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George N Tzanakakis - One of the best experts on this subject based on the ideXlab platform.
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igf i regulates ht1080 Fibrosarcoma Cell migration through a syndecan 2 erk ezrin signaling axis
Experimental Cell Research, 2017Co-Authors: Maria Mytilinaiou, Dragana Nikitovic, Aikaterini Berdiaki, Antonis Papoutsidakis, Dionysios J Papachristou, Aristidis Tsatsakis, George N TzanakakisAbstract:Fibrosarcoma is a tumor of mesenchymal origin, originating from fibroblasts. IGF-I is an anabolic growth factor which exhibits significant involvement in cancer progression. In this study, we investigated the possible participation of syndecan-2 (SDC-2), a Cell membrane heparan sulfate (HS) proteoglycan on IGF-I dependent Fibrosarcoma Cell motility. Our results demonstrate that SDC-2-deficient HT1080 Cells exhibit attenuated IGF-I-dependent chemotactic migration (p < 0.001). SDC-2 was found to co-localize to IGF-I receptor (IGF-IR) in a manner dependent on IGF-I activity (P ≤ 0.01). In parallel, the downregulation of SDC-2 significantly inhibited both basal and due to IGF-I action ERK1/2 activation, (p < 0.001). The phosphorylation levels of ezrin (Thr567), which is suggested to act as a signaling bridge between the Cellular membrane receptors and actin cytoskeleton, were strongly enhanced by IGF-I at both 1h and 24h (p < 0.05; p < 0.01). The formation of an immunoprecipitative complex revealed an association between SDC2 and ezrin which was enhanced through IGF-I action (p < 0.05). Immunoflourescence demonstrated a co-localization of IGF-IR, SDC2 and ezrin upregulated by IGF-I action. IGF-I enhanced actin polymerization and ezrin/actin specific localization to Cell membranes. Finally, treatment with IGF-I strongly increased SDC2 expression at both the mRNA and protein level (p < 0.001). Therefore, we propose a novel SDC2-dependent mechanism, where SDC2 is co-localized with IGF-IR and enhances its' IGFI-dependent downstream signaling. SDC2 mediates directly IGFI-induced ERK1/2 activation, it recruits ezrin, contributes to actin polymerization and ezrin/actin specific localization to Cell membranes, ultimately facilitating the progression of IGFI-dependent Fibrosarcoma Cell migration.
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receptor for hyaluronic acid mediated motility rhamm regulates ht1080 Fibrosarcoma Cell proliferation via a β catenin c myc signaling axis
Biochimica et Biophysica Acta, 2016Co-Authors: Katerina Kouvidi, Evgenia Karousou, Dragana Nikitovic, Aikaterini Berdiaki, Alberto Passi, Maria Tzardi, George N TzanakakisAbstract:Abstract Background High levels of hyaluronan (HA) synthesis in various cancer tissues, including sarcomas, are correlated with tumorigenesis and malignant transformation. RHAMM (receptor for hyaluronic acid-mediated motility) is overexpressed during tumor development in different malignancies. β-Catenin is a crucial downstream mediator of the Wnt signaling cascade which facilitates carcinogenic events characterized by deregulated Cell proliferation. Methods Real-time PCR, in vitro Cell proliferation assay, siRNA transfection, flow cytometry, immunoprecipitation, western blotting and immunofluorescence were utilized. Results The reduction of RHAMM expression was strongly correlated with an inhibition of HT1080 Fibrosarcoma Cell growth (p ≤ 0.01). LMWHA, in a RHAMM-dependent manner increases Cell growth of HT1080 Cells ((p ≤ 0.01). Both basal and LMWHA dependent growth of HT1080 Cells was attenuated by β-catenin deficiency (p ≤ 0.01). β-Catenin cytoplasmatic deposition is positively regulated by RHAMM (p ≤ 0.01). Immunoflourescence and immunoprecipitation suggest that RHAMM/β-catenin form an intraCellular complex. Transfection experiments identified c-myc as candidate downstream mediator of RHAMM/β-catenin effects on HT1080 Fibrosarcoma Cell proliferation. Conclusions LMWHA/RHAMM downstream signaling regulates Fibrosarcoma Cell growth in a β-catenin/c-myc dependent manner. General significance The present study suggests that RHAMM is a novel β-catenin intraCellular binding partner, protecting β-catenin from degradation and supporting the nuclear translocation of this key Cellular mediator, which results in c-myc activation and enhanced Fibrosarcoma Cell growth.
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role of receptor for hyaluronic acid mediated motility rhamm in low molecular weight hyaluronan lmwha mediated Fibrosarcoma Cell adhesion
Journal of Biological Chemistry, 2011Co-Authors: Katerina Kouvidi, Dragana Nikitovic, Aikaterini Berdiaki, Pavlos Katonis, Nikos K. Karamanos, Nikos Afratis, Vincent C Hascall, George N TzanakakisAbstract:Hyaluronan (HA) modulates key cancer Cell functions through interaction with its CD44 and receptor for hyaluronic acid-mediated motility (RHAMM) receptors. HA was recently found to regulate the migration of Fibrosarcoma Cells in a manner specifically dependent on its size. Here, we investigated the effect of HA/RHAMM signaling on the ability of HT1080 Fibrosarcoma Cells to adhere onto fibronectin. Low molecular weight HA (LMWHA) significantly increased (p ≤ 0.01) the adhesion capacity of HT1080 Cells, which high molecular weight HA inhibited. The ability of HT1080 RHAMM-deficient Cells, but not of CD44-deficient ones, to adhere was significantly decreased (p ≤ 0.001) as compared with control Cells. Importantly, the effect of LMWHA on HT1080 Cell adhesion was completely attenuated in RHAMM-deficient Cells. In contrast, adhesion of RHAMM-deficient Cells was not sensitive to high molecular weight HA treatment, which identifies RHAMM as a specific conduit of the LMWHA effect. Western blot and real time-PCR analyses indicated that LMWHA significantly increased RHAMM transcript (p ≤ 0.05) and protein isoform levels (53%, 95 kDa; 37%, 73 kDa) in Fibrosarcoma Cells. Moreover, Western blot analyses showed that LMWHA in a RHAMM-dependent manner enhanced basal and adhesion-dependent ERK1/2 and focal adhesion kinase (FAK) phosphorylation in HT1080 Cells. Utilization of a specific ERK1/2 inhibitor completely inhibited (p ≤ 0.001) LMWHA-dependent adhesion, suggesting that ERK1/2 is a downstream effector of LMWHA/RHAMM signaling. Likewise, the utilization of the specific ERK1 inhibitor resulted in a strong down-regulation of FAK activation in HT1080 Cells, which identifies ERK1/2 as a FAK upstream activator. In conclusion, our results suggest that RHAMM/HA interaction regulates Fibrosarcoma Cell adhesion via the activation of FAK and ERK1/2 signaling pathways.
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bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of Fibrosarcoma Cells.
Biochimica et biophysica acta, 2009Co-Authors: Aikaterini Berdiaki, Dragana Nikitovic, Pavlos Katonis, Nikos K. Karamanos, Aristeidis M. Tsatsakis, George N TzanakakisAbstract:article i nfo Article history: Background: Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extraCellular matrix derived signals to regulate Cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS Fibrosarcoma Cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration. Methods: Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized. Results: bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 Cells (p=0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high mole- cular weight HA deposition in the periCellular matrix. Increased endogenous HA production (p=0.0022) and treatment with exogenous high molecular weight HA (p=0.0268) correlated with a significant decrease of HT1080 Cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 Cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 Cells' motility (pb0.01). In contrast, no effects on B6FS Fibrosarcoma Cell motility were observed. Conclusions: bFGF regulates, in a Cell-specific manner the migration capability of Fibrosarcoma Cells by modulating their HA metabolism. HA metabolism is suggested to be a potential therapeutic target in Fibrosarcoma.
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chondroitin sulfate a regulates Fibrosarcoma Cell adhesion motility and migration through jnk and tyrosine kinase signaling pathways
in Vivo, 2009Co-Authors: Eleni Fthenou, Fang Zong, Alexandros Zafiropoulos, Katalin Dobra, Anders Hjerpe, George N TzanakakisAbstract:Fibrosarcoma is an uncommon soft tissue tumor with a complex Cell microenvironment, particularly rich in glycosaminoglycans/proteoglycans (GAGs/PGs). Chondroitin sulfate proteoglycans (CSPGs) participate in the modulation of various Cellular functions, including adhesion and migration. The role of chondroitin sulfate (CS) chains on adhesion, chemotaxis and migration of poorly differentiated Fibrosarcoma B6FS Cell was studied, utilizing exogenous CS treatment and chondroitinase digestions as well as specific modulators of CS synthesis. Cleavage of Cell-associated CS chains and specific inhibition of endogenous CS production severely impaired these Fibrosarcoma Cell functions. These results show that the reduction of endogenous CSPG expression as well as cleavage of the CS chain inhibited Fibrosarcoma Cell motility, migration and adhesion. Treatment with free CS chains enhanced Cell chemotaxis and migration, whereas adhesion was inhibited. CS chains were found to upregulate Cell motility through the MAPK pathway, specifically through JNK, whereas CS-induced migration was found to require tyrosine kinase dependent pathways. This study suggests a new role of CS on tumor Cell adhesion, chemotaxis and migration. The interactions between Cells and the surrounding extraCellular matrix (ECM) play crucial roles in various processes such as Cell adhesion, migration and tumor growth. Cellular adhesion to ECM is a complex, tightly regulated process in which glycosaminoglycans/proteoglycans (GAGs/PGs) can participate in the formation of the specific adhesion sites. Cell motility and
Aikaterini Berdiaki - One of the best experts on this subject based on the ideXlab platform.
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igf i regulates ht1080 Fibrosarcoma Cell migration through a syndecan 2 erk ezrin signaling axis
Experimental Cell Research, 2017Co-Authors: Maria Mytilinaiou, Dragana Nikitovic, Aikaterini Berdiaki, Antonis Papoutsidakis, Dionysios J Papachristou, Aristidis Tsatsakis, George N TzanakakisAbstract:Fibrosarcoma is a tumor of mesenchymal origin, originating from fibroblasts. IGF-I is an anabolic growth factor which exhibits significant involvement in cancer progression. In this study, we investigated the possible participation of syndecan-2 (SDC-2), a Cell membrane heparan sulfate (HS) proteoglycan on IGF-I dependent Fibrosarcoma Cell motility. Our results demonstrate that SDC-2-deficient HT1080 Cells exhibit attenuated IGF-I-dependent chemotactic migration (p < 0.001). SDC-2 was found to co-localize to IGF-I receptor (IGF-IR) in a manner dependent on IGF-I activity (P ≤ 0.01). In parallel, the downregulation of SDC-2 significantly inhibited both basal and due to IGF-I action ERK1/2 activation, (p < 0.001). The phosphorylation levels of ezrin (Thr567), which is suggested to act as a signaling bridge between the Cellular membrane receptors and actin cytoskeleton, were strongly enhanced by IGF-I at both 1h and 24h (p < 0.05; p < 0.01). The formation of an immunoprecipitative complex revealed an association between SDC2 and ezrin which was enhanced through IGF-I action (p < 0.05). Immunoflourescence demonstrated a co-localization of IGF-IR, SDC2 and ezrin upregulated by IGF-I action. IGF-I enhanced actin polymerization and ezrin/actin specific localization to Cell membranes. Finally, treatment with IGF-I strongly increased SDC2 expression at both the mRNA and protein level (p < 0.001). Therefore, we propose a novel SDC2-dependent mechanism, where SDC2 is co-localized with IGF-IR and enhances its' IGFI-dependent downstream signaling. SDC2 mediates directly IGFI-induced ERK1/2 activation, it recruits ezrin, contributes to actin polymerization and ezrin/actin specific localization to Cell membranes, ultimately facilitating the progression of IGFI-dependent Fibrosarcoma Cell migration.
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receptor for hyaluronic acid mediated motility rhamm regulates ht1080 Fibrosarcoma Cell proliferation via a β catenin c myc signaling axis
Biochimica et Biophysica Acta, 2016Co-Authors: Katerina Kouvidi, Evgenia Karousou, Dragana Nikitovic, Aikaterini Berdiaki, Alberto Passi, Maria Tzardi, George N TzanakakisAbstract:Abstract Background High levels of hyaluronan (HA) synthesis in various cancer tissues, including sarcomas, are correlated with tumorigenesis and malignant transformation. RHAMM (receptor for hyaluronic acid-mediated motility) is overexpressed during tumor development in different malignancies. β-Catenin is a crucial downstream mediator of the Wnt signaling cascade which facilitates carcinogenic events characterized by deregulated Cell proliferation. Methods Real-time PCR, in vitro Cell proliferation assay, siRNA transfection, flow cytometry, immunoprecipitation, western blotting and immunofluorescence were utilized. Results The reduction of RHAMM expression was strongly correlated with an inhibition of HT1080 Fibrosarcoma Cell growth (p ≤ 0.01). LMWHA, in a RHAMM-dependent manner increases Cell growth of HT1080 Cells ((p ≤ 0.01). Both basal and LMWHA dependent growth of HT1080 Cells was attenuated by β-catenin deficiency (p ≤ 0.01). β-Catenin cytoplasmatic deposition is positively regulated by RHAMM (p ≤ 0.01). Immunoflourescence and immunoprecipitation suggest that RHAMM/β-catenin form an intraCellular complex. Transfection experiments identified c-myc as candidate downstream mediator of RHAMM/β-catenin effects on HT1080 Fibrosarcoma Cell proliferation. Conclusions LMWHA/RHAMM downstream signaling regulates Fibrosarcoma Cell growth in a β-catenin/c-myc dependent manner. General significance The present study suggests that RHAMM is a novel β-catenin intraCellular binding partner, protecting β-catenin from degradation and supporting the nuclear translocation of this key Cellular mediator, which results in c-myc activation and enhanced Fibrosarcoma Cell growth.
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role of receptor for hyaluronic acid mediated motility rhamm in low molecular weight hyaluronan lmwha mediated Fibrosarcoma Cell adhesion
Journal of Biological Chemistry, 2011Co-Authors: Katerina Kouvidi, Dragana Nikitovic, Aikaterini Berdiaki, Pavlos Katonis, Nikos K. Karamanos, Nikos Afratis, Vincent C Hascall, George N TzanakakisAbstract:Hyaluronan (HA) modulates key cancer Cell functions through interaction with its CD44 and receptor for hyaluronic acid-mediated motility (RHAMM) receptors. HA was recently found to regulate the migration of Fibrosarcoma Cells in a manner specifically dependent on its size. Here, we investigated the effect of HA/RHAMM signaling on the ability of HT1080 Fibrosarcoma Cells to adhere onto fibronectin. Low molecular weight HA (LMWHA) significantly increased (p ≤ 0.01) the adhesion capacity of HT1080 Cells, which high molecular weight HA inhibited. The ability of HT1080 RHAMM-deficient Cells, but not of CD44-deficient ones, to adhere was significantly decreased (p ≤ 0.001) as compared with control Cells. Importantly, the effect of LMWHA on HT1080 Cell adhesion was completely attenuated in RHAMM-deficient Cells. In contrast, adhesion of RHAMM-deficient Cells was not sensitive to high molecular weight HA treatment, which identifies RHAMM as a specific conduit of the LMWHA effect. Western blot and real time-PCR analyses indicated that LMWHA significantly increased RHAMM transcript (p ≤ 0.05) and protein isoform levels (53%, 95 kDa; 37%, 73 kDa) in Fibrosarcoma Cells. Moreover, Western blot analyses showed that LMWHA in a RHAMM-dependent manner enhanced basal and adhesion-dependent ERK1/2 and focal adhesion kinase (FAK) phosphorylation in HT1080 Cells. Utilization of a specific ERK1/2 inhibitor completely inhibited (p ≤ 0.001) LMWHA-dependent adhesion, suggesting that ERK1/2 is a downstream effector of LMWHA/RHAMM signaling. Likewise, the utilization of the specific ERK1 inhibitor resulted in a strong down-regulation of FAK activation in HT1080 Cells, which identifies ERK1/2 as a FAK upstream activator. In conclusion, our results suggest that RHAMM/HA interaction regulates Fibrosarcoma Cell adhesion via the activation of FAK and ERK1/2 signaling pathways.
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bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of Fibrosarcoma Cells.
Biochimica et biophysica acta, 2009Co-Authors: Aikaterini Berdiaki, Dragana Nikitovic, Pavlos Katonis, Nikos K. Karamanos, Aristeidis M. Tsatsakis, George N TzanakakisAbstract:article i nfo Article history: Background: Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extraCellular matrix derived signals to regulate Cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS Fibrosarcoma Cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration. Methods: Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized. Results: bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 Cells (p=0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high mole- cular weight HA deposition in the periCellular matrix. Increased endogenous HA production (p=0.0022) and treatment with exogenous high molecular weight HA (p=0.0268) correlated with a significant decrease of HT1080 Cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 Cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 Cells' motility (pb0.01). In contrast, no effects on B6FS Fibrosarcoma Cell motility were observed. Conclusions: bFGF regulates, in a Cell-specific manner the migration capability of Fibrosarcoma Cells by modulating their HA metabolism. HA metabolism is suggested to be a potential therapeutic target in Fibrosarcoma.
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Regulation of hyaluronan and versican deposition by growth factors in Fibrosarcoma Cell lines.
Biochimica et biophysica acta, 2007Co-Authors: Aikaterini Berdiaki, Eleni Fthenou, Alexandros Zafiropoulos, Aristidis Tsatsakis, Pavlos Katonis, Nikos K. Karamanos, George N TzanakakisAbstract:Versican, a large chondroitin sulphate proteoglycan and hyaluronan (HA), a non-sulphated glycosaminoglycan are major constituents of the periCellular matrix. In many neoplastic tissues, changes in the expression of versican and HA affect tumour progression. Here, we analyse the synthesis of versican and hyaluronan by Fibrosarcoma Cells, and document how the latter is affected by PDGF-BB, bFGF and TGFB2, growth factors endogenously produced by these Cells. Fibrosarcoma Cell lines B6FS and HT1080 were utilised and compared with normal lung fibroblasts (DLF). The major versican isoforms expressed by DLF and B6FS Cells were V0 and V1. Treatment of B6FS Cells with TGFB2 showed a significant increase of V0 and V1 mRNAs. Versican expression in HT1080 Cells was not significantly affected by any of the growth factors. In addition, TGFB2 treatment increased versican protein in DLF Cells. HA, showed approximately a 2-fold and a 9-fold higher production in DLF Cells compared to B6FS and HT1080 Cells, respectively. In HT1080 Cells, HA biosynthesis was significantly increased by bFGF, whereas, in B6FS Cells it was increased by TGFB2 and PDGF-BB. Furthermore, analysis of HA synthases (HAS) expression indicated that HT1080 expressed similar levels of all three HAS isoforms in the following order: HAS2> HAS3> HAS1. bFGF shifted that balance by increasing the abundance of HAS1. The major HAS isoform expressed by B6FS Cells was HAS2. PDGF-BB and TGFB2 showed the most prominent effects by increasing both HAS2 and HAS1 isoforms. In conclusion, these growth factors modulated, through upregulation of specific HAS isoforms, HA synthesis, secretion and net deposition to the periCellular matrix.
K. Zabielska - One of the best experts on this subject based on the ideXlab platform.
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enhancing anti tumor efficacy of doxorubicin by non covalent conjugation to gold nanoparticles in vitro studies on feline Fibrosarcoma Cell lines
PLOS ONE, 2015Co-Authors: Michal Wojcik, R Lechowski, K M Pawlowski, Wiktor Lewandowski, Magdalena Krol, Jozef Mieczkowski, K. ZabielskaAbstract:Background Feline injection-site sarcomas are malignant skin tumors of mesenchymal origin, the treatment of which is a challenge for veterinary practitioners. Methods of treatment include radical surgery, radiotherapy and chemotherapy. The most commonly used cytostatic drugs are cyclophosphamide, doxorubicin and vincristine. However, the use of cytostatics as adjunctive treatment is limited due to their adverse side-effects, low biodistribution after intravenous administration and multidrug resistance. Colloid gold nanoparticles are promising drug delivery systems to overcome multidrug resistance, which is a main cause of ineffective chemotherapy treatment. The use of colloid gold nanoparticles as building blocks for drug delivery systems is preferred due to ease of surface functionalization with various molecules, chemical stability and their low toxicity. Methods Stability and structure of the glutathione-stabilized gold nanoparticles non-covalently modified with doxorubicin (Au-GSH-Dox) was confirmed using XPS, TEM, FT-IR, SAXRD and SAXS analyses. MTT assay, Annexin V and Propidium Iodide Apoptosis assay and Rhodamine 123 and Verapamil assay were performed on 4 feline Fibrosarcoma Cell lines (FFS1WAW, FFS1, FFS3, FFS5). Statistical analyses were performed using Graph Pad Prism 5.0 (USA). Results A novel approach, glutathione-stabilized gold nanoparticles (4.3 +/- 1.1 nm in diameter) non-covalently modified with doxorubicin (Au-GSH-Dox) was designed and synthesized. A higher cytotoxic effect (p<0.01) of Au-GSH-Dox than that of free doxorubicin has been observed in 3 (FFS1, FFS3, FFS1WAW) out of 4 feline Fibrosarcoma Cell lines. The effect has been correlated to the activity of glycoprotein P (main efflux pump responsible for multidrug resistance). Conclusions The results indicate that Au-GSH-Dox may be a potent new therapeutic agent to increase the efficacy of the drug by overcoming the resistance to doxorubicin in feline Fibrosarcoma Cell lines. Moreover, as doxorubicin is non-covalently attached to glutathione coated nanoparticles the synthesized system is potentially suitable to a wealth of different drug molecules.
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Derivation of feline vaccine-associated Fibrosarcoma Cell line and its growth on chick embryo chorioallantoic membrane – a new in vivo model for veterinary oncological studies
Veterinary Research Communications, 2012Co-Authors: K. Zabielska, K. M. Pawłowski, T. Motyl, R Lechowski, Izabella Dolka, M Krol, Artur żbikowskiAbstract:Feline vaccine associated Fibrosarcomas are the second most common skin tumor in cats. Methods of treatment are: surgery, chemotherapy and radiotherapy. Nevertheless, the usage of cytostatics in feline vaccine associated sarcoma therapy is limited due to their adverse side effects, high toxicity and low biodistribution after i.v. injection. Therefore, much research on new therapeutic drugs is being conducted. In human medicine, the chick embryo chorioallantoic membrane (CAM) model is used as a cheap and easy to perform assay to assess new drug effectiveness in cancer treatment. Various human Cell lines have different tumors growth on CAM. In veterinary medicine such model has not been described yet. In the present article derivation of feline vaccine associated Fibrosarcoma Cell line and its growth on CAM is described. The Cell line and the tumor grown were confirmed by histopathological and immunohistochemical examination. As far as we believe, this is the first attempt to create such model, which may be used for further in vivo studies in veterinary oncology.
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derivation of feline vaccine associated Fibrosarcoma Cell line and its growth on chick embryo chorioallantoic membrane a new in vivo model for veterinary oncological studies
Veterinary Research Communications, 2012Co-Authors: K. Zabielska, T. Motyl, R Lechowski, Izabella Dolka, K M Pawlowski, M Krol, Artur żbikowskiAbstract:Feline vaccine associated Fibrosarcomas are the second most common skin tumor in cats. Methods of treatment are: surgery, chemotherapy and radiotherapy. Nevertheless, the usage of cytostatics in feline vaccine associated sarcoma therapy is limited due to their adverse side effects, high toxicity and low biodistribution after i.v. injection. Therefore, much research on new therapeutic drugs is being conducted. In human medicine, the chick embryo chorioallantoic membrane (CAM) model is used as a cheap and easy to perform assay to assess new drug effectiveness in cancer treatment. Various human Cell lines have different tumors growth on CAM. In veterinary medicine such model has not been described yet. In the present article derivation of feline vaccine associated Fibrosarcoma Cell line and its growth on CAM is described. The Cell line and the tumor grown were confirmed by histopathological and immunohistochemical examination. As far as we believe, this is the first attempt to create such model, which may be used for further in vivo studies in veterinary oncology.
Dragana Nikitovic - One of the best experts on this subject based on the ideXlab platform.
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igf i regulates ht1080 Fibrosarcoma Cell migration through a syndecan 2 erk ezrin signaling axis
Experimental Cell Research, 2017Co-Authors: Maria Mytilinaiou, Dragana Nikitovic, Aikaterini Berdiaki, Antonis Papoutsidakis, Dionysios J Papachristou, Aristidis Tsatsakis, George N TzanakakisAbstract:Fibrosarcoma is a tumor of mesenchymal origin, originating from fibroblasts. IGF-I is an anabolic growth factor which exhibits significant involvement in cancer progression. In this study, we investigated the possible participation of syndecan-2 (SDC-2), a Cell membrane heparan sulfate (HS) proteoglycan on IGF-I dependent Fibrosarcoma Cell motility. Our results demonstrate that SDC-2-deficient HT1080 Cells exhibit attenuated IGF-I-dependent chemotactic migration (p < 0.001). SDC-2 was found to co-localize to IGF-I receptor (IGF-IR) in a manner dependent on IGF-I activity (P ≤ 0.01). In parallel, the downregulation of SDC-2 significantly inhibited both basal and due to IGF-I action ERK1/2 activation, (p < 0.001). The phosphorylation levels of ezrin (Thr567), which is suggested to act as a signaling bridge between the Cellular membrane receptors and actin cytoskeleton, were strongly enhanced by IGF-I at both 1h and 24h (p < 0.05; p < 0.01). The formation of an immunoprecipitative complex revealed an association between SDC2 and ezrin which was enhanced through IGF-I action (p < 0.05). Immunoflourescence demonstrated a co-localization of IGF-IR, SDC2 and ezrin upregulated by IGF-I action. IGF-I enhanced actin polymerization and ezrin/actin specific localization to Cell membranes. Finally, treatment with IGF-I strongly increased SDC2 expression at both the mRNA and protein level (p < 0.001). Therefore, we propose a novel SDC2-dependent mechanism, where SDC2 is co-localized with IGF-IR and enhances its' IGFI-dependent downstream signaling. SDC2 mediates directly IGFI-induced ERK1/2 activation, it recruits ezrin, contributes to actin polymerization and ezrin/actin specific localization to Cell membranes, ultimately facilitating the progression of IGFI-dependent Fibrosarcoma Cell migration.
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receptor for hyaluronic acid mediated motility rhamm regulates ht1080 Fibrosarcoma Cell proliferation via a β catenin c myc signaling axis
Biochimica et Biophysica Acta, 2016Co-Authors: Katerina Kouvidi, Evgenia Karousou, Dragana Nikitovic, Aikaterini Berdiaki, Alberto Passi, Maria Tzardi, George N TzanakakisAbstract:Abstract Background High levels of hyaluronan (HA) synthesis in various cancer tissues, including sarcomas, are correlated with tumorigenesis and malignant transformation. RHAMM (receptor for hyaluronic acid-mediated motility) is overexpressed during tumor development in different malignancies. β-Catenin is a crucial downstream mediator of the Wnt signaling cascade which facilitates carcinogenic events characterized by deregulated Cell proliferation. Methods Real-time PCR, in vitro Cell proliferation assay, siRNA transfection, flow cytometry, immunoprecipitation, western blotting and immunofluorescence were utilized. Results The reduction of RHAMM expression was strongly correlated with an inhibition of HT1080 Fibrosarcoma Cell growth (p ≤ 0.01). LMWHA, in a RHAMM-dependent manner increases Cell growth of HT1080 Cells ((p ≤ 0.01). Both basal and LMWHA dependent growth of HT1080 Cells was attenuated by β-catenin deficiency (p ≤ 0.01). β-Catenin cytoplasmatic deposition is positively regulated by RHAMM (p ≤ 0.01). Immunoflourescence and immunoprecipitation suggest that RHAMM/β-catenin form an intraCellular complex. Transfection experiments identified c-myc as candidate downstream mediator of RHAMM/β-catenin effects on HT1080 Fibrosarcoma Cell proliferation. Conclusions LMWHA/RHAMM downstream signaling regulates Fibrosarcoma Cell growth in a β-catenin/c-myc dependent manner. General significance The present study suggests that RHAMM is a novel β-catenin intraCellular binding partner, protecting β-catenin from degradation and supporting the nuclear translocation of this key Cellular mediator, which results in c-myc activation and enhanced Fibrosarcoma Cell growth.
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role of receptor for hyaluronic acid mediated motility rhamm in low molecular weight hyaluronan lmwha mediated Fibrosarcoma Cell adhesion
Journal of Biological Chemistry, 2011Co-Authors: Katerina Kouvidi, Dragana Nikitovic, Aikaterini Berdiaki, Pavlos Katonis, Nikos K. Karamanos, Nikos Afratis, Vincent C Hascall, George N TzanakakisAbstract:Hyaluronan (HA) modulates key cancer Cell functions through interaction with its CD44 and receptor for hyaluronic acid-mediated motility (RHAMM) receptors. HA was recently found to regulate the migration of Fibrosarcoma Cells in a manner specifically dependent on its size. Here, we investigated the effect of HA/RHAMM signaling on the ability of HT1080 Fibrosarcoma Cells to adhere onto fibronectin. Low molecular weight HA (LMWHA) significantly increased (p ≤ 0.01) the adhesion capacity of HT1080 Cells, which high molecular weight HA inhibited. The ability of HT1080 RHAMM-deficient Cells, but not of CD44-deficient ones, to adhere was significantly decreased (p ≤ 0.001) as compared with control Cells. Importantly, the effect of LMWHA on HT1080 Cell adhesion was completely attenuated in RHAMM-deficient Cells. In contrast, adhesion of RHAMM-deficient Cells was not sensitive to high molecular weight HA treatment, which identifies RHAMM as a specific conduit of the LMWHA effect. Western blot and real time-PCR analyses indicated that LMWHA significantly increased RHAMM transcript (p ≤ 0.05) and protein isoform levels (53%, 95 kDa; 37%, 73 kDa) in Fibrosarcoma Cells. Moreover, Western blot analyses showed that LMWHA in a RHAMM-dependent manner enhanced basal and adhesion-dependent ERK1/2 and focal adhesion kinase (FAK) phosphorylation in HT1080 Cells. Utilization of a specific ERK1/2 inhibitor completely inhibited (p ≤ 0.001) LMWHA-dependent adhesion, suggesting that ERK1/2 is a downstream effector of LMWHA/RHAMM signaling. Likewise, the utilization of the specific ERK1 inhibitor resulted in a strong down-regulation of FAK activation in HT1080 Cells, which identifies ERK1/2 as a FAK upstream activator. In conclusion, our results suggest that RHAMM/HA interaction regulates Fibrosarcoma Cell adhesion via the activation of FAK and ERK1/2 signaling pathways.
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bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of Fibrosarcoma Cells.
Biochimica et biophysica acta, 2009Co-Authors: Aikaterini Berdiaki, Dragana Nikitovic, Pavlos Katonis, Nikos K. Karamanos, Aristeidis M. Tsatsakis, George N TzanakakisAbstract:article i nfo Article history: Background: Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extraCellular matrix derived signals to regulate Cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS Fibrosarcoma Cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration. Methods: Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized. Results: bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 Cells (p=0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high mole- cular weight HA deposition in the periCellular matrix. Increased endogenous HA production (p=0.0022) and treatment with exogenous high molecular weight HA (p=0.0268) correlated with a significant decrease of HT1080 Cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 Cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 Cells' motility (pb0.01). In contrast, no effects on B6FS Fibrosarcoma Cell motility were observed. Conclusions: bFGF regulates, in a Cell-specific manner the migration capability of Fibrosarcoma Cells by modulating their HA metabolism. HA metabolism is suggested to be a potential therapeutic target in Fibrosarcoma.
Altomare Di Benedetto - One of the best experts on this subject based on the ideXlab platform.
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targeting a newly established spontaneous feline Fibrosarcoma Cell line by gene transfer
PLOS ONE, 2012Co-Authors: Rounak Nande, Altomare Di Benedetto, Pierpaolo Aimola, Flavia De Carlo, Miranda B CarperAbstract:Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) Fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS Fibrosarcoma. We isolated and characterized a NVS Fibrosarcoma Cell line (Cocca-6A) from a spontaneous Fibrosarcoma that occurred in a domestic calico cat. The feline Cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study Cell-cycle changes and Cell death of transduced Cells. Cocca-6A Fibrosarcoma Cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A Cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A Cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A Cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline Fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline Fibrosarcomas.