chondroitin-sulfates and Astrocytoma

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is the sulfotransferase responsible for biosynthesis of highly sulfated chondroitin sulfate CS-E. Although involvements of CS-E in neuronal cell functions have been extensively analyzed, the role of GalNAc4S-6ST in astrocytic tumor progression remains unknown. Here, we reveal that GalNAc4S-6ST transcripts were detected in astrocytic tumors derived from all 30 patients examined using quantitative reverse transcription-PCR analysis. Patients with high GalNAc4S-6ST mRNA expression had significantly worse outcome compared with patients with low expression, and multivariate survival analysis disclosed that GalNAc4S-6ST is an independent poor prognostic factor for astrocytic tumors. We then tested whether CS-E enhanced haptotaxic migration of glioblastoma U251-MG cells that endogenously express both the CS-E's scaffold tyrosine phosphatase ζ (PTPζ) and GalNAc4S-6ST, in the presence of CS-E's preferred ligands, pleiotrophin (PTN) or midkine (MK), using a modified Boyden chamber method. Haptotaxic stimulation of cell migration by PTN was most robust on control siRNA-transfected U251-MG cells, while that enhancing effect was cancelled following transduction of GalNAc4S-6ST siRNA. Similar results were obtained using MK, suggesting that both PTN and MK enhance migration of U251-MG cells by binding to CS-E. We also found that PTPζ as well as PTN and MK were frequently expressed in astrocytic tumor cells. Thus, our findings indicate that GalNAc4S-6ST mRNA expressed by astrocytic tumor cells is associated with poor patient prognosis likely by enhancing CS-E-mediated tumor cell motility in the presence of PTN and/or MK.

Topics: Adolescent; Adult; Aged; Astrocytoma; Brain Neoplasms; Carrier Proteins; Cell Line, Tumor; Child; Child, Preschool; Chondroitin Sulfates; Cytokines; Disease Progression; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Male; Middle Aged; Midkine; Multivariate Analysis; Prognosis; Receptor-Like Protein Tyrosine Phosphatases, Class 5; Reverse Transcriptase Polymerase Chain Reaction; Sulfotransferases; Survival Analysis

Proteoglycans (PGs) have been suggested to work as receptors in lipoprotein uptake mechanisms. An interaction between apolipoprotein E (apoE) and glucosaminoglycans (GAG), polysaccharides linked to proteoglycans, has been proposed in this pathway. At the same time, proteoglycans, apoE as well as lipoprotein receptors have been reported to be constituents of amyloid plaques, one hallmark of Alzheimer's disease. With this study, we are the first to investigate the interaction between beta very low density lipoprotein (beta-VLDL) and a neuronal highly abundant GAG, chondroitin sulphate (CS), comparing hippocampal neurons, expressing high levels of low density lipoprotein receptor related protein (LRP) and U373 astrocytoma cells, highly positive for the low density lipoprotein receptor (LDLR). We were able demonstrate that degradation of chondroitin sulphate proteoglycans (CSPGs) with chondroitinase ABC resulted in reduced (125)I-beta-VLDL uptake. We showed that externally added CSs compete with internalization of beta-VLDL. The effect was found to be dose-dependent, but was influenced neither by cell type, nor receptor type. The position of sulphation of added CSs showed only a slight influence. The data generated suggested an interaction between apolipoproteins and soluble CSs; therefore, 3H-cholesterol linked to apoE was coadministered with CSs to the cells. The results revealed that apoE bound, but no unbound cholesterol, was reduced in cellular internalization, suggesting that CSPGs may be involved in lipoprotein uptake in the intact brain, mediated, at least in part, by apoE.

Topics: Animals; Apolipoproteins E; Astrocytoma; Brain; Brain Chemistry; Cell Line; Cholesterol; Chondroitin ABC Lyase; Chondroitin Sulfates; Female; Hippocampus; Immunoblotting; Lipoproteins, VLDL; Neurons; Pregnancy; Rats; Receptors, Lipoprotein; Spectrometry, Fluorescence

Based on the hypothesis that the attachment of neuroectodermal cells to thrombospondin-1 (TSP-1) may affect tumor spread and play a role in the anti-tumor effects of retinoic acid, we investigated the expression of TSP-1 in these cells in situ and the effect of retinoic acid on the morphology of TSP-1-adherent neuroblastoma (SK-N-SH) and malignant astrocytoma (U-251MG) cells in vitro. TSP-1-adherent SK-N-SH cells demonstrated process outgrowth, with further neuronal differentiation after retinoic acid treatment, consistent with the in situ studies showing that TSP-1 expression occurs in a differentiation-specific manner in neuroblastic tumors. TSP-1-adherent U-251MG cells failed to spread; however, after retinoic acid treatment the cells demonstrated broad lamellipodia containing radial actin fibers and organization of integrins alpha3beta1 and alpha5beta1 in clusters in lamellipodia and filopodia. The attachment of both SK-N-SH and U-251MG cells to TSP-1 was found to be mediated by heparan sulfate proteoglycans, integrins, and the CLESH-1 adhesion domain first identified in CD36. Heparin and heparitinase treatment inhibited TSP-1 attachment. Integrins alpha3beta1 and alpha5beta1 mediated TSP-1 attachment of SK-N-SH cells, and integrins alpha3beta1, alpha5beta1, and alphavbeta3 mediated TSP-1 attachment of U-251MG cells. Attachment was dependent on the RGD sequence which is located in the carboxy-terminus of TSP-1. Treatment with a pharmacologic dosage of retinoic acid altered the TSP-1 cell adhesion mechanism in both cell lines in that neither heparin nor micromolar concentrations of the RGD peptide inhibited attachment; after treatment, attachment was inhibited by the CSVTCG peptide located in the type I repeat domain of TSP-1 and a recombinant adhesion domain (CLESH-1) from CD36. Expression of CD36 was found in the retinoic acid-treated U-251MG cells. These data indicate that neuroectodermally derived cells utilize several mechanisms to attach to TSP-1, and these are differentially modulated by treatment with retinoic acid. These data also suggest that the CSVTCG sequence of TSP-1 modulates or directs cytoskeletal organization in neuroblastoma and astrocytoma cells.

Topics: Astrocytes; Astrocytoma; Brain; CD36 Antigens; Cell Adhesion; Cell Differentiation; Chondroitin ABC Lyase; Chondroitin Sulfates; Cytoskeleton; Endothelium; Ganglioneuroblastoma; Ganglioneuroma; Glioblastoma; Heparin; Humans; Integrin alpha3beta1; Integrins; Neuroblastoma; Neurons; Oligopeptides; Peptide Fragments; Polysaccharide-Lyases; Receptors, Fibronectin; Recombinant Proteins; Thrombospondin 1; Tretinoin; Tumor Cells, Cultured

A polyclonal rabbit antiserum was utilized to localize chondroitin sulfate in human gliomas. Tissue sections were digested with chondroitinase ABC to create the antigenic determinant on the chondroitin sulfate proteoglycan molecule. Normal CNS tissue showed a positive immunohistochemical staining both in white and gray matter, sparing the cytoplasm of glial and neuronal cells. Differentiated astrocytomas presented the same pattern as the normal CNS. Anaplastic astrocytomas and glioblastomas showed progressive reduction of parenchymal positivity as anaplasia increased. These data suggest that chondroitin sulfate is a character expressed by differentiated CNS cells and that it is lost with dedifferentiation. Vascular structures presented positive material in the adventitia in all the oncotypes. A discontinuous positivity was observed in the basal membrane zone of the vessels.

Topics: Astrocytoma; Brain Neoplasms; Chondroitin; Chondroitin Sulfates; Glioma; Histocytochemistry; Humans; Immunologic Techniques