Activation of platelet protease-activated receptor-1 induces epithelial-mesenchymal transition and chemotaxis of colon cancer cell line SW620

Abstract

The aim of the present study was to examine the role of protease-activated receptor-1 (PAR1)-stimulated platelet activation in the epithelial-mesenchymal transition (EMT) and migration of colon cancer cells, and to identify the underlying mechanisms. TFLLR-NH2, a PAR1 agonist, was used to activate platelets and the platelet supernatants were used to treat the SW620 colon cancer cell line. Expression of E-cadherin and vimentin on SW620 cells was detected by immunofluorescence and western blotting, and the level of the transforming growth factor β1 (TGF-β1) was measured using ELISA following the activation of platelets by TFLLR-NH2. miR-200b expression was detected using quantitative PCR in SW620 cells. In order to investigate the chemotactic ability of the SW620 cells, the expression of CXC chemokine receptor type 4 (CXCR4) was measured by flow cytometry. Transwell migration assays were performed following exposure of the cells to the supernatant of PAR1-activated platelets. SW620 cells cultured in the supernatant of TFLLR-NH2-activated platelets upregulated E-cadherin expression and downregulated the vimentin expression. In the in vitro platelet culture system, a TFLLR-NH2 dose-dependent increase of secreted TGF-β1 was detected in the supernatant. The activation of PAR1 on the platelets led to the inhibition of miR-200b expression in the SW620 cells that were cultured in platelet-conditioned media. The number of SW620 cells that penetrated through the Transwell membrane increased with the dose of TFLLR-NH2 used to treat the platelets. The percentage of CXCR4-positive SW620 cells was significantly higher when they were exposed to the supernatant of platelets cultured for 24 h with PAR1 agonist than when cultured in non-conditioned media (40.89 ± 6.74 vs. 3.47 ± 1.40%, P < 0.01). Platelet activation with a PAR1 agonist triggered TGF-β secretion, which induced EMT of SW620 human colon cancer cells via the downregulation of miR-200b expression, and activated platelets had a chemotactic effect on colon cancer cells mediated by the upregulation of CXCR4 on the cell surface.

Figure 1

Flow cytometry detection of CD62P in the platelets treated with the PAR1 agonist TFLLR-NH₂. (a) CD62P expression in the platelets treated with different concentrations of TFLLR-NH₂ as detected by flow cytometry (mean ± standard deviation of three independent experiments). *P<0.05 and **P<0.01. (B) Representative FACS plots for CD62P expression in the platelets activated by (a) 0, (b) 1, (c) 3 and (d) 10 μM TFLLR-NH₂. M1, CD62P-negative region; M2, CD62P-positive region. PAR1, protease-activated receptor-1.

Figure 2

(A) Confocal immunofluorescence images showing the expression of vimentin (red) and E-cadherin (green) in the SW620 cells after 24 h treatment with (a) PAR1-activated platelet supernatant, (b) non-activated platelet supernatant and (c) no treatment. SP magnification, ×400. (B) Expression of E-cadherin or vimentin protein in the SW620 cells detected by western blotting. (a) PAR1-activated platelets supernatant, (b) non-activated platelets supernatant and (c) medium control. PAR1, protease-activated receptor-1.

Figure 2

(A) Confocal immunofluorescence images showing the expression of vimentin (red) and E-cadherin (green) in the SW620 cells after 24 h treatment with (a) PAR1-activated platelet supernatant, (b) non-activated platelet supernatant and (c) no treatment. SP magnification, ×400. (B) Expression of E-cadherin or vimentin protein in the SW620 cells detected by western blotting. (a) PAR1-activated platelets supernatant, (b) non-activated platelets supernatant and (c) medium control. PAR1, protease-activated receptor-1.

Figure 3

(A) Migration of the SW620 cells induced by platelet activation examined by Transwell migration assay. The number of the migrated SW620 cells induced by platelet supernatants extracted from the platelets activated by different concentrations of TFLLR-NH₂. *P<0.05 compared to the control. (B) Fluorescence microscopy image of the upper Transwell chamber after migration of the SW620 cells towards the activated platelet supernatant. The nuclei were stained with DAPI. (a) Media only control, (b) no TFLLR-NH₂ in the platelet culture, (c) 1 μM TFLLR-NH₂ and (d) 7 μM TFLLR-NH₂.

Figure 3

(A) Migration of the SW620 cells induced by platelet activation examined by Transwell migration assay. The number of the migrated SW620 cells induced by platelet supernatants extracted from the platelets activated by different concentrations of TFLLR-NH₂. *P<0.05 compared to the control. (B) Fluorescence microscopy image of the upper Transwell chamber after migration of the SW620 cells towards the activated platelet supernatant. The nuclei were stained with DAPI. (a) Media only control, (b) no TFLLR-NH₂ in the platelet culture, (c) 1 μM TFLLR-NH₂ and (d) 7 μM TFLLR-NH₂.

Figure 4

The levels of TGF-β1 released by platelets treated with different concentrations of TFLLR-NH₂ as detected by ELISA. The TGF-β1 secretion was significantly higher in the activated platelet group than that in the control, and the TGF-β1 level increased in a dose-dependent manner in the activated platelet groups. *P<0.05 and **P<0.01 vs. the control group. TGF-β1, transforming growth factor β1.

Figure 5

Expression of miR-200b of the SW620 treated with different volumes of the PAR1-activated platelet supernatant detected by qPCR at (A) 24 or (B) 48 h. (a) after 24 h the groups treated with 60 and 120 μl of platelet supernatant did not indicate significant difference (P>0.05) compared to the control group, whereas those treated with 240 and 480 μl showed a significantly lower miR-200b expression. *P<0.05 and **P<0.01. PAR1, protease-activated receptor-1.

Figure 5

Expression of miR-200b of the SW620 treated with different volumes of the PAR1-activated platelet supernatant detected by qPCR at (A) 24 or (B) 48 h. (a) after 24 h the groups treated with 60 and 120 μl of platelet supernatant did not indicate significant difference (P>0.05) compared to the control group, whereas those treated with 240 and 480 μl showed a significantly lower miR-200b expression. *P<0.05 and **P<0.01. PAR1, protease-activated receptor-1.

Figure 6

CXCR4 expression of SW620 cells treated with culture medium (control) or with supernatants of the platelets cultured in 3 μM TFLLR-NH₂ as detected by flow cytometry. **P<0.01. CXCR4, CXC chemokine receptor type 4.