Dabigatran antagonizes growth, cell-cycle progression, migration, and endothelial tube formation induced by thrombin in breast and glioblastoma cell lines

Abstract

Thrombin activates its G-coupled seven transmembrane protease-activated receptor (PAR-1) by cleaving the receptor's N-terminal end. In several human cancers, PAR1 expression and activation correlates with tumor progression and metastatization. This provides compelling evidence for the effectiveness of an appropriate antithrombin agent for the adjuvant treatment of patients with cancer. Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin. In this study, we aimed to explore if dabigatran may affect mechanisms favoring tumor growth by interfering with thrombin-induced PAR-1 activation. We confirmed that exposure of tumor cells to thrombin significantly increased cell proliferation and this was coupled with downregulation of p27 and concomitant induction of cyclin D1. Dabigatran was consistently effective in antagonizing thrombin-induced proliferation as well as it restored the baseline pattern of cell cycle protein expression. Thrombin significantly upregulated the expression of proangiogenetic proteins like Twist and GRO-α in human umbilical vascular endothelial cells (HUVEC) cells and their expression was significantly brought down to control levels when dabigatran was added to culture. We also found that the chemoattractant effect of thrombin on tumor cells was lost in the presence of dabigatran, and that the thrombin antagonist was effective in dampening vascular tube formation induced by thrombin. Our data support a role of thrombin in inducing the proliferation, migration, and proangiogenetic effects of tumor cells in vitro. Dabigatran has activity in antagonizing all these effects, thereby impairing tumor growth and progression. In vivo models may help to understand the relevance of this pathway.

Keywords: Cancer; PAR-1; dabigatran; thrombin.

Figure 1

In vitro proliferation of MDA‐MB231 (A) and U87 (B) cells cultured with thrombin (A, B), with thrombin and dabigatran (C, D) and with dabigatran only. Proliferation was measured by measuring the absorbance of culture media following generation of formazan from reduction of MTS tetrazolium. The reaction is dependent on cell metabolism. Each condition had three replicates. Shown are the results of three experiments, with data expressed as mean fold increase of proliferation compared to T0. Error bars represent sd. T: thrombin 0.5 UI; D1: dabigatran 100 nmol/L; D2, dabigatran 500 nmol/L; D3, dabigatran 1000 nmol/L. *P < 0.05 (control vs. thrombin panels A,B or thrombin vs. thrombin/dabigatran panels C, D).

Figure 2

Western blot showing cyclin D1, p27, Twist, and Gro‐α level in MDA‐MB231 (A) and U87 cells (B) exposed to thrombin compared to untreated cells. (C, D) Cumulative results from two independent experiments with MDA‐MB231 (C) or U87 (D) cells are shown. *P < 0.05.

Figure 3

Western blot analysis of cyclin D1, p27, Twist, and Gro‐α level in MDA‐MB231 (A) and U87 (B) cells exposed to thrombin, thrombin and dabigatran or dabigatran. (C) Histograms showing cumulative results from two independent experiments. *P < 0.05.

Figure 4

Cell cycle analysis of MDA‐MB231cells by BrdU. Panel A: MDA‐MB231cells were treated with thrombin, thrombin and dabigatran or dabigatran for 24 h and pulsed with BrdU for 1 h followed by staining with anti‐BrdU FITC and PI. BrdU‐labeled G0/G1, S, and G2/M populations are shown. Panel B: histograms of cells at S phase for each different treatment. *P < 0.05 control versus thrombin; ^§ P < 0.05 thrombin versus thrombin/dabigatran.

Figure 5

Angiogenetic properties of thrombin and dabigatran in Human umbilical vascular endothelial cells (HUVEC) cells. (A) mRNA expression of Gro‐α and Twist measured by real‐time PCR. Relative expression normalized to β‐actin is shown. (B) Effect of thrombin and dabigatran on tube formation by (HUVECs). Cells were cultured on Matrigel with medium containing 2% serum. Representative photomicrographs (×100, C) and quantitation (D) of branching points are shown. *P < 0.05. Scale bar, 250 μm.

Figure 6

Dabigatran antagonizes thrombin‐driven chemotaxis of MDA‐MB231. Cells were seeded in the upper chamber of a Boyden transmigration system. After 4 h incubation, cells migrated to the lower surface of the filters were fixed, stained, and quantitated. Representative pictures (A) and the average number of cells counted in five microscopy fields (B, ×100) are shown. Scale bar, 100 μm.