Tumor-stroma interactions play a pivotal role in regulation tumor progression and malignance. We had shown previously that undersulfation of GAGs reduces the proliferation of the leukemic cell line Wehi 3B and C6 glioma cells suggesting a regulatory role of proteoglycans on cellular processes involved in tumor progression. We report here that GAGs are also involved in the control of glioma cell adhesion, proliferation and migration on ECM proteins.
It has long been appreciated that some of the earliest steps of neoplastic transformation involve changes in tissue interaction, including alterations in cell adhesion. Loss of cell adhesion was postulated as involved in genomic instability and related to the invasive behavior and motility of malignant cells. Among ECM proteins, laminin was shown as the most potent stimulator of glioma migration and invasion[10, 20]. In particular, laminin mediate C6 glioma cells invasion both in vitro and in vivo . Mahesparan and co-workers reported that laminin was the most efficient ECM protein in inducing cell migration of three different human glioma cell lines (U-373 MG, A-172 MG and HF-66), followed by fibronectin, while type IV collagen did not promote cell migration. In our experiments, however, we observed an inverse correlation with rat C6 glioma regarding both adhesion and migration: type IV collagen was the most potent substrate, followed by fibronectin and somewhat weaker attachment and migration on laminin. It is possible that the different adhesion ability of C6 cells to ECM proteins may be related to its migratory capacity.
For cell adhesion and migration, specific cell surface receptors that interact with ECM components are needed. Cell-surface heparan sulfate or chondroitin sulfate proteoglycans have been implicated in cell attachment via interactions with ECM proteins, such as laminin and fibronectin, and integrins. The syndecan family of transmembrane proteoglycans can act as co-receptors to modulate integrin-mediated cell-matrix adhesion. Together with integrins, they form a dual receptor system active in cell-matrix adhesion. Cell attachment and spreading can be promoted through integrin interactions with the integrin-binding domain of fibronectin. Additional binding of a heparin-binding domain of fibronectin to a cell surface heparan sulfate proteoglycans (HSPG) promotes focal adhesion formation by activating PKC. The HSPG involved appears to be exclusively syndecan-4 that binds to fibronectin via its GAG side chains.
Several studies have suggested that cell surface proteoglycans are involved in the control of glioma cell growth, adhesion and invasion. The chondroitin sulfate lectican BEHAB is up-regulated in malignant gliomas and derived cell lines, and was proposed to play a role in brain tumor invasion. C6 glioma cells transfected with appican display dramatic change in their phenotypic appearance as well as increased cell adhesion . In addition, we have previously demonstrated that sodium chlorate treatment, an inhibitor of GAG's sulfation, reduces C6 cells proliferation and adhesion to ECM proteins further suggesting that cell surface highly sulfated components act as co-receptor for glioma cell attachment to ECM. In the present paper, we observed that the addition of heparin or chondroitin sulfate to the culture dish together within cells impaired C6 adhesion affecting the earliest steps of C6 cell adhesion, possibly due to the competition with GAG chains of cell surface proteoglycans to fibronectin and laminin binding sites. We could suggest that the specific interactions of GAGs with fibronectin and laminin affect the organization of focal adhesions necessary to promote cell adhesion. In contrast, addition of heparin or chondroitin sulfate after cells completely attached to substrate did not promote cell detachment possibly because focal adhesion points were already established and the addition of GAGs were not sufficient to disturb them. In fact, it has been reported that ECM ligands bind to heparan sulfate proteoglycans and produce actin filament reorganization even in the absence of integrin occupancy. The interactions of heparan sulfate chains with extracellular ligands may help the formation of proteoglycan clusters, which appears to be critical for focal adhesion formation.
Moreover, it was reported that cell growth is anchorage dependent. Thus, the reduction in C6 cell proliferation on fibronectin promoted by heparin and chondroitin sulfate observed in our experiments could be consequence of the altered cell adhesion. In addition, highly sulfated GAGs, heparin and heparan sulfate, and sulfated polysaccharide, fucoidan, have been reported stimulating tumor cell invasion in vitro, due to a stimulation of the proteolytic cascade of plasminogen activation. This suggests that sulfated GAGs liberated by tumor cells, mediate ECM degradation amplifying pericellular plasminogen activation and locally enhancing tumor cell invasion in a positive feedback. In our experiments we observed that both chondroitin sulfate and heparin induced C6 cell migration on laminin substrate in different proportions according to the dose, with the most prominent effect at the fist 6 hours. These results are consistent with the influence of the GAGs on the earliest steps of cell adhesion to ECM proteins (Figure 1 and Table 1). It is possible that GAGs induce the organization of adhesion contacts in C6 cells that specifically lead to proliferation or migration when maintained on fibronectin or laminin, respectively.