TR47, a PAR1-based peptide, inhibits melanoma cell migration in vitro and metastasis in vivo

Abstract

Activated Protein C (APC) is a serine-protease that displays antithrombotic and anti-inflammatory properties. In addition, cleavage of protease-activated receptor 1 (PAR1) by APC exerts endothelial cytoprotective actions. The effects of APC on endothelial cells may be reproduced by TR47, a PAR1-based peptide that mimics the novel N-terminus of PAR1 generated upon cleavage at Arg-46 by APC. In this study we demonstrate that wild-type APC and its signaling-proficient mutant, APC-2Cys (which has dramatically reduced anticoagulant activity), display similar inhibitory effects towards the transendothelial migration of A375 human melanoma cells. Consistent with this observation, APC and APC-2Cys significantly reduced the in vivo metastatic potential of the B16F10 murine melanoma cells. TR47 recapitulated the in vitro and in vivo protective profiles of APC and APC-2Cys. Treatment of EA.hy926 endothelial cells with TR47 (20 μM) significantly decreased the A375 cell migration. In addition, treatment of C57/BL6 mice with a single TR47 dose (125 μg/animal) strongly reduced the metastatic burden of B16F10 cells. Together, our results suggest that protection of the endothelial barrier by APC/TR47-mediated signaling pathways might be a valuable therapeutic approach to prevent metastasis.

Activated protein C and PAR1‐derived and PAR3‐derived peptides are anti‐inflammatory by suppressing macrophage NLRP3 inflammasomes

“…49,50 Studies demonstrated that shortening the PAR1-derived peptide P1(47-66) to an 8-or 9-mer, respectively, retains cytoprotective activities. 22,51 Here, we show that a single mutation of Asn47 to…”

“…Subsets of wells were selected and cells were treated for 60 minutes at 37°C in serum-free RPMI with: Although PAR3 itself is generally thought to be a nonsignaling receptor, it is speculated that PAR3 can modulate PAR1 signaling through PAR1:PAR3 dimerization. 24,[27][28][29] Thus, we hypothesized that although low concentrations of either P1 (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66) Figure 3A). Next, we sought to determine whether this reduction in caspase-1 activity by APC, PAR1, and PAR3 peptides might be conserved in human primary monocytes as it is known that APC can effect human primary monocytes.…”

“…In addition, since protease activated receptor (PAR)1-and PAR3derived peptides that mimic APC's noncanonical cleavages in these two G-protein-coupled-receptors (GPCRs) are biased agonists that reduce vascular leakage in vivo, we also evaluated whether PAR1and PAR3-derived biased agonist peptides, namely P1(47-66) (also known as TR47) and P3(42-65) (also known as P3R), respectively, can mimic APC's anti-inflammatory action and exert anti-inflammatory activity. 17,18 Herein, we report for the human THP-1 cell line and for human primary monocytes that APC, P1 (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66), and P3(42-65) each can restrict the NLRP3 inflammasome as measured by caspase-1 activity and that the two PAR agonist peptides or their derivatives can synergistically suppress inflammasome activity. All other reagents were purchased from Fisher Scientific (Hampton, NH).…”

“…NLRP3 could be detected via fluorescent staining in response to treatment by APC, P1(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66), P3, or the combination of peptides. Fluorescent staining of NLRP3 was increased with LPS and ATP treatment, whereas significant decreases in the number of cells with positive staining for NLRP3 was observed for APC, P1(47-66), P3, and the combination of peptides (Figure 5).…”

Endothelial Protein C Receptor (EPCR), Protease Activated Receptor-1 (PAR-1) and Their Interplay in Cancer Growth and Metastatic Dissemination

“…Transgenic mice overexpressing EPCR (Tie2-EPCR) had a 50–92% decrease in liver and lung metastases compared with wildtype animals. Additionally, treatment of the mouse with recombinant human APC or a signaling-proficient mutant, APC-2Cys (with reduced anticoagulant activity), also led to a reduction of metastatic melanoma foci by inhibiting transendothelial migration of malignant cells [11,48]. The APC/EPCR interaction, e.g., in melanoma cells, likely decreases expression of endothelial adhesion molecules, such as P-selectin that is essential for tumor cell-endothelium interactions during extravasation, ultimately hindering dissemination [11].…”