Unfractionated and Low Molecular Weight Heparin Reduce Platelet Induced Epithelial-Mesenchymal Transition in Pancreatic and Prostate Cancer Cells

Abstract

The interaction with platelets is of crucial importance for tumor cells passing through hematogenous metastasis. Platelets protect cancer cells from immune surveillance and exhibit many other prometastatic effects. Notably, platelets can change the epithelial tumor phenotype, a process termed epithelial-mesenchymal transition (EMT), which confers stem cell-like properties onto tumor cells associated with an increased motility and drug resistance. The aim of the study is to investigate the impact of heparin on the platelet induced EMT program in pancreatic and prostate tumor cells. Platelet activation and interaction with cancer cells were determined by static adhesion assays. Applying ELISAs, the platelet release of EMT inducing mediators was quantified. EMT marker protein expression by tumor cells was explored by western blot and qPCR. Our data show that different tumor cell entities have different platelet binding capacities and also that a weak interaction is sufficient to change tumor cell phenotype. Additionally, unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) reduced tumor cell platelet interaction. Subsequently, attenuated platelet-derived mediator release resulted in reduced EMT marker protein and transcription factor expression by the cancer cells and decreased cell migration. These data suggest that heparin reduces platelet induced EMT program and prevents the formation of cancer cells with stem cell-like properties. This additional mechanism argues for the use of heparin in oncological applications.

Keywords: EMT; cancer stem cells; enoxaparin; epithelial-mesenchymal transition; heparin; platelet; tumor.

Figure 1

(a) Representative pictures of Calcein-AM labeled platelets interacting with confluent layers of AsPC-1, MDA-MB-231, MV3 and PC-3 cells. Prior to interaction platelets were coincubated with 1 IU/mL UFH or 1 IU/mL enoxaparin. Scale bar corresponds to 50 µm. (b) Quantification of platelet (Calcein-AM labeled) tumor cell (AsPC-1, MDA-MB-231, MV3 and PC-3) by fluorescent plate reader assay. Data are means of at least n = 3 (±SD), asterisks indicate statistical significance: * p ≤ 0.05; *** p ≤ 0.001.

Figure 2

Impact of heparin on platelet derived HGF and PDGF release. (a) Impact of UFH or Enoxaparin on AsPC-1 cell induced HGF release from platelets. (b) Impact of UFH or enoxaparin on PC-3 cell induced HGF release from platelets. (c) Impact of UFH or enoxaparin on AsPC-1 cell induced PDGF release from platelets. (d) Impact of UFH or enoxaparin on PC-3 cell induced PDGF release from platelets. Data are means of at least n = 3 (±SD), asterisks indicate statistical significance: *** p ≤ 0.001.

Figure 3

Impact of heparin on platelet derived EGF and TGF-β1 release. (a) Impact of UFH or enoxaparin on AsPC-1 cell induced EGF release from platelets. (b) Impact of UFH or enoxaparin on PC-3 cell induced EGF release from platelets. (c) Impact of UFH or enoxaparin on AsPC-1 cell induced TGF-β1 release from platelets. (d) Impact of UFH or enoxaparin on PC-3 cell induced TGF-β1 release from platelets Data are means of at least n = 3 (±SD), asterisks indicate statistical significance: * p ≤ 0.05; ** p ≤ 0.01.

Figure 4

Relative fold change in mRNA expression in AsPC-1 or PC-3 cells treated with buffer, platelets, platelets and UFH, or platelets and enoxaparin for 72 h, respectively. Values are normalized to Gapdh, β-actin and RPL13 expression. Statistical significance was evaluated by two-way ANOVA test. Data are means of at least n = 3 (±SD), asterisks indicate statistical significance: ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

Western blotting of the expression profile of E-cadherin and vimentin in AsPC-1 and PC-3 cells. (a) Expression of E-cadherin and vimentin in AsPC-1 cells after treatment with platelets or platelets and UFH or platelets and enoxaparin for 72 h, respectively. (b) Quantification of vimentin expression by pixel density measurements in AsPC-1 cells, data of three independent experiments. Asterisks indicate statistical significance: ** p ≤ 0.01. (c) Quantification of E-cadherin expression by pixel density measurements in AsPC-1 cells, data of three independent experiments. (d) Expression of E-cadherin and vimentin in PC-3 cells after treatment with platelets or platelets and UFH or platelets and enoxaparin for 72 h. (e) Quantification of vimentin expression by pixel density measurements in PC-3 cells, data of three independent experiments. (f) Quantification of E-cadherin expression by pixel density measurements in AsPC-1 cells, data of three independent experiments.

Figure 6

Representative images from wound healing assays of AsPC-1 cell and PC-3 cell cultures treated with buffer or platelets or platelets and heparins. Scale bar corresponds to 50 µm. Quantification of scratch width decrease in percent is based on data of three independent experiments. (a) Wound healing assay after 72 h coincubation of AsPC-1 cells with buffer, platelets, platelets and UFH, or platelets and enoxaparin and 16 h serum-starvation. Images show the invasion of AsPC-1 cells into the cell-free region at the beginning (0 h), after 24 h and after 72 h. Histogram shows the decrease of the scratch size after 72 h (right part of the figure). (b) Wound healing assay after 72 h coincubation of PC-3 cells with buffer, platelets, platelets and UFH or platelets and enoxaparin and 16 h serum-starvation. Images show the invasion of PC-3 cells into the cell-free region at the beginning (0 h), after 8 h and after 24 h. Histogram shows the decrease of the scratch size after 24 h (right part of the figure).

Figure 6

Representative images from wound healing assays of AsPC-1 cell and PC-3 cell cultures treated with buffer or platelets or platelets and heparins. Scale bar corresponds to 50 µm. Quantification of scratch width decrease in percent is based on data of three independent experiments. (a) Wound healing assay after 72 h coincubation of AsPC-1 cells with buffer, platelets, platelets and UFH, or platelets and enoxaparin and 16 h serum-starvation. Images show the invasion of AsPC-1 cells into the cell-free region at the beginning (0 h), after 24 h and after 72 h. Histogram shows the decrease of the scratch size after 72 h (right part of the figure). (b) Wound healing assay after 72 h coincubation of PC-3 cells with buffer, platelets, platelets and UFH or platelets and enoxaparin and 16 h serum-starvation. Images show the invasion of PC-3 cells into the cell-free region at the beginning (0 h), after 8 h and after 24 h. Histogram shows the decrease of the scratch size after 24 h (right part of the figure).