| Abstract |
Osteosarcoma is the most common bone tumor associated with childhood and adolescence. The main characteristics of the osteosarcoma are the malignant transformation of the osteoblasts and the excessive accumulation of the partially mineralized extracellular matrix (ECM). The abundant content of the ECM modulates the microenvironment of the bone tissue and affects the normal function of the osteoblasts as well as the differentiation of the malignant cells.
Proteoglycans (PGs) are one of the major classes of macromolecules located both at the cell membrane and the ECM. They consist of a protein core to which one or more sulfated glycosaminoglycan (GAG) chains are covalently bound. GAG chains are linear polymers composed of repeating dissacharide units consisting of a hexosamine and an uronic acid. The specific physicochemical characteristics with which PGs are endowed enable them to affect the organization of the ECM, as well as to participate in the regulation of several cellular events, including cell proliferation, adhesion and migration. These roles of PGs are perpetrated through specific interactions with target macromolecules or growth factors and these interactions may involve both their GAG chains and/or protein cores.
PGs are major non-collagen component of the bone ECM. They are synthesized and secreted by osteoblasts and participate in the regulation of bone calcification. Furthermore, it has been suggested that cell membrane (mainly heparan sulfate containing) as well as secreted PGs participate in cell-cell and cell-ECM interactions and play important roles in the in the formation and the renewal of the bone tissue.
Both the physicochemical characteristics and the alternations in the composition of the osteosarcoma ECM as well as the association of these changes with its differentiation and prognosis have not been adequately studied. The role of PGs in the mechanisms of osteosarcoma development is suggested to be important. The fine
structural analysis of the proteoglycans’ GAG chains may contribute to the better understanding of these glycocomplexes, which participate in the regulation of numerous cellular events as well as in the cell malignant transformation. Therefore, the aim of this study was to contribute to the understanding of the role specific PGs / GAGs have in the pathology of the osteosarcoma, which is one of the most important human primary bone tumors.
MG-63 and Saos 2 human osteosarcoma cell lines, endowed with high and low metastatic capability, and differing in their differentiation level, being medium and well differentiated, respectively were utilized. The synthesis and the distribution of GAGs among the cell membrane and the culture medium by these cell lines were studied. The obtained results showed that both cell lines synthesized extracellular hyaluronan (HA) and both extracellular and cell-associated galactosaminoglycans (GalAGs) and heparin sulfate (HS). Even though both cell lines synthesized considerable amounts of PGs, the Saos 2 cells produced HA, GalAGs and HS at considerably lower rates than the MG-63 cells.
The role of genistein on the synthesis of these macromolecules has also been studied. Genistein is a well known specific inhibitor of the protein tyrosine kinase (PTK) and affects the proliferation of both cancer and normal cells. The inhibitory effect of genistein on the synthesis of both extracellularly secreted and cell-associated GAGs / PGs in Saos 2 cells was found to be dose-dependent and mediated most probably through a PTK mechanism. The synthesis of GAGs / PGs by the MG-63 cells in the presence of genistein was dependent on their type and localization suggesting that a more complex mechanism regulates their PG synthesis.
It has been shown that growth factors such as the transforming growth factor (TGF-β2), basic fibroblast growth factor (bFGF) and the platelet derived growth factor
support the growth of osteosarcoma. Therefore, we examined the actions of TGF-β2, bFGF and PDGF-BB on the synthesis and the distribution of the GAGs / PG between the two cell lines. The obtained results showed that the action of the growth factors differs between the two cell lines and that the regulation of the GAG synthesis depends on their type and location.
Alternations in the structural composition of the GAG/PG component may have important consequences on the cell proliferation and / or differentiation. Human osteoblasts and the two osteosarcoma cell lines, able to produce galactosaminoglycan (GalAGs) and heparan sulfate (HS)-containing proteoglycans, were treated with their main GAG chain types (i.e. chondtroitin sulfate (CS), dermatan sulfate (DS) and heparin) and the their effects on cell growth were examined. The obtained results showed that exogenous GAGs affect the growth of both normal and cancer cell in a dose-dependent fashion. This effect is closely related the fine chemical structure of GAGs, i.e. the presence of the L-iduronic acid and the degree of sulfation.
Versican, large sized chondroitin sulfate PG and its binding partner hyaluronan (HA) are extracellular matrix components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in migration and proliferation of cancer cells. Likewise the disruption of HA synthesis by inhibiting hyaluronan synthase-2 (HAS2) expression in osteosarcoma cells inhibits cell proliferation, migration and invasiveness. Considering that growth factors, such as TGF-β2, bFGF and PDGF-BB, are important regulators for the expression of the ECM molecules, 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). Our results show that TGF-β2 was the major stimulator of HAS2 isoform
expression as well as HA synthesis in osteosarcoma cells, while PDGF-BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. These results indicate that TGF-β2 may account for the metastatic potential of these cells.
The small, leucine rich proteoglycans such as biglycan, decorin, fibromodulin, lumican and osteoadherin are the most abundant proteoglycans of the osteoid. The characteristic horse-shoe shape of their protein core enables them to interact with the other ECM components (mainly with collagen fibers) as well as with the inorganic phase during bone mineralization. The expression of lumican by osteosarcoma cells has not previously been reported.
We examined the expression of lumican by MG-63 and Saos 2 cell osteosarcoma cells. The two human osteosarcoma cell lines were found to express and to secrete lumican which was partly substituted with keratan sulfate chains glycosaminoglycans. Importantly, the non-metastatic, well differentiated Saos 2 cells produced lumican at rates that were up to seven-fold higher tan the highly metastatic MG-63 cells. The utilization of short interfering RNA specific for the lumican gene resulted in efficient down-regulation of its mRNA levels in both cell lines. The gowth of Saos 2 cells was inhibited by lumican, whereas their migration and chemotactic response to fibronectin were found to be promoted. Lumican expression was negatively correlated with the basal level of Smad activation in these cells, suggesting that lumican may affect the bioavailability of Smad 2 activators. By contrast, these cellular functions of highly aggressive MG-63 cells were demonstrated not to be sensitive to a decrease in their low endogenous lumican levels. These results suggest that lumican expression may be positively correlated with the differentiation and negatively to the progression of the osteosarcoma.
Decorin is a well studied SLRP, reported to have inhibitory action on the growth of numerous cell lines. It has been shown that decorin by acting through the EFGR pathway causes apoptosis/ cell growth inhibition. The aim of the present phase of our study was to examine the regulation of decorin synthesis by the MG-63 and Saos 2 cell line as well as by human osteoblasts, after treatment with TGF-β2, PDGF-BB and bFGF. Likewise, the effect of decorin on these cells’ growth was examined. We show that the MG-63 cells in contrast to the Saos 2 cells and the normal osteoblasts express decorin. Furthermore, the expression of decorin by the MG-63 cells was stimulated by all utilized growth factors while none of the treatments induced decorin expression in Saos 2 cell and the normal osteoblasts. Treatment with exogenous decorin inhibited Saos 2 cell growth while neither exogenous decorin nor downregulation of the endogenous decorin synthesis did affect MG-63 cell growth. On the contrary, treatment with decorin seemed to be beneficial to osteosarcoma cells, since it was necessary for MG-63 cell migration and acted as a mediator, counteracting the TGF-β2-induced cytostatic function. In contrast to the established model decorin in MG-63 ce3lls did not induce p21 expression nor cause protracted retraction and inactivation of the EGFR. Conversely, EFGR appeared to be overexpressed and continuously phosphorylated. These results provide new data on pathways that cancer cell might emply to overcome the established decorin-induced growth suppression.
N-acetylneuraminic acid and N-glycolylneuraminic acid are the dominant sialic acids in mammals usually found in the non-reducing terminal of oligosaccharide side chains in glycoproteins or glycolipid form. Their expression and distribution pattern has been correlated both with the malignant phenotype and tumor grade of human cancers. Our aim was to examine the synthesis and distribution of sialic acid containing glycoconjugates in human osteosarcoma cell lines and particularly whether the type
and content of N-acetylneuraminic acid and N-glycolylneuraminic acid may be correlated to osteosarcoma metastatic potential. It was determined that the MG-63 and Saos 2 cell lines differ both in the content and distribution of the two sialic acid types. MG-63 cells produce up to 5-fold more total sialic acid as compared to the Saos 2 cells. Neu5Gc is the predominant sialic acid present on the MG-63 cells membrane and represents 40 % of the determined sialic acid by these cells. Saos 2 cell fraction was found to be poor in sialic acid content as only traces of Neu5Ac were detected. However these cells secrete considerable amounts of Neu5Ac to culture media. In conclusion both the total sialic acid and specifically the Neu5Gc content may be correlated to osteosarcoma metastatic potential.
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