selective par1 antagonists sch 530348  (Axon Medchem LLC)

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: PAR1 mRNA expression

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Microarray

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: PAR1 is overexpressed in glioma tumor progenitor cells (TPCs) (a), Expression of PAR1 mRNA using real-time PCR, in freshly sorted glioma-derived A2B5+ TPCs (n = 12) relative to normal A2B5+ glial progenitor cells (GPCs) (n = 4) and unsorted cells (UNS) (n = 4), reveals that PAR1 mRNA was significantly upregulated at all stages of glioma development. (b–c) Relative quantification of PAR1 gene (b) and protein expression (c) using RT-PCR (b) and flow cytometry (c) detection in GBM-derived glioma-initiating cell lines (GICLs) established from unsorted or A2B5+ cells (*) maintained in serum-free media (SFM) supplemented with FGF, EGF (20 ng/ml) and PDGF (10 ng/ml) for less than 10 passages; and commercially available adherent GBM cells U87 and U251 (c) cultured in 10% serum culture conditions. Comparable quantities of cDNA were ensured by amplification of GAPDH (b). (d–f) Flow cytometry analysis of PAR1, A2B5 and CD133 expression in GBM-derived GICLs. Representative scatter plot of GICL8 stained with A2B5 (bottom right), PAR1 (upper left), both (upper right), or their corresponding isotype controls (bottom left) (D). Error bars indicate Means ± s.e.m.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cell Culture, Amplification, Staining

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: Analysis of PAR1 expression in relation with TCGA-defined glioblastoma subtypes and copy number variations (a–b), PAR1 gene expression was enriched in the CL subtype of GBM defined by the TCGA (n= 483) relative to normal brain tissue (n = 10) (a) but was not correlated with the glioma-Cpg Island Methylator Phenotype (G-CIMP) subtype. (b, c) Expression value of PAR1 gene expression in low-grade gliomas (n = 468) derived from the TCGA showing a significant enrichment of PAR1 in anaplastic AST, relative to grade II OLG and OA. Black lines in each group indicate mean ± s.e.m. 1 way ANOVA, P < 0.0001; *P < 0.05; **P < 0.01; ***P < 0.001 after Tukey’s multiple test comparison. (d) PAR1 expression was significantly correlated with EGFR, PTEN and CDKN2A copy number variations. AST, astrocytoma; CL, classical; OLG,: oligodendroglioma; OA, oligoastrocytoma; II and III: WHO grades II and III, respectively; MES, mesenchymal; NL, neural; PN: proneural.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Expressing, Gene Expression, Derivative Assay, Comparison

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: Lentiviral induced PAR1 knockdown (KD) validation. Validation of PAR1 gene (a, b, e) and protein (c–e) induced silencing using quantitative RT-PCR (a, b) and flow cytometry (c, d) A2B5+ derived glioma-initiating cell lines (GICLs), 6 days after transduction with two different PAR1 KD lentiviruses, compared with glioma cells transduced with a SCR. Gene expression levels normalized to GAPDH. One-way ANOVA with repeated measures; *P < 0.05; **P < 0.01; ***P < 0.001 after Tukey post-hoc comparisons. Means ± s.e.m.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Knockdown, Biomarker Discovery, Quantitative RT-PCR, Flow Cytometry, Derivative Assay, Transduction, Gene Expression

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: PAR1 silencing inhibits the growth and self-renewal of glioma TPCs. (a) Representative photomicrograph illustrating the number of A2B5+ TPCs derived from GICL-8 6 days after transduction with either PAR1-KD or control lentiviruses. Scale bar, 100 µm. (b–g) Effects of PAR1 silencing on the in vitro expansion (b, c), proliferation (d, e), survival (f, g) and clonal sphere formation (h, i) of A2B5+ TPCs derived from two glioma-initiating cell lines (GICL8 and GICL9), 6 days after transduction with different PAR1 knockdown (KD) lentiviruses, compared with scrambled lentivirus (SCR) and control (CT) untransduced cells. (b, c) Lentiviral KD of PAR1 significantly reduced the number of A2B5+ GICL relative to both SCR shRNAi-transduced and non-transduced CT cells. (d, e) EdU incorporation in association with propidium iodide (PI) staining revealed that GICLs subjected to PAR1 KD manifested fewer cells in S phase relative to SCR and CT cells. (f, g) PAR1 KD significantly increased the percentage of apoptotic cells as determined by flow cytometry analysis of Annexin V, relative to SCR and CT cells. KD, Knockdown. Means ± s.e.m. One-way ANOVA with repeated measures (P < 0.0001) with Tukey post-hoc comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. Post-hoc comparisons between PAR1 KD cells and SCR and non-transduced control (CT) cells are illustrated by orange and black stars, respectively; three independent experiments for each cell line. (h, i) GICLs were plated into a 96-well plates for limiting dilution sphere formation assay, and counted 14 days later; wells were then scored for the presence or absence of sphere growth. The log fraction of the negative wells (non-responding) was plotted as a function of cell density per well.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Derivative Assay, Transduction, Control, In Vitro, Knockdown, Staining, Flow Cytometry, Tube Formation Assay

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: PAR1 silencing suppresses the in vivo growth of TPC-derived tumors and prolongs survival Effects of PAR1 silencing on the in vivo expansion of A2B5+ GICLs derived from two different GBM (28 000 cells per animal, n = 5–6 mice per group), 4 weeks after transduction with PAR1 knockdown (KD) lentiviruses, compared with scrambled lentivirus (SCR) and control (CT) untransduced cells. (a) Hematoxylin-eosin stained sections of xenografts following intracranial implantation of A2B5+ TPCs. (b–g) Graphs representing the stereological analysis of the tumor extension, measured along the antero-posterior axis of xenograft mice brain (b, c); tumor volume (d, e); and proliferation of glioma TPCs as shown by the number of xenografted cells stained with the anti-human nuclei antigen (HNA) co-expressing the mitotic marker Ki67 (f, g), demonstrating a prominent inhibitory effect of PAR1 silencing on the tumorigenicity and mitotic activity of glioma A2B5+ TPCs relative to SCR and CT cells. Means ± s.e.m. P-values calculated using one-way ANOVA (P < 0.0001) followed by Tukey post-hoc comparisons with *P < 0.05; **P < 0.01; ***P < 0.001. Post-hoc comparisons between cells transduced with PAR1 KD shRNAi, compared with those transduced with SCR shRNAi untransduced CT cells are illustrated by orange and black stars, respectively. (h–i) Kaplan–Meier curves show an increase in median survival of mice bearing intracranial glioma TPCs transduced with PAR1 shRNA relative to SCR and CT mice. Log-rank analysis, (P < 0.0001) followed by pairwise comparison between all groups with **P < 0.01 and ***P < 0.001.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: In Vivo, Derivative Assay, Transduction, Knockdown, Control, Staining, Expressing, Marker, Activity Assay, shRNA, Comparison

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: Pharmacological inhibition of PAR1 impedes glioma TPC expansion and migration in vitro. Effects of the specific PAR1 inhibitors SCH79797 and SCH530348 on the in vitro expansion (a–d) and migration (e–h) of A2B5+ GICL derived from GBMs. Dose-dependent growth of GBM-derived TPCs was measured by counting the number of cells 4 days after administration of SCH79797 (a, b) and SCH530348 (c, d) relative to highest concentration of vehicle control (DMSO). P-values were calculated using one-way ANOVA with repeated measures followed by Tukey post-hoc comparisons with *P < 0.05; **P < 0.01; ***P < 0.001. Results were obtained from three independent experiments for each cell line. Migration was measured by assessing the average cell speed of A2B5+ GICLs in a dose-dependent manner, 24 h after administration of SCH79797 (e, f) and SCH530348 (g, h), relative to 1% DMSO vehicle control solution. P-values were calculated using Kruskal–Wallis test followed by Dunn’s multiple comparison test post-hoc comparisons with **P < 0.01; ***P < 0.001. Means ± s.e.m.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Inhibition, Migration, In Vitro, Derivative Assay, Concentration Assay, Control, Comparison