| Abstract |
Fibrosarcoma is an uncommon tumor with a rich extracellular matrix, there are no
specific biomarkers for its diagnosis. Versican, a large chondroitin sulphate proteoglycan and
hyaluronan (HA), a non-sulphated glycosaminoglycan are major constituents of the
pericellular matrix. Inmany neoplastic tissues, changes in the expression of versican and
HAaffect tumour progression. In this study, it was analysed 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 HT1080were 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 ofV0 and V1mRNAs. 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: HAS2N HAS3N HAS1. bFGF shifted that
balance by increasing the abundance of HAS1. The major HAS isoform expressed by B6FS
cells was HAS2. PDGFBB 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.
Syndecan-1 is suggested to participate in the regulation of a number of cellular
processes such as cell proliferation and migration. Recent studies have suggested the putative
mechanisms of synmdecan-1 cytoplasmic, transmembrane and extracellular domain action on
cell functions and signal transduction. The aim of this study was to investigate the effect of
specific syndecan-1 domains on fibrosarcoma cell migration and proliferation. Transfected
B6FS fibrosarcoma cells with full length (FL/EGFP)of syndecan-1 construct, a variant
coding for the endodomain (77/EGFP) and a variant coding for the RMKKK cytoplasmic
sequence, were used. The expression and cellular localization of the FL, 77 and RMKKK
recombinant proteins was analysed using confocal microscopy, after six weeks selection
using geneticin (400μg/ml). Recombinant FL/EGFP showed cell membrane reactivity while
RMKKK/EGFP showed distinct nuclear localization. The overexpression of 77/EGFP that
lacks the ectodomain resulted in altered morphology, the cells became smaller and rounded.
The proliferation of transfected fibrosarcoma cells with the respective constructs was
measured using WST-1 colorimetric reagent. Our results demonstrated that the expression of
both full length and the two truncated recombinant proteins, compared to vector transfected
cells, significantly inhibited fibrosarcoma cell proliferation. B6FS transfected with the 77/
EGFP and the RMKKK/EGFP constructs lack the ectodomain of syndecan-1, enhancing the
suggestion that the syndecan-1 ectodomain is responsible for the stimulation of
proliferation.Furthermore, we studied the effect of FL/EGFP, the endodomain 77/EGFP and
the cytoplasmic RMKKK/EGFP recombinant proteins on the migrative response of B6FS
fibrosarcoma cells. Our results demonstrated that B6FS cells transfected with FL/EGFP and
RMKKK/EGFP constructs had an enhanced migration capability compared to EGFP vector
transfected cells. In contrast, the expression of 77/EGFP recombinant protein inhibited B6FS
cell migration. This study suggets that syndecan-1 ectodomain may have a “switch”
function , resulting in activation of the cytoplasmic domain, after ligand binding.
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 β-
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. β-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.
Additionally, the effect of chondtoitin sulphate A on PDGF-BB induced
proliferation, and migration of normal fibroblasts was examined.The aim of the present study
was to examine the involvement of CS on PDGF-BB induced proliferative responses and
receptor activation in human lung fibroblasts. The addition of exogenous free CS chains
caused a significant downregulation of the PDGF-BB mediated mitogenic and chemotactic
responses. Similar results were obtained by the increase of endogenous CS biosynthesis after
β-D-xyloside treatment. Furthermore, removal of the membrane-bound CS chains by
selective enzymatic treatment significantly increased the proliferative capacity of human
fibroblasts. Analysis of PDGF-R phosphorylation in the presence of CS or β-D-xyloside,
demonstrated a reduction of PDGF-Rβ phosphorylation in the tyrosine residue 1021. These
results demonstrate, for the first time, that CS either soluble or surface bound downregulates
the mitogenic responses of PDGF-BB in normal human lung fibroblasts through the
reduction of PDGF-Rβ phosphorylation.
Matrix adhesion signals by regulating the assembly of the actin cytoskeleton and the
associated integrin adhesion complexes play a key role in cell motility and chemotaxis.
Fibrosarcoma is an uncommon soft tissue tumor whose cell microenvironment is rich in
ECM components and particularly in glycosaminoglycans/proteoglycans (GAGs/ PGs). In
this study the role of chondroitin sulfate (CS) was investigated on fibrosarcoma cell
adhesion, motility and migration, utilizing exogenous CS treatment, chondroitinase
digestions as well as specific modulators of CS synthesis. Cleavage of cell-associated CS as
well as specific inhibition of endogenous CS production severely impaired these
fibrosarcoma cell functions. Treatment with free CS chains enhanced cell motility and
chemotaxis, whereas adhesion was inhibited. When inhibitors of the main cellular signaling
pathways regulating actin cytoskeleton rearrangements were applied, CS chains were found
to upregulate cell motility through the MAPK pathway, specifically through JNK, whereas
CS-induced chemotaxis was found to require tyrosine kinase dependent pathways. This study
introduces a new crucial role of CS chains on tumor cell adhesion, motility and chemotaxis.
Summarizing, this study introduces a new role of chondroitin sulfate A in the
regulation of proliferation,adhesion, migration and chemotaxis of both fibrosarcoma and
normal fibroblasts through tyrosine kinases and JNK signaling pathways.
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