9 research outputs found
Primary Ovarian Carcinomas and Abdominal Metastasis Contain 4,6-Disulfated Chondroitin Sulfate Rich Regions, Which Provide Adhesive Properties to Tumour Cells
<div><p>High mortality in ovarian cancer patients is primarily caused through rapid metastasis of the tumour, but the underlying mechanisms are poorly understood. Glycosaminoglycans, are abundantly present in tumours and chondroitin sulfate-E (CSE), a highly 4,6-sulfated glycosaminoglycan, has been indicated to play a role in carcinogenesis. In this study we investigated the presence of CSE in ovarian cancer metastasis and studied its role in tumour cell adhesiveness and migration. CSE was studied immunohistochemically in primary ovarian carcinomas and abdominal metastases using the single chain antibody GD3G7. The role of CSE was studied in 2D (scratch assays) and 3D (collagen matrices, spheroids) systems using SKOV3 cells applying 1: overexpression of CSE by stable transfection with DNA encoding GalNAc4S-6 sulfotransferase, 2: enzymatic removal of CS, and 3: addition of CSE. In ovarian cancer tissue, CSE expression was predominantly seen in the stromal compartment of both primary ovarian carcinomas and metastases, with a comparable degree of intensity and extent. Overexpression of CSE disaccharide units by tumour cells increased their adhesive properties which was especially seen in tumour spheroid formation. Increased expression of CSE reduced cell migration. Addition of free CSE had similar effects. The data presented here indicate that CSE is associated with metastatic lesions and that it provides tumours with adhesive properties. CSE rich motifs are put forward as a potential target for ovarian cancer therapy.</p></div
Reversed phase high performance liquid chromatography (RP-HPLC) for CS disaccharides.
<p>Chondroitin sulfate disaccharide composition of wild type, cells transfected with an empty vector (C5, C6), and cells transfected with a vector containing GalNAc4S-6ST (F5, F7, F9). Significantly higher CSE disaccharide content (*) was found in cells transfected with GalNAc4S-6ST (one-way ANOVA; level of significance p<0.05).</p
Increase of staining for CSE in SKOV3 cells transfected with GalNAc4S-6ST (antibody GD3G7).
<p>SKOV3 wildtype (A) and transfection control (empty vector) SKOV3(-) cells (C5 (B) and C6 (C)) showed weak (peri)cellular expression of CSE, whereas SKOV3 cells transfected with DNA encoding GalNAc4S-6ST (F5(D), F7(E), F9(F)) showed strong expression for CSE expression, especially in more dense regions. Bar: 100 µm.</p
Strong staining for CSE in transcoelomic, ovarian metastasis (antibody GD3G7).
<p>A) Primary tumour, B) contralateral ovary, C) omentum, D) Douglas' pouch all displayed strong staining for CSE. Note the staining at sites where the mesothelial lining and its underlying basal membrane were associated with intruding tumour cells (B, arrow). Bar: 100 µm.</p
CSE inhibits artificial wound closure in 2D scratch assays.
<p>A) Wildtype SKOV3 cells and cell transfected with empty vector closed the artificial wound within 24 hours, whereas cells transfected with GalNAc4S-6ST cells did not. B) Addition of CSE to wildtype SKOV3 cells inhibited cell migration, showing limited gap-closure after 24 hours. Bar: 200 µm.</p
Spheroid formation by SKOV3 cells depends on chondroitin sulfate.
<p>A) Spheroid formation (tumour cell aggregation) was inhibited by enzymatic digestion using either of chondroitinase-ABC or –AC but not by chondroitinase B. Bar: 200 µm B) Spheroids formed by wild type SKOV3 cells and cells transfected with GalNAc4S-6ST and empty vector. Note the smooth and roundish appearance of GalNAc4S-6ST transfected cells. Bar: 100 µm.</p
CSE inhibits SKOV3 cell migration out of spheroids in 3D collagen matrices.
<p>A) Wildtype and cells transfected with an empty vector (-) showed cell migration out of spheroids, whereas migration of cells transfected with GalNAc4S-6STwas almost absent. B) Strong restriction of migration of wildtype SKOV3 cells out of spheroids in CSE decorated collagen matrices. Bar: 200 µm.</p
Interfering with UDP-GlcNAc Metabolism and Heparan Sulfate Expression Using a Sugar Analogue Reduces Angiogenesis
Heparan sulfate (HS), a long linear
polysaccharide, is implicated
in various steps of tumorigenesis, including angiogenesis. We successfully
interfered with HS biosynthesis using a peracetylated 4-deoxy analogue
of the HS constituent GlcNAc and studied the compound’s metabolic
fate and its effect on angiogenesis. The 4-deoxy analogue was activated
intracellularly into UDP-4-deoxy-GlcNAc, and HS expression was inhibited
up to ∼96% (IC<sub>50</sub> = 16 μM). HS chain size was
reduced, without detectable incorporation of the 4-deoxy analogue,
likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase
activity. Comprehensive gene expression analysis revealed reduced
expression of genes regulated by HS binding growth factors such as
FGF-2 and VEGF. Cellular binding and signaling of these angiogenic
factors was inhibited. Microinjection in zebrafish embryos strongly
reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish
and chicken model systems. All of these data identify 4-deoxy-GlcNAc
as a potent inhibitor of HS synthesis, which hampers pro-angiogenic
signaling and neo-vessel formation