Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 3. Proliferative diabetic retinopathy epiretinal membranes. Negative control slide that was treated identically with an irrelevant antibody showing no labeling (A). Immunohistochemical staining for CD31 showing blood vessels positive for CD31 (B). Immunohistochemical staining for protease-activated receptor-1 (PAR1) showing immunoreactivity in the vascular endothelium (arrows), in stromal cells (arrowheads) (C), in intravascular leukocytes (arrowheads) (D), and in stromal spindle-shaped cells (E). Immunohistochemical staining for CD45 showing stromal cells positive for CD45 (F). Immunohistochemical staining for α-smooth muscle actin showing immunoreactivity in spindle-shaped myofibroblasts (G). Double immunohistochemistry for CD45 (brown) and PAR1 (red) showing stromal cells and intravascular leukocytes (arrowheads) co-expressing CD45 and PAR1 (H) (original magnification X40).

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Negative Control, Labeling, Immunohistochemical staining, Staining, Immunohistochemistry, Expressing

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 5. Western blot analysis of protease-activated receptor-1 (PAR1) (A) and thrombin (B) in rat retinas. Significant increases in the ratios of the cleaved PAR1 over the intact protein and of the proteolytically active thrombin over the zymogen prothrombin in the retinas of diabetic rats (D) compared to the nondiabetic control rats (C). Each experiment was repeated three times with fresh samples (n = 7). *p < 0.05 compared to control.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Western Blot, Analysis, Control

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 4. Human retinal microvascular endothelial cells were left untreated or were treated with either IL-1β (10 ng/ml), TNF-α (30 ng/ml), or a combination of cytokines containing IL-1β (10 ng/ml) plus TNF-α (30 ng/ml) for 24 h. The levels of expression of protease-activated receptor-1 (PAR1) were compared by Western blot analysis. Western blots are representative of at least three independent experiments, each is performed in duplicate and bar graphs are representative of all three experiments. *p < 0.05 compared to untreated control. #p < 0.05 compared to IL-1β.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Western Blot, Analysis, Control

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 7. Effects of thrombin (A) and matrix metalloproteinase-1 (MMP-1) (B) on the migration of human retinal microvascular endothelial cells (HRMEC). Thrombin- induced migration of HRMEC was inhibited by the protease-activated receptor-1 (PAR1) inhibitor vorapaxar. HRMECs were left untreated or treated either with 1 nM recombinant thrombin, with 10 µM PAR1 inhibitor vorapaxar for 30 min before addition of 1 nM recombinant thrombin or with 5 nM recombinant MMP-1. Cells were visualized using inverted microscope (Olympus 1X 81, Olympus Corporation, Tokyo, Japan). Three independent experiments were performed. Six independent field images were taken from each treated group for migration analysis, which was performed using Image J software. One image from each group is illustrated and the bar graphs show the analysis of all six images from each group. *p < 0.05 compared to untreated control. #p < 0.05 compared to vorapaxar + thrombin.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Migration, Recombinant, Inverted Microscopy, Analysis, Software, Control

Journal: The Journal of Biological Chemistry

Article Title: In Vivo Nano-imaging of Membrane Dynamics in Metastatic Tumor Cells Using Quantum Dots *

doi: 10.1074/jbc.M109.075374

Figure Lengend Snippet: Preparation and characterization of the anti-PAR1 monoclonal antibody. A, frame format of molecular structure of PAR1. Matrix metalloprotease 1 activates PAR1 by cleaving its exodomain at the Arg41–Ser42 peptide bond. This activation promotes PAR1-dependent Ca2+ signaling following cancer cell migration and invasion. The peptide CNATLDPRSFLL, including a sequence from Asn35 to Leu45 of PAR1, was synthesized and used as an antigen to make an anti-PAR1 antibody capable of functioning as both a cancer cell tracer and a PAR1-inhibitor (anticancer agent). B, epitope mapping for the PAR1 antibody. (1)-(18) represent peptides of 12 amino acids, including part or all of the 35NATLDPRSFLL45 sequence, were used for epitope mapping by enzyme-linked immunosorbent assay. OD492 indicates the absorbance of peroxidase-conjugated antibodies at 492 nm. The PAR1 antibody specifically reacted with the ninth peptide, TNATLDPRSFLL, but not with the tenth peptide, NATLDPRSFLLR, despite the fact that both peptides included the same NATLDPRSFLL sequence. The reason for this might be that an amino group from Asn in the tenth peptide inhibited the binding of the anti-PAR1 antibody because another peptide, Cys-bound NATLDPRSFLL, was used for the antigen (A). Additionally, Arg46 in the tenth peptide might inhibit the binding of antibody and peptide. The results indicate that the epitope for the antibody is 35NATLDPRSFLL45. C, purification of PAR1 antibody. To obtain high purity PAR1 antibody, the PAR1 antibody was purified from the ascites with protein G beads and checked using 10% SDS-PAGE. Lane 1 shows the pattern of a crude sample of the ascites. Lanes 2 and 3 show the patterns of samples unbound by Protein G and released from Protein G after binding, respectively. T, A, HC, and LC show the transferrin, albumin, heavy chain of IgG, and light chain of IgG, respectively. The sample of purified PAR1 antibody contained no transferrin or albumin (lane 3).

Article Snippet: To obtain high purity anti-PAR1 antibody, the cloned hybridoma cells were injected into the abdominal cavity of a severe combined immunodeficient (SCID) mouse (Charles River), which is an immunodeficient mouse without immunoglobulins, and ascites were prepared from the mouse.

Techniques: Activation Assay, Migration, Sequencing, Synthesized, Enzyme-linked Immunosorbent Assay, Binding Assay, Purification, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: In Vivo Nano-imaging of Membrane Dynamics in Metastatic Tumor Cells Using Quantum Dots *

doi: 10.1074/jbc.M109.075374

Figure Lengend Snippet: Materials and methods for in vivo nano-imaging. A, fluorescent image of PAR1-KPL cells. Fluorescence of PAR1-GFP was clearly observed in the cell membrane and endocytotic vesicles in PAR1-KPL cells (excitation, 488 nm; emission, 500–550 nm; and exposure time, 3 s). B, imaging of KPL and PAR1-KPL cells with anti-PAR1-QDs (40 nm). QDs on the cell membrane located near the glass surface were visualized in vitro. Yellow lines show the cellular outline delineated using phase-contrast images. Excitation, 532 nm; emission, 685–725 nm; and exposure time, 0.1 s. C, fluorescence intensity of QDs measured on the cell membrane of KPL and PAR1-KPL cells in vitro. Error bars indicate ±S.E. KPL cells, n = 13. PAR1-KPL cells, n = 11. D, schematic drawing of in vivo nano-imaging. Tumors were stripped, turned over, and fixed with thread (see supplemental Fig. S2). A polyvinyl chloride plate containing a small window (10 mm × 10 mm) was mounted on the exposed tumor, and then the skin around the tumor and the plate were bonded by instant adhesive. The plate was fixed to a handmade aluminum stage designed to stabilize the plate with screws. In the optical schematic for in vivo nano-imaging, L, M, and DM show the lens, mirror, and dichroic mirror, respectively. An example of a tumor-bearing nude mouse is provided. E, spatial precision of in vivo nano-imaging. Immobile QDs were tracked in tumor stroma. The standard deviation of the position of QDs was 7.4 nm in the x-axis and 9.4 nm in the y-axis. F, fluorescent imaging of PAR1-GFP and anti-PAR1-QDs on the edge of tumor cells in mice. Fluorescence of anti-PAR1-QDs was compared with that of PAR1-GFP on tumor cell membrane. Only the QDs that colocalized with PAR1-GFP on the cell edge (white squares) were analyzed. The laser-excited fluorescence of PAR1-GFP and anti-PAR1-QDs were filtered with a 500–550 nm bandpass filter and a 685–725 nm bandpass filter, respectively. G, determination of the cellular outline by superimposed images of anti-PAR1-QDs fluorescence on tumor cells in mice. An outline of a cell (yellow lines) determined by superimposed images (bottom panel) was applied to a cell image in a single frame (top panel). QDs on cell edges (white squares) were analyzed. H, labeling of blood cells in SCID mice with anti-PAR1-QDs. Bright field and fluorescent images of whole blood cells and leukocyte cells are shown. In whole blood cells, most cells were red blood cells that were autofluorescent. Yellow lines show the outline of leukocytes. Although there was low level binding of a few QDs per cell (yellow arrowhead), high affinity binding of QDs as seen in Figs. 3 and ​and44 was not observed. Excitation, 532 nm; emission, >580 nm; and exposure time, 0.2 s. I, graphic representation of fluorescent images of whole blood cells (red symbol) and leukocyte cells (blue symbol). The number of the cells binding QDs was fitted to an exponential function. Approximation formulas for the fitting were 16,000 × e−3x (red symbols) and 1,100 × e−2.3x (blue symbols), where x is the number of attached particles. Bars, 10 μm.

Article Snippet: To obtain high purity anti-PAR1 antibody, the cloned hybridoma cells were injected into the abdominal cavity of a severe combined immunodeficient (SCID) mouse (Charles River), which is an immunodeficient mouse without immunoglobulins, and ascites were prepared from the mouse.

Techniques: In Vivo, Imaging, Fluorescence, In Vitro, Standard Deviation, Labeling, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: In Vivo Nano-imaging of Membrane Dynamics in Metastatic Tumor Cells Using Quantum Dots *

doi: 10.1074/jbc.M109.075374

Figure Lengend Snippet: Membrane dynamics in metastatic cancer cells outside of vessels. A, QD fluorescent image of cells far from tumor vessels. The PAR1 diffusion constant as represented by blue squares was analyzed in E and F. B, QD fluorescent image of a cell near a vessel. Orange squares show a cancer cell forming membrane protrusions in the direction of the vessel. Red dotted lines represent the outline of a vessel determined by superimposed images of auto-fluorescence of blood cells. C, magnified view of B. QDs on the membrane facing the vessel, represented by green squares, and QDs on the membrane on the opposite side of the cell, represented by red squares, were differentially selected and analyzed in E and F. D, cell image superimposed for 3.6 s. The yellow line shows an outline of the cell. The red arrowhead indicates a clearly imaged membrane protrusion. E, traces of blue, orange, and green squares as shown with arrowheads in A and C. Numbers show the tracking order. F, MSD plots of QDs on membrane in A (blue), on the membrane facing the vessel in C (green), and on the membrane in the tail of the cell in C (red). D, diffusion constant. Error bars indicate ±S.E. Blue data, n = 24 (6 trajectories/cell × 4 cells). Red data, n = 39 (13 trajectories/cell × 3 cells). Green data, n = 39 (13 trajectories/cell × 3 cells). Squares in A and C show typical QDs on the edges of cells. Excitation, 532 nm; emission, >580 nm. The exposure time in A–C was 0.2 s. Bars, 10 μm.

Article Snippet: To obtain high purity anti-PAR1 antibody, the cloned hybridoma cells were injected into the abdominal cavity of a severe combined immunodeficient (SCID) mouse (Charles River), which is an immunodeficient mouse without immunoglobulins, and ascites were prepared from the mouse.

Techniques: Diffusion-based Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: In Vivo Nano-imaging of Membrane Dynamics in Metastatic Tumor Cells Using Quantum Dots *

doi: 10.1074/jbc.M109.075374

Figure Lengend Snippet: A model for membrane dynamics in metastatic cancer cells. A–F, cells in vivo during metastasis. A, cells far from vessels. B, cell near vessels. C, cell in the bloodstream. C′, cells in narrow vessels. D, cells adhering to the inner vascular surface without directional migration. E, cells migrating directionally on the surface. F, cells after extravasation. Numerical values show the diffusion (nm2/s) of PAR1 labeled with anti-PAR1-QDs. See text for details.

Article Snippet: To obtain high purity anti-PAR1 antibody, the cloned hybridoma cells were injected into the abdominal cavity of a severe combined immunodeficient (SCID) mouse (Charles River), which is an immunodeficient mouse without immunoglobulins, and ascites were prepared from the mouse.

Techniques: In Vivo, Migration, Diffusion-based Assay, Labeling

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice

doi: 10.1055/s-0042-1758855

Figure Lengend Snippet: Primers used for the semi-quantification of mRNA levels of coagulation and inflammatory factors in cultured Colon26 cells

Article Snippet: The concentrations of IL-6, matrix metalloproteinase-2 (MMP-2), TF, and PAI-1 in heparinized plasma and those of PAR1, PAR2, STAT3, cyclin D1, Ki67, p53, VEGF-A, and VEGFR-1 in normal or tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA) using mouse IL-6 quantikine ELISA kit (M6000B; R&D Systems, Minneapolis, Minnesota, United States), mouse MMP-2 ELISA kit (ab254516; Abcam, Cambridge, UK), mouse TF ELISA kit (SEA524Ra; Cloud-Clone, Houston, Texas, United States), mouse PAI-1 ELISA kit (ab197752; Abcam), mouse PAR1 ELISA kit (MBS753326; MyBioSource, San Diego, California, United States), mouse PAR2 ELISA kit (MBS4501658; MyBioSource), mouse STAT3 ELISA kit (OKEH02995; Aviva System Biology, San Diego, California, United States), cyclin D1 ELISA kit (NOV596744; Novus, Centennial, California, United States), mouse Ki67 ELISA kit (EM1473; Wuhan Fine Biotech, Hubei, China), mouse p53 ELISA kit (ab224878; Abcam), mouse VEGF-A ELISA kit (BMS619–2; Thermo Fisher Scientific, Waltham, Massachusetts, United States), and mouse VEGFR-1 ELISA kit (CSB-E04762m; CUSABIO, Houston, Texas, United States) respectively, according to the manufacturer's instructions.

Techniques: Coagulation, Cell Culture

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice

doi: 10.1055/s-0042-1758855

Figure Lengend Snippet: ( A ) mRNA expression of coagulation and inflammation factor genes in cultured Colon26 cells determined using PCR. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-6, interleukin-6; MW, molecular weight markers; PAI-1, plasminogen activator inhibitor-1; PAR1, protease-activated receptor-1; PAR2, protease-activated receptor-2;; TF, tissue factor. ( B ) Western blot image of TF protein in total protein 0.25 to 2.5 μg/lane of a cell extract of cultured Colon26 cells. The data indicate that the molecular weight of TF is approximately 48 kDa.

Article Snippet: The concentrations of IL-6, matrix metalloproteinase-2 (MMP-2), TF, and PAI-1 in heparinized plasma and those of PAR1, PAR2, STAT3, cyclin D1, Ki67, p53, VEGF-A, and VEGFR-1 in normal or tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA) using mouse IL-6 quantikine ELISA kit (M6000B; R&D Systems, Minneapolis, Minnesota, United States), mouse MMP-2 ELISA kit (ab254516; Abcam, Cambridge, UK), mouse TF ELISA kit (SEA524Ra; Cloud-Clone, Houston, Texas, United States), mouse PAI-1 ELISA kit (ab197752; Abcam), mouse PAR1 ELISA kit (MBS753326; MyBioSource, San Diego, California, United States), mouse PAR2 ELISA kit (MBS4501658; MyBioSource), mouse STAT3 ELISA kit (OKEH02995; Aviva System Biology, San Diego, California, United States), cyclin D1 ELISA kit (NOV596744; Novus, Centennial, California, United States), mouse Ki67 ELISA kit (EM1473; Wuhan Fine Biotech, Hubei, China), mouse p53 ELISA kit (ab224878; Abcam), mouse VEGF-A ELISA kit (BMS619–2; Thermo Fisher Scientific, Waltham, Massachusetts, United States), and mouse VEGFR-1 ELISA kit (CSB-E04762m; CUSABIO, Houston, Texas, United States) respectively, according to the manufacturer's instructions.

Techniques: Expressing, Coagulation, Cell Culture, Molecular Weight, Western Blot

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice

doi: 10.1055/s-0042-1758855

Figure Lengend Snippet: Tissue levels of coagulation- and tumor-related factors in untreated (UTR) mice and Colon26-inoculated mice orally treated with water or edoxaban (EDX) daily for 21 d

Article Snippet: The concentrations of IL-6, matrix metalloproteinase-2 (MMP-2), TF, and PAI-1 in heparinized plasma and those of PAR1, PAR2, STAT3, cyclin D1, Ki67, p53, VEGF-A, and VEGFR-1 in normal or tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA) using mouse IL-6 quantikine ELISA kit (M6000B; R&D Systems, Minneapolis, Minnesota, United States), mouse MMP-2 ELISA kit (ab254516; Abcam, Cambridge, UK), mouse TF ELISA kit (SEA524Ra; Cloud-Clone, Houston, Texas, United States), mouse PAI-1 ELISA kit (ab197752; Abcam), mouse PAR1 ELISA kit (MBS753326; MyBioSource, San Diego, California, United States), mouse PAR2 ELISA kit (MBS4501658; MyBioSource), mouse STAT3 ELISA kit (OKEH02995; Aviva System Biology, San Diego, California, United States), cyclin D1 ELISA kit (NOV596744; Novus, Centennial, California, United States), mouse Ki67 ELISA kit (EM1473; Wuhan Fine Biotech, Hubei, China), mouse p53 ELISA kit (ab224878; Abcam), mouse VEGF-A ELISA kit (BMS619–2; Thermo Fisher Scientific, Waltham, Massachusetts, United States), and mouse VEGFR-1 ELISA kit (CSB-E04762m; CUSABIO, Houston, Texas, United States) respectively, according to the manufacturer's instructions.

Techniques: Coagulation

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice

doi: 10.1055/s-0042-1758855

Figure Lengend Snippet: ( A ) Immunofluorescence data for PAR1 and PAR2 expression in the normal thigh tissues of untreated (UTR) mice and tumor tissues of the water-treated group or edoxaban (EDX) (10 mg/kg/d)-treated group of Colon26-inoculated mice on day 21. ( B ) Immunofluorescence data for STAT3, cyclin D1, and Ki67 expression in normal thigh tissues of untreated (UTR) mice and tumor tissues of the water-treated group or EDX-treated group of Colon26-inoculated mice on day 21.

Article Snippet: The concentrations of IL-6, matrix metalloproteinase-2 (MMP-2), TF, and PAI-1 in heparinized plasma and those of PAR1, PAR2, STAT3, cyclin D1, Ki67, p53, VEGF-A, and VEGFR-1 in normal or tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA) using mouse IL-6 quantikine ELISA kit (M6000B; R&D Systems, Minneapolis, Minnesota, United States), mouse MMP-2 ELISA kit (ab254516; Abcam, Cambridge, UK), mouse TF ELISA kit (SEA524Ra; Cloud-Clone, Houston, Texas, United States), mouse PAI-1 ELISA kit (ab197752; Abcam), mouse PAR1 ELISA kit (MBS753326; MyBioSource, San Diego, California, United States), mouse PAR2 ELISA kit (MBS4501658; MyBioSource), mouse STAT3 ELISA kit (OKEH02995; Aviva System Biology, San Diego, California, United States), cyclin D1 ELISA kit (NOV596744; Novus, Centennial, California, United States), mouse Ki67 ELISA kit (EM1473; Wuhan Fine Biotech, Hubei, China), mouse p53 ELISA kit (ab224878; Abcam), mouse VEGF-A ELISA kit (BMS619–2; Thermo Fisher Scientific, Waltham, Massachusetts, United States), and mouse VEGFR-1 ELISA kit (CSB-E04762m; CUSABIO, Houston, Texas, United States) respectively, according to the manufacturer's instructions.

Techniques: Immunofluorescence, Expressing

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice

doi: 10.1055/s-0042-1758855

Figure Lengend Snippet: ( A ) Images of immunofluorescence analysis of TUNEL-positive cells and control DAPI staining in normal thigh tissue of untreated (UTR) mice and tumor tissue in the water-treated group or edoxaban (EDX) (10 mg/kg/d)-treated group of Colon26-inoculated mice on day 21. ( B ) TUNEL-positive cells/mm 2 in each group of tissues shown in Fig. 5A were counted as described in the text. The data are shown as mean ± SD. Significant differences in TUNEL-positive cells between groups are shown as ** p <0.01. ( C ) The concentration of p53 protein, determined by ELISA, in solubilized samples (100 mg) of normal thigh tissue from untreated (UTR) mice and tumor tissues from the water-treated group or EDX-treated group of Colon26-inoculated mice on day 21. The data are shown as mean ± SD. Significant differences in p53 protein concentrations between groups are shown as ** p <0.01.

Article Snippet: The concentrations of IL-6, matrix metalloproteinase-2 (MMP-2), TF, and PAI-1 in heparinized plasma and those of PAR1, PAR2, STAT3, cyclin D1, Ki67, p53, VEGF-A, and VEGFR-1 in normal or tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA) using mouse IL-6 quantikine ELISA kit (M6000B; R&D Systems, Minneapolis, Minnesota, United States), mouse MMP-2 ELISA kit (ab254516; Abcam, Cambridge, UK), mouse TF ELISA kit (SEA524Ra; Cloud-Clone, Houston, Texas, United States), mouse PAI-1 ELISA kit (ab197752; Abcam), mouse PAR1 ELISA kit (MBS753326; MyBioSource, San Diego, California, United States), mouse PAR2 ELISA kit (MBS4501658; MyBioSource), mouse STAT3 ELISA kit (OKEH02995; Aviva System Biology, San Diego, California, United States), cyclin D1 ELISA kit (NOV596744; Novus, Centennial, California, United States), mouse Ki67 ELISA kit (EM1473; Wuhan Fine Biotech, Hubei, China), mouse p53 ELISA kit (ab224878; Abcam), mouse VEGF-A ELISA kit (BMS619–2; Thermo Fisher Scientific, Waltham, Massachusetts, United States), and mouse VEGFR-1 ELISA kit (CSB-E04762m; CUSABIO, Houston, Texas, United States) respectively, according to the manufacturer's instructions.

Techniques: Immunofluorescence, TUNEL Assay, Control, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice

doi: 10.1055/s-0042-1758855

Figure Lengend Snippet: ( A ) Concentrations of VEGF-A and ( B ) VEGFR-1, determined by ELISA, in solubilized samples (100 mg) of normal thigh tissue from untreated (UTR) mice and tumor tissues from water-treated group or EDX-treated group of Colon26-inoculated mice on day 21. The data are shown as mean ± SD. Significant differences in concentrations of the respective proteins between groups are shown as ** p <0.01.

Article Snippet: The concentrations of IL-6, matrix metalloproteinase-2 (MMP-2), TF, and PAI-1 in heparinized plasma and those of PAR1, PAR2, STAT3, cyclin D1, Ki67, p53, VEGF-A, and VEGFR-1 in normal or tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA) using mouse IL-6 quantikine ELISA kit (M6000B; R&D Systems, Minneapolis, Minnesota, United States), mouse MMP-2 ELISA kit (ab254516; Abcam, Cambridge, UK), mouse TF ELISA kit (SEA524Ra; Cloud-Clone, Houston, Texas, United States), mouse PAI-1 ELISA kit (ab197752; Abcam), mouse PAR1 ELISA kit (MBS753326; MyBioSource, San Diego, California, United States), mouse PAR2 ELISA kit (MBS4501658; MyBioSource), mouse STAT3 ELISA kit (OKEH02995; Aviva System Biology, San Diego, California, United States), cyclin D1 ELISA kit (NOV596744; Novus, Centennial, California, United States), mouse Ki67 ELISA kit (EM1473; Wuhan Fine Biotech, Hubei, China), mouse p53 ELISA kit (ab224878; Abcam), mouse VEGF-A ELISA kit (BMS619–2; Thermo Fisher Scientific, Waltham, Massachusetts, United States), and mouse VEGFR-1 ELISA kit (CSB-E04762m; CUSABIO, Houston, Texas, United States) respectively, according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice

doi: 10.1055/s-0042-1758855

Figure Lengend Snippet: Effect of edoxaban (EDX) on plasma thrombin-antithrombin complex (TAT) levels in untreated (UTR) control mice and Colon26-inoculated mice. Seven days (day 0) after inoculating mice with Colon26 cells, water or EDX (10 mg/kg/d) was orally administered daily for 21 days; citrated plasma samples were prepared from blood drawn from the heart on days -7, 0, 7, 14, and 21 of UTR mice ( n = 5) (○), water-treated group ( n = 5) (●), or EDX-treated group ( n = 5) (▪) of Colon26-inoculated mice. Plasma TAT concentration was determined by ELISA. Significant differences in plasma TAT levels between the water-treated group or EDX-treated group of Colon26-inoculated mice and the UTR mouse group are shown as ** p <0.01. Significant differences in plasma TAT levels between the water-treated group and EDX-treated group of Colon26-inoculated mice are shown as † p <0.05.

Article Snippet: The concentrations of IL-6, matrix metalloproteinase-2 (MMP-2), TF, and PAI-1 in heparinized plasma and those of PAR1, PAR2, STAT3, cyclin D1, Ki67, p53, VEGF-A, and VEGFR-1 in normal or tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA) using mouse IL-6 quantikine ELISA kit (M6000B; R&D Systems, Minneapolis, Minnesota, United States), mouse MMP-2 ELISA kit (ab254516; Abcam, Cambridge, UK), mouse TF ELISA kit (SEA524Ra; Cloud-Clone, Houston, Texas, United States), mouse PAI-1 ELISA kit (ab197752; Abcam), mouse PAR1 ELISA kit (MBS753326; MyBioSource, San Diego, California, United States), mouse PAR2 ELISA kit (MBS4501658; MyBioSource), mouse STAT3 ELISA kit (OKEH02995; Aviva System Biology, San Diego, California, United States), cyclin D1 ELISA kit (NOV596744; Novus, Centennial, California, United States), mouse Ki67 ELISA kit (EM1473; Wuhan Fine Biotech, Hubei, China), mouse p53 ELISA kit (ab224878; Abcam), mouse VEGF-A ELISA kit (BMS619–2; Thermo Fisher Scientific, Waltham, Massachusetts, United States), and mouse VEGFR-1 ELISA kit (CSB-E04762m; CUSABIO, Houston, Texas, United States) respectively, according to the manufacturer's instructions.

Techniques: Clinical Proteomics, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice

doi: 10.1055/s-0042-1758855

Figure Lengend Snippet: The proposed basic mechanism of the effect of edoxaban (EDX) on tumor growth and apoptosis mediated by factor Xa-PAR2. ( A ) In the absence of EDX, tissue factor (TF) expressed in inoculated Colon26 cells activates blood coagulation and inflammation in host mice, and generated factor Xa activates PAR2, whereas generated thrombin possibly activates PAR1/PAR4 on Colon26 cells and various stroma cells in the tumor microenvironment. PAR2 activation stimulates TF expression, STAT3-mediated IL-6, PAI-1, and MMP-2 production in peri-cancer cells, and RAS/PI3K/SyK/Ki67/cyclin D1 signaling pathways in tumor cells ( black upward arrows ). These affect tumor growth. In addition, thrombin-mediated PAR1 and PAR4 pathways induce p53 expression, leading to apoptosis, which may be regulated by the PAR2-dependent system. ( B ) EDX administration to mice suppresses factor Xa-mediated PAR2 activation, reducing PAR2 levels, followed by a decrease in plasma TF, IL-6, PAI-1, and MMP-2 levels and RAS/PI3K/SyK/Ki67/cyclin D1 signaling pathways ( white downward arrows ), leading to tumor growth suppression. Furthermore, suppression of PAR2 pathway activation by EDX may reduce the regulatory system against the PAR1/PAR4 pathway and may increase the p53-dependent apoptosis ( black upward arrows ).

Article Snippet: The concentrations of IL-6, matrix metalloproteinase-2 (MMP-2), TF, and PAI-1 in heparinized plasma and those of PAR1, PAR2, STAT3, cyclin D1, Ki67, p53, VEGF-A, and VEGFR-1 in normal or tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA) using mouse IL-6 quantikine ELISA kit (M6000B; R&D Systems, Minneapolis, Minnesota, United States), mouse MMP-2 ELISA kit (ab254516; Abcam, Cambridge, UK), mouse TF ELISA kit (SEA524Ra; Cloud-Clone, Houston, Texas, United States), mouse PAI-1 ELISA kit (ab197752; Abcam), mouse PAR1 ELISA kit (MBS753326; MyBioSource, San Diego, California, United States), mouse PAR2 ELISA kit (MBS4501658; MyBioSource), mouse STAT3 ELISA kit (OKEH02995; Aviva System Biology, San Diego, California, United States), cyclin D1 ELISA kit (NOV596744; Novus, Centennial, California, United States), mouse Ki67 ELISA kit (EM1473; Wuhan Fine Biotech, Hubei, China), mouse p53 ELISA kit (ab224878; Abcam), mouse VEGF-A ELISA kit (BMS619–2; Thermo Fisher Scientific, Waltham, Massachusetts, United States), and mouse VEGFR-1 ELISA kit (CSB-E04762m; CUSABIO, Houston, Texas, United States) respectively, according to the manufacturer's instructions.

Techniques: Coagulation, Generated, Activation Assay, Expressing, Protein-Protein interactions, Clinical Proteomics