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

Abstract

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.

Keywords: 2,5-DHBA–gelatin conjugate; 2,5-dihydroxybenzoic acid (2,5-DHBA); cell migration and invasion; extracellular Hsp90; heparin-like polymer.

Figure 1

The 2,5-dihydroxybenzoic acid (2,5-DHBA)–gelatin conjugate inhibited the adsorption of pseudorabies virus (PRV) to cells. The degree of inhibition was determined by the plaque assay. The mean values of four to six repeats ± SD are presented. The statistical difference from control cells: * p < 0.05; ** p < 0.01.

Figure 2

The 2,5-DHBA–gelatin conjugate inhibited the basal unstimulated cell migration and invasion. (A,B) The migration/invasion of cells was determined in the presence of 2,5-DHBA–gelatin at concentrations of 10–50 μg/mL and of heparin, dermatan sulfate (DS), chondroitin sulfate A (ChS), and polyclonal anti-heat shock protein 90 (Hsp90) antibodies (concentration of 50 μg/mL for all reagents). The migration/invasion of untreated cells (control) was assumed to be 100%. The mean values of three to five repeats ± SD are presented. Asterisks indicate the statistical difference from control cells: * p < 0.05, ** p < 0.01.

Figure 3

The 2,5-DHBA–gelatin conjugate dissociated Hsp90α and Hsp90β from the cell plasma membrane. Cells were treated with 2,5-DHBA–gelatin at concentrations of 10–100 μg/mL and with heparin, dermatan sulfate (DS), or chondroitin sulfate A (ChS) (a concentration of 50 μg/mL for all substances). Incubation in all experiments was performed for 1 h at 37 °C, except one experiment in which cells were incubated at 4 °C (indicated in the graph). After the treatment, the expression of Hsp90 isoforms on the plasma membrane was determined by flow cytometry using Hsp90α- and Hsp90β-specific antibodies. (A,C) Representative flow cytometry histograms for A-172 and HT1080 cells. Control (untreated) cells (black lines), 2,5-DHBA–gelatin-treated cells (red lines), and cells treated with heparin (blue lines) were probed with antibodies directed to Hsp90α and Hsp90β; control cells were also probed with the isotype control antibody (green lines). (B,D) Quantification of membrane-associated Hsp90α and Hsp90β levels after different treatments. The Hsp90 isoform-specific mean fluorescence intensity (MFI) are presented; the MFIs of control cells were assumed to be 100%. The mean values of three to five repeats ± SD are presented. The representative results from two to four experiments are presented.

Figure 4

Treatment of cells with 2,5-DHBA–gelatin decreased the Hsp90-mediated stimulation of migration and invasion of cells. (A) Soluble native bovine Hsp90 stimulated cell motility. Cell migration/invasion was expressed in percent; the migration/invasion of control cells without Hsp90 was taken as 100%. (B) Hsp90-induced stimulation of cell migration. (C) Hsp90-induced stimulation of cell invasion. The degree of stimulation of cell migration/invasion by soluble Hsp90 was determined in the presence of the conjugate in the medium at concentrations of 10–50 μg/mL or in the presence of heparin, dermatan sulfate (DS), and chondroitin sulfate A (ChS) in the medium (50 μg/mL for all substances). The stimulation of migration/invasion induced by soluble Hsp90 was expressed in percent relative to control cells. (A–C) The mean values of three to five repeats ± SD are presented. Statistical difference from control cells: * p < 0.05, ** p < 0.01.

Figure 5

The 2,5-DHBA–gelatin polymeric conjugate impaired the binding of FITC-labelled native Hsp90 to cells. Cells were incubated with Hsp90–FITC for 1 h at 4 °C in the presence of the 2,5-DHBA–gelatin conjugate at concentrations in the range of 10–50 μg/mL and heparin (50 μg/mL). The binding of Hsp90–FITC was analyzed by flow cytometry. (A) Representative flow cytometry histograms. Cells incubated with Hsp90–FITC were treated with 2,5-DHBA–gelatin (green lines) and heparin (red lines); untreated (control) cells are represented as black lines, whereas blue lines represent cells incubated without Hsp90–FITC (autofluorescence). (B) Quantification of the binding of Hsp90–FITC to cells. The MFI of 2,5-DHBA–gelatin-treated and untreated control cells is presented; the MFIs of control untreated cells were taken as 100%. The mean values of three to five repeats ± SD are presented. Asterisks indicate the statistical difference from control cells: * p < 0.05, ** p < 0.01.

Figure 6

A hypothetical model of the action of the 2,5-DHBA–gelatin conjugate, heparin, and heparin-like substances on the Hsp90-mediated migration and invasion of tumor cells. Heparan sulfate (HS) glycosaminoglycan chains of cell surface heparan sulfate proteoglycans (HSPGs) bind with extracellular Hsp90s and facilitate Hsp90-mediated signaling via cell receptors LRP1 and Her2 associated with cell migration and invasion. The 2,5-DHBA–gelatin conjugate and heparin compete with HS for the binding with extracellular Hsp90, which results in the detachment of HSPG-associated Hsp90 from the cell surface HSPGs and the inhibition of binding of soluble Hsp90 to cell surface HSPGs. Both processes lead to the impairment of Hsp90-mediated motility-related signaling, which results in an inhibition of the migration and invasion of tumor cells.