoglycan synthesis (sodium chlorate as selective inhibitor and beta-D-xyloside as a stimulator) and specific glycosidases to cleave cell-associated glycosaminoglycans, were utilized. Platelet derived growth factor demonstrated a growth stimulating effect on B6FS, whereas no effect was evident on HT1080 fibrosarcoma cells. Beta-D-xyloside had no effect on the basal level or the platelet derived growth factor-induced cell proliferation, whereas sodium chlorate severely reduced the basal level of proliferation in both cell lines. Significant co-stimulatory effects of chondroitin sulfate A in combination with platelet derived growth factor BB on the growth of HT1080 and B6FS cells were found. The co-stimulatory effect of chondroitin sulfate A was not due to transcriptional up regulation of platelet derived growth factor receptors genes, but rather to more efficient signalling of tyrosine kinase receptors. In conclusion, this study shows that chondroitin sulfate A can enhance the mitogenic activity of platelet-derived growth factor in fibrosarcoma cells utilizing a pathway which involves tyrosine kinases. This result introduces a new modulating role for chondroitin sulfate in signalling pathways critical for cancer growth.

Becaplermin↗
PubMed2006

Determination and distribution of N-acetyl- and N-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells.

Sialic acids containing glycoconjugates are very common in human neoplasias and their expression frequently correlates with malignant phenotype and the tumor grade. The majority of tumor markers containing sialic acids in man involve changes in the amount of total sialic acids and in the presence of the two main sialic acid types, Neu5Ac and Neu5Gc, and their derivatives. The aim of the present study was to examine whether malignant mesothelioma cell lines synthesize sialic acid containing glycoconjugates at both the extracellular and cell membrane levels and particularly whether the type and the content of Neu5Ac and Neu5Gc are of biological importance for mesothelioma cell differentiation and evaluation of its prognosis. The study was performed in three human malignant mesothelioma cell lines, two with a fibroblast like phenotype (STAV-FCS and Vester) and one of epithelial differentiation (STAV-AB), which developed from the pleural effusions of patients with malignant mesothelioma and in one human adenocarcinoma cell line (Wart). Neu5Ac and Neu5Gc were determined following a mild hydrolysis step and a sample clean-up procedure. The determination was performed by reversed-phase HPLC after the NeuAc and NeuGc had been converted to per-O-benzoylated derivatives. It was found that Neu5Gc is the major sialic acid in the culture media of all cell lines examined. Molar ratios of Neu5Ac to Neu5Gc showed that Neu5Gc is the predominant sialic acid in the culture medium of the fibroblast-like mesothelioma cells. Neu5Ac is almost undetectable in the cell membrane, whereas Neu5Gc is present in considerable amounts. The obtained results suggest that the type and the content of Neu5Ac and Neu5Gc in culture media are of biological importance for mesothelioma cell differentiation and may be of value in the evaluation of prognosis.

Adenocarcinoma↗
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

Lumican, a small leucine-rich proteoglycan substituted with keratan sulfate chains is expressed and secreted by human melanoma cells and not normal melanocytes.

Melanoma is a frequent and therapy-resistant human disease. Malignant melanocytes modulate their microenvironment in order to penetrate the dermal/epidermal junction and eventually invade the dermis. The small leucine-rich proteoglycans (SLRPs) constitute important constituents of the dermis extracellular matrix (ECM), participating in both the structural and the functional organization of the skin. The role of a keratan sulphate SLRP lumican, has recently been investigated in the growth and metastasis of several cancers. In this study, the expression of lumican was studied in two human melanoma cell lines (WM9, M5) as well as in normal neonatal human melanocytes (HEMN) using real time PCR, western blotting with antibodies against the protein core and keratan sulfate, and treatments with specific enzymes. Both human metastatic melanoma cell lines were found to express lumican mRNA and effectively secrete lumican in a proteoglycan form, characterized to be substituted mostly with keratan sulfate chains. Lumican mRNA was not detected in normal melanocytes. This is the first time that the synthesis and secretion of lumican in human melanoma cell lines is reported. The role of this proteoglycan in the development and progression of malignant melanoma has to be further investigated.

Cell Line, Tumor↗
PubMed2006

Effects of the natural isoflavonoid genistein on growth, signaling pathways and gene expression of matrix macromolecules by breast cancer cells.

Genistein is a well known protein tyrosine kinase inhibitor. It is structurally similar to 17beta-estradiol and exerts antiestrogenic effects. It also affects the signal transduction components Akt, FAK, ErbB-2 and Bcl-2. Key enzymes implicated in cancer invasion are also affected by genistein. A critical evaluation of the effects of genistein on breast cancer growth, signaling and gene expression is presented in this review.

Antineoplastic Agents↗
PubMed2002

Severe fenthion intoxications due to ingestion and inhalation with survival outcome.

Two cases of severe fenthion intoxication are presented. The first is a case of a psychiatric patient who attempted suicide with ingestion of the compound, and the second case was of a child exposed to the chemical agent by air spraying. Both patients were treated in the intensive care unit with atropine and pralidoxime and finally survived. Fenthion blood levels on admission were 2.7 and 0.95 microg/mL, respectively. Different concentrations of pralidoxime were added to the first patient's poisoned serum in order to assess in vitro the effect of pralidoxime on cholinesterase reactivation. The clinical and toxicological data of the poisonings are discussed, as well as the potential therapeutic use of pralidoxime in organophosphate intoxication.

Administration, Oral↗
PubMed1999

Proteoglycans in human malignant mesothelioma. Stimulation of their synthesis induced by epidermal, insulin and platelet-derived growth factors involves receptors with tyrosine kinase activity.

Identification of proteoglycans in two human malignant mesothelioma cell lines, one with epithelial differentiation and the other with fibroblast-like phenotype, and the effects of epidermal (EGF), insulin-like (IGF-I) and platelet-derived (PDGF-BB) growth factors on the synthesis of hyaluronan (HA) and proteoglycans (PGs) were studied. Both cell lines synthesize HA and PGs: these last were recovered both as secreted and cell-associated compounds. Chondroitin sulfate (CS) containing PGs are mainly organized as versican in the extracellular medium and as thrombomodulin and syndecan in the cell membrane. Heparan sulfate (HS) containing PGs are mainly in the form of perlecan in the culture medium, whereas cell-associated HSPGs were recovered mainly as syndecan-1, -2 and -4. Receptors for EGF, IGF-I and PDGF-BB were identified in both cell lines. In addition to cell proliferation, these growth factors stimulated the synthesis of HA and PGs, the pattern of stimulation being unique for each of them and depending on the cell phenotype. EGF increased the synthesis of HA and PGs. IGF-I showed similar stimulatory effects on the synthesis of CSPGs, whereas higher amounts were needed to influence the synthesis of HA and HSPGs, the latter only being stimulated in the epithelial cell line. PDGF-BB stimulated the synthesis of HA, HSPGs and CSPGs at low concentrations, while the stimulatory effect was abolished at higher levels. Incubation with genistein inhibited the HA and PG synthesis induced by growth factors in a mode depending on both growth factor and genistein concentrations. The results clearly suggest that the stimulatory effects of EGF, IGF-I and PDGF-BB on matrix synthesis, expressed as proteoglycan synthesis, are mediated via receptor-growth factor complexes and the protein tyrosine kinase intracellular pathway.

Chromatography, Ion Exchange↗
PubMed1997

Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate.

In this report we describe a convenient and sensitive HPLC method for separating and determining the non- and variously sulphated delta-disaccharides derived from heparan sulphate, heparin and Fragmin, using heparin- and heparan sulphate lyases. This method is superior to others since it can separate and determine twelve different non-, mono-, di- and trisulphated delta-disaccharides containing either N-sulphated, N-acetylated or unsubstituted glucosamine in a single HPLC run. The various types of delta-disaccharides are separated by an ion-pair reversed-phase chromatographic procedure on a Supelcosil LC-18 column, using a binary acetonitrile gradient system with tetrabutylammonium as the ion-pairing reagent. The eluted peaks were recorded by dual wavelength at 232 and 226 nm and a linear detector response was obtained over the entire interval tested, i.e., to 50 micrograms of delta-disaccharides. As little as 0.8-5 ng of delta-disaccharides can be reliably detected and accurately determined. Following separate digestion with the heparin- and heparan sulphate lyases (heparin lyases I, II and III), the characteristic heparin delta-disaccharides in the heparan sulphate chain, as well as the heparan sulphate delta-disaccharides in the heparin polymer, can be identified. Using combined digestions with these three lyases, the glycosaminoglycan chains are degraded almost completely (> 90%) to delta-disaccharides, which are then determined by direct injections into the HPLC system and thus an almost complete spectrum of disaccharide composition can be obtained. By this method, it is possible to analyse and confirm that the heparan sulphate chain is defined as a glycosaminoglycan dominated by GlcNAc(+/- 6S)-GlcA disaccharides and by some copolymeric disaccharides, such as GlcNS-IdoA2S and GlcNS6S-IdoA2S, otherwise most common in heparin. Fragmin, which is a controlled cepolymerized heparin fragment of M(r) 5000, is made up mainly of trisulphated disaccharides of the GlcNS6S-IdoA2S type (88.8%). Using separate digestions with the specific heparin lyases, one can also distinguish between heparin and heparan sulphate.

Acetonitriles↗
PubMed1997

Proteoglycan synthesis induced by transforming and basic fibroblast growth factors in human malignant mesothelioma is mediated through specific receptors and the tyrosine kinase intracellular pathway.

The effects and the mechanisms of action of transforming (TGF-beta 2) and basic fibroblast (bFGF) growth factors on the synthesis of hyaluronan and proteoglycans (PGs) in human malignant mesothelioma cells have been studied. Two cell lines, one with an epithelial differentiation (STAV-AB) and the other with a fibroblast-like phenotype (STAV-FCS) have been examined. Using monoclonal antibodies to growth factor receptors, the presence of a high density of the specific receptors for TGF-beta 2 and bFGF was immunochemically demonstrated in both mesothelioma cell lines. These cell lines synthesize hyaluronan, galactosaminoglycan containing PGs (GalAGPGs) and heparan sulphate containing PGs (HSPGs) at different levels; the epithelial differentiated cells produce a 6-8 times higher amount than those with the fibroblast-like morphology. In both cell lines the rates of proteoglycan synthesis were affected in a dose-dependent mode by TGF-beta 2 and bFGF. Maximal synthetic levels of both secreted and cell-bound proteoglycans were reached at 10 ng/mL whereafter they remained constant. TGF-beta 2 stimulated the synthesis of hyaluronan, GalAGPGs and of HSPGs in STAV-FCS, whereas this effect was pronounced only for GalAGPGs in STAV-AB. bFGF showed stimulatory effects for the synthesis of hyaluronan and cell associated GalAGPGs in STAV-FCS, whereas no significant enhancement was recorded for HSPGs. In the STAV-AB cell line the synthesis of hyaluronan and GalAGPGs remained unaltered by the addition of both growth factors. Although the synthesis of total HSPGs remained constant, this was due to a decrease in the secreted product and a similar increase of the cell-associated proteoglycan. The stimulatory mechanisms of both growth factors was examined by using the specific protein tyrosine kinase inhibitor genistein. Incubation of both cell lines with this isoflavonoid inhibited the enhanced synthesis of hyaluronan and all PGs induced by TGF-beta 2 and bFGF. It is suggested that most, if not all, of the stimulatory effects on the hyaluronan and PGs synthesis are mediated via protein tyrosine kinase activity elicited by receptor-ligand complexes. Decreased synthetic rates obtained when giving genistein to unstimulated mesothelioma cells may indicate the relation of hyaluronan and PGs synthesis with an autocrine stimulatory mechanisms.

Fibroblast Growth Factor 2↗
PubMed1996

High performance capillary electrophoresis method to characterize heparin and heparan sulfate disaccharides.

A rapid, sensitive and accurate high-performance capillary electrophoresis method is described for the determination of the sulfation pattern of heparin and heparan sulfate disaccharides. The analysis, performed after enzymic degradation of the polysaccharides with heparinase and heparinases II and III in combination, yields highly UV-absorbing delta-disaccharides. The separation is performed with reversed polarity using 15 mM phosphate buffer, pH 3.50. This method is superior to others since all known 12 disaccharides carrying N-acetylated, N-sulfated or unsubstituent glucosamine can be separated in a single run of 15 min. At the highest sensitivity the analysis consumes only a few femtograms of glycosaminoglycan and allows a determination of delta-disaccharides at the attomole level.

Carbohydrate Sequence↗
PubMed1996

Effect of insulin and epidermal growth factors on the synthesis of glycosaminoglycans/proteoglycans in cultured human malignant mesothelioma cells of different phenotypic morphology.

Two human malignant mesothelioma cell sublines, one with a fibroblast-like and the other with an epithelial differentiation, were examined for their capacity to synthesize glycosaminoglycans in the presence of IGF-I, EGF, and their combination. This synthesis depends on the morphology of the mesothelioma cells, with many-fold higher amounts of both hyaluronan and proteoglycans being produced by the cells with epithelial morphology than by those of the fibroblast phenotype. In both cell lines this synthesis was affected in a dose-dependent fashion by the exogenously added growth factors and exposure to IGF-I and EGF in combination showed a synergistic effect. This effect of those factors seems to be mediated via protein tyrosin kinase-dependent receptors and was different in the fibroblast-like and epithelial cells. The synthetic rates of the various glycosaminoglycans formed (hyaluronan, galactosaminoglycans and heparan sulfate) were also variously affected by these factors, indicating differences in how the synthesis of the various glycosaminoglycans is regulated. The results obtained suggest a close correlation between the presence of the appropriate growth factor(s), the process of cell differentiation and the synthesis of glycosaminoglycans in these cells.

Chondroitin Sulfates↗
PubMed1995

Determination of hyaluronan and galactosaminoglycan disaccharides by high-performance capillary electrophoresis at the attomole level. Applications to analyses of tissue and cell culture proteoglycans.

A rapid, highly sensitive and reproducible HPCE method is described for the determination of all non- and variously sulphated disaccharides present in hyaluronan and vertebrate chondroitin sulphates and dermatan sulphates. Following chondroitinase digestion of glycosaminoglycans or proteoglycans, the non-, di- and tri-sulphated delta-disaccharides are completely separated and readily determined within 14 min on a fused-silica capillary in 15 mM sodium dihydrogen orthophosphate, pH 3.00, using reversed polarity at 20 kV and detection at 232 nm. The determination of the various delta-disaccharides derived from either glucuronic or iduronic acid and the presence of glucuronic and iduronic clustered structures in dermatan sulphate can also easily be made, using digests with chondroitinase AC or B. A linear detector response was obtained for the entire interval tested (up to 10 mg/l of delta-disaccharides). Concentrations as small as 32, 65, 100 and 250 pmol/l (22, 38, 50 and 98 ng/l) of tri-, di- and nonsulphated delta-disaccharides, respectively, can be reliably detected.

Animals↗
PubMed1995

Effects on glycosaminoglycan synthesis in cultured human mesothelioma cells of transforming, epidermal, and fibroblast growth factors and their combinations with platelet-derived growth factor.

Two human mesothelioma cell sublines with fibroblast-like and epithelial morphology produce hyaluronan, galactosaminoglycans, and heparan sulfate in amounts varying with their cell phenotype. The epithelially differentiated cells synthesize these glycosaminoglycans in 6- to 8-fold higher amounts than the fibroblast-like cells. Hyaluronan is mainly a secretory product (> 90%), a considerable proportion of galactosaminoglycans is present in the extracellular medium (> 80%), while more of the heparan sulfate (50-70%) is cell-associated. In both cell lines the rates of synthesis of glycosaminoglycans are affected by the addition of the exogenous growth factors. In fibroblast phenotype cells, TGF-beta 2, EGF, and bFGF increase the production of glycosaminoglycans from 1.6- to 2.0-fold, with the exception of HS, which is suppressed by the addition of bFGF. The combination of these growth factors with PDGF-BB showed that only EGF causes a synergistic action in the synthesis of all glycosaminoglycans and that no additive effect is obtained when PDGF-BB is combined with TGF-beta 2 and bFGF. In epithelially differentiated cells, the addition of exogenous TGF-beta 2 and bFGF has no significant effect on hyaluronan synthesis, which is increased by EGF to 45%. The synthesis of galactosaminoglycans is stimulated by EGF and TGF-beta 2 approximately 35%, whereas bFGF has no significant effect. Heparan sulfate production is considerably increased by the addition of EGF by 50%, whereas bFGF has no significant effect and TGF-beta 2 suppresses this synthesis. The combination of the various growth factors with PDGF-BB showed that only heparan sulfate synthesis is affected. Thus, combining PDGF-BB with TGF-beta 2 and EGF this synthesis is increased by 35 and 25%, whereas the combination of bFGF with PDGF-BB has no further effect. The remarkable differences found between the two mesothelioma sublines may well be related to the importance of glycosaminoglycan-growth factor interactions as a key factor in phenotypic cell differentiation.

Epidermal Growth Factor↗
PubMed1993

Prevention of human pancreatic cancer cell-induced hepatic metastasis in nude mice by dipyridamole and its analog RA-233.

BACKGROUND: Several studies have provided evidence suggesting that platelets play a key role in tumor metastasis. A number of antiplatelet agents have been used to prevent tumor metastasis in animal models and humans. Antiplatelet agents, dipyridamole (adenosine transport inhibitor), and RA-233 (inhibitor of cAMP PDE) were used to prevent tumor-cell-platelet interactions both in in vitro and in vivo systems; however, the data were not very conclusive. METHODS: Our studies used dipyridamole and RA-233 alone and in combination to investigate their effects on human pancreatic tumor cells (RWP-2)-induced platelet aggregation in human blood and on hepatic metastasis in nude mice. To examine effects of dipyridamole and RA-233 on liver metastasis, the tumor cells (RWP-2) were injected intrasplenically in nude mice grouped into control, dipyridamole (8 mg/kg), RA-233 (8 mg/kg), and dipyridamole plus RA-233 (8 mg/kg each). The agents were administered intraperitoneally 1 hour before and 24 hours after the tumor cell injection. RESULTS: When dipyridamole and RA-233 were used alone, only weak to moderate effects were seen on RWP-2 tumor cell-induced platelet aggregation. However, these agents, when combined, strongly inhibited the tumor cell-induced aggregation in human platelet-rich plasma. In tumor metastasis experiments, reductions of approximately 70% in hepatic nodules and 90% in surface area occupied by the tumor were seen with the combination treatment (dipyridamole plus RA-233) as compared with the control group of mice. CONCLUSIONS: This study suggests that the combination of dipyridamole and RA-233 provides an effective intervention for the antithrombotic approach to the treatment of cancer metastases.

Adenosine↗
PubMed1993

Liver metastases with 10 human colon carcinoma cell lines in nude mice and association with carcinoembryonic antigen production.

BACKGROUND: The secretion of carcinoembryonic antigen (CEA) by many colorectal tumors is associated with a worse prognosis and a greater likelihood of metastases. The exact biologic function of CEA is not known. In the literature, it has been postulated CEA may be a tumorigenicity-enhancing factor. METHODS: Ten different human colonic adenocarcinoma cell lines (RW-7213, RW-2982, LS174T, SW1116, RW-5928, DLD-2, SW-48, DLD-1, SW-480, and HCT-8) with a wide range of CEA production (from undetectable to 5200 ng/ml in culture medium) were injected into the spleens of groups of nude mice as a model for experimental hepatic metastasis. RESULTS: There was a wide range in local tumorigenicity in the spleen (from 0-90%) and in liver metastases (from 0-70%). The capacity to grow in both liver and spleen was associated with CEA production. The four cell lines that secreted the highest amounts of CEA produced the highest tumorigenicity in the spleen (67-90%) with frequent liver colonization (25-70%). The two cell lines that secreted no detectable CEA produced neither splenic tumors nor hepatic colonies. Low-level CEA production was associated with intermediate and more variable tumorigenicity. CONCLUSIONS: There was an association between CEA secretion and the ability of 10 different colorectal cell lines to grow in nude mouse spleen and liver models.

Adenocarcinoma↗
PubMed1992

In vivo selection of a highly metastatic cell line from a human pancreatic carcinoma in the nude mouse.

A cell line with high metastatic capacity to the liver was established by sequential passages of a human pancreatic cancer cell line through the nude mouse liver. A subline, L3.5, established after five passages of the fast-growing variant (FG) of the human pancreatic cancer COLO 357 through the nude mouse liver produced extensive hepatic metastases in 100% of experimental animals when injected into the spleen. The incidence of pulmonary metastases decreased from 43% for FG to 9% for L3.5. The L3.5 cell line showed aggressive growth with almost complete replacement of the hepatic parenchyma in one third of the mean time required for the development of macroscopic metastases of FG in the liver after splenic injections of tumor cells. This study indicates that the nude mouse provides a good model for in vivo selection of metastatic cells from human pancreatic cancer. The L3.5 cell line will be valuable in the study of human pancreatic cancer metastasis, particularly in the area of survival and growth of metastatic cells in the microenvironment of the liver.

Adenocarcinoma↗
PubMed1992

Light microscopy of dried saliva in the evaluation of xerostomia of the sicca syndrome. A preliminary report.

Dried, freshly produced saliva from 21 patients with xerostomia related to the sicca syndrome [15 with primary Sjögren's syndrome (pSS), 3 with rheumatoid arthritis (RA) and secondary Sjögren's syndrome (sSS), and 3 with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE)] and 21 age and sex matched controls, was examined by light microscopy. A typical fern-like pattern was demonstrated by the crystallized mucus of the healthy individuals. In contrast, much thicker, shorter, irregular and densely arranged branches of crystallized mucus, sometimes giving a reindeer horn appearance, were observed in the patients' saliva. Given the lack of a reliable clinical measure for the objective evaluation of xerostomia, light salivary microscopy, simple and easy as it is, may fill this deficit, if its sensitivity and specificity are documented.

Aged↗