A 2,5-Dihydroxybenzoic Acid-Gelatin Conjugate Inhibits the Basal and Hsp90-Stimulated Migration and Invasion of Tumor Cells.

The extracellular cell surface-associated and soluble heat shock protein 90 (Hsp90) is known to participate in the migration and invasion of tumor cells. Earlier, we demonstrated that plasma membrane-associated heparan sulfate proteoglycans (HSPGs) bind the extracellular Hsp90 and thereby promote the Hsp90-mediated motility of tumor cells. Here, we showed that a conjugate of 2,5-dihydroxybenzoic acid with gelatin (2,5-DHBA-gelatin), a synthetic polymer with heparin-like properties, suppressed the basal (unstimulated) migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells, which was accompanied by the detachment of a fraction of Hsp90 from cell surface HSPGs. The polymeric conjugate also inhibited the migration/invasion of cells stimulated by exogenous soluble native Hsp90, which correlated with the inhibition of the attachment of soluble Hsp90 to cell surface HSPGs. The action of the 2,5-DHBA-gelatin conjugate on the motility of A-172 and HT1080 cells was similar to that of heparin. The results demonstrate a potential of the 2,5-DHBA-gelatin polymer for the development of antimetastatic drugs targeting cell motility and a possible role of extracellular Hsp90 in the suppression of the migration and invasion of tumor cells mediated by the 2,5-DHBA-gelatin conjugate and heparin.

Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells.

The extracellular heat shock protein 90 (Hsp90) is known to participate in cell migration and invasion. Recently, we have shown that cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and anchoring of extracellular Hsp90 to the plasma membrane, but the biological relevance of this finding was unclear. Here, we demonstrated that the digestion of heparan sulfate (HS) moieties of HSPGs with a heparinase I/III blend and the metabolic inhibition of the sulfation of HS chains by sodium chlorate considerably impair the migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells stimulated by extracellular native Hsp90. Heparin, a polysaccharide closely related to HS, also reduced the Hsp90-stimulated migration and invasion of cells. Phorbol 12-myristate 13-acetate, an intracellular inducer of cell motility bypassing the ligand activation of receptors, restored the basal migration of heparinase- and chlorate-treated cells almost to the control level, suggesting that the cell motility machinery was insignificantly affected in cells with degraded and undersulfated HS chains. On the other hand, the downstream phosphorylation of AKT in response to extracellular Hsp90 was substantially impaired in heparinase- and chlorate-treated cells as compared to untreated cells. Taken together, our results demonstrated for the first time that cell surface HSPGs play an important role in the migration and invasion of cancer cells stimulated by extracellular Hsp90 and that plasma membrane-associated HSPGs are required for the efficient transmission of signal from extracellular Hsp90 into the cell.

A sensitive and specific lateral flow assay for rapid detection of antibodies against glycoprotein B of Aujeszky's disease virus.

A direct double antibody lateral flow assay (DDA-gB-LFA) for the detection of antibodies against the glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera was developed. A native ADV gB was used for the preparation of a conjugate with colloidal gold particles and the immobilization on the strip membrane. The gB purified from ADV virions by immunoaffinity chromatography retained its native epitope structure after adsorption on the nitrocellulose membrane and the surface of colloidal gold particles. The diagnostic specificity and sensitivity of the DDA-gB-LFA were evaluated using 236 field swine sera. The diagnostic specificity and sensitivity of the DDA-gB-LFA compared to a commercially available gB-based ELISA were 98.0% and 98.6%, respectively, when determined with the use of the reader-detection mode, and 98.0% and 93.5%, respectively, when determined using visual detection. The DDA-gB-LFA provides a rapid, sensitive, and specific determination of ADV gB-directed antibodies in sera and can be used for the detection of ADV-exposed swine.

Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90ß to the cell plasma membrane.

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90ß, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90ß are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90ß to the plasma membrane.

Cell-binding properties of glycoprotein B of Aujeszky's disease virus.

The glycoprotein B (gB) of Aujeszky's disease virus (ADV) has a role in virus entry and cell-to-cell spread. In this report we examined the cell-binding properties of native ADV gB purified from the virus envelope by affinity chromatography. The binding of gB to the surface of susceptible cells BHK-21 and MDBK was specific, dose-dependent, and nearly saturable, which is characteristic of conventional receptor-ligand interactions. The purified gB was shown to specifically bind to immobilised heparin. The addition of soluble exogenous heparin and heparinase treatment of cells inhibited the binding of gB to the cells. Cell-associated gB could also be dissociated from the cells by soluble heparin. The results indicated that ADV gB binds specifically to cellular heparan sulphate. The binding of gB to cells inhibited the attachment of virus to cells and thus the formation of viral plaques. The results suggest that ADV gB may have a function in the initial attachment of ADV to the surface of susceptible cells.