gpcr antagonists psb1115  (Tocris)

Journal: Cancer immunology research

Article Title: Netrin-1 Promotes the Immunosuppressive Activity of MDSCs in Colorectal Cancer.

doi: 10.1158/2326-6066.CIR-22-0658

Figure Lengend Snippet: Figure 4. The netrin-1/A2BR axis activates the cAMP/PKA/CREB signaling pathway. A, Intracellular cAMP concentrations in different treated MDSCs were detected by a colorimetric competitive ELISA method as described in Materials and Methods (n ¼ 3). B, With or without netrin-1 treatment, p-CREB/CREB protein levels in MDSCs were detected at different time points by Western blot analysis. C, The Arg1 activity of MDSCs was measured after treatment with a PKA inhibitor (H89, 20 mmol/L), adenylyl cyclase (AC) agonist (forskolin, 10 mmol/L), and netrin-1 for 24 hours (n ¼ 3). D, MDSCs were incubated with netrin-1, the A2BR antagonist PSB1115 (PSB, 10 mmol/L), 20 mmol/L H89, and 10 mmol/L forskolin for 30 minutes, and cells were then assessed for phosphorylation of CREB via Western blot analysis. Results are representative of at least three independent experiments. Data are represented as means SEM. One-way ANOVA with Tukey multiple comparisons was used for statistical analysis. , P < 0.001; , P < 0.01.

Article Snippet: For inhibiting A2BR activity, the A2BR antagonist PSB1115 (Tocris Bioscience, 2009) was added at 10 mmol/L for 30 minutes before netrin-1 treatment for 24 hours.

Techniques: Competitive ELISA, Western Blot, Activity Assay, Incubation, Phospho-proteomics

Journal: Cancer immunology research

Article Title: Netrin-1 Promotes the Immunosuppressive Activity of MDSCs in Colorectal Cancer.

doi: 10.1158/2326-6066.CIR-22-0658

Figure Lengend Snippet: Figure 7. Tumor-derived Netrin-1 affects the immunosuppressive function of MDSCs. Within the tumor microenvironment, netrin-1 secreted by tumor cells enhances the immunosuppressive activity of the MDSC population through the A2BR receptor and the downstream cAMP/PKA/CREB signaling pathway to induce T-cell anergy.

Article Snippet: For inhibiting A2BR activity, the A2BR antagonist PSB1115 (Tocris Bioscience, 2009) was added at 10 mmol/L for 30 minutes before netrin-1 treatment for 24 hours.

Techniques: Derivative Assay, Activity Assay

Journal: JCI Insight

Article Title: Identification of an ATP/P2X7/mast cell pathway mediating ozone-induced bronchial hyperresponsiveness

doi: 10.1172/jci.insight.140207

Figure Lengend Snippet: ( A ) ATP induces murine airway mast cell degranulation. WT mice (females, aged 9–25 weeks) were exposed to aerosolized ATP (50 mg/mL) or PBS; BALF collected after 30 minutes for histamine analysis. Black and red circles represent histamine levels from PBS- and ATP-treated mice, respectively. n = 3; * P < 0.05 by Student’s t test. ( B ) ATP-induced BHR is mast cell dependent. C57BL/6 WT and C57BL/6Kit W-sh/W-sh mast cell–deficient mice (females, aged 9–25 weeks) were exposed to aerosolized ATP (50 mg/mL), then methacholine 30 minutes later. Black and red circles represent PBS-treated ( n = 4) and ATP-treated WT mice ( n = 16), respectively; black and red squares represent PBS-treated ( n = 3) and ATP-treated mast cell–deficient mice ( n = 8), respectively; * P < 0.05 by mixed effects analysis between ATP-treated groups. ( C and D ) ATP induces degranulation in murine BMMCs ( C ) and human CBMCs ( D ) in vitro. Mast cells were treated with ATP (300 μM, 1000 μM) or PBS for 30 minutes prior to hexosaminidase measurement. Black and red circles represent PBS- and ATP-treated cells, respectively. n = 4; * P < 0.05 by Student’s t test. ( E ) ATP potentiates antigen-induced degranulation in CBMCs. CBMCs were incubated for 3 days at 1 × 10 6 cells/mL at 37°C in medium with 10 ng/mL recombinant human IL-4. Cells were incubated with antagonist (PSB1115) or vehicle (DMSO) for 30 minutes prior to ATP or adenosine addition and centrifuged after 30 minutes, and β-hexosaminidase release was assessed. Red and purple circles represent ATP- and adenosine-treated cells, respectively. n = 4; * P < 0.05 by Student’s t test. ( F ) ATP but not adenosine induces BHR in A 3 –/– mice. A 3 –/– mice (females, aged 9–25 weeks) were exposed to aerosolized ATP (50 mg/mL), adenosine (50 mg/mL), or PBS and then methacholine 30 minutes later. Purple and red triangles represent adenosine-treated ( n = 7) and ATP-treated mice ( n = 8), respectively. * P < 0.05 by mixed effects analysis. Data are shown as mean ± SEM.

Article Snippet: In experiments using A2B antagonist PSB1115 (Tocris), cells were incubated with antagonist or vehicle (DMSO) in Siraganian buffer for 30 minutes prior to ATP or adenosine addition.

Techniques: In Vitro, Incubation, Recombinant

Journal: Cancers

Article Title: Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1

doi: 10.3390/cancers13215508

Figure Lengend Snippet: The adenosine pathway in the LLC1 tumor microenvironment. ( A ) RNA was extracted from ASB-XIV and LLC1 tumor cells and subjected to RNA-Seq analysis. The mRNA expressions of immunosuppressive molecules in these two cell lines were shown. ( B ) The expressions of CD38, CD39, and CD73 on LLC1 were examined by flow cytometry in vitro. ( C ) C57BL/6 mice ( n = 5) received a subcutaneous injection of 5 × 10 5 LLC1. The combination of 2 mg/Kg A2A and 1 mg/Kg A2B adenosine receptor antagonists (SCH 58261 and PSB 1115) was administered on days 7, 9, 11, 13, 15, 17, and 19. ( D ) LLC1 tumor growth was compared in mice ( n = 8) that received 30μg CpG monotherapy, L82 LP-pulsed DC vaccination (1 × 10 6 ), and CpG, or the triple combination of DC vaccination, CpG, and A2R antagonists. DC vaccines were prophylactically administered twice at a 2-week interval.

Article Snippet: A2A receptor antagonist (SCH58261) and A2B receptor antagonist (PSB1115) were obtained from Tokyo Chemical Industry (Tokyo, Japan) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: RNA Sequencing Assay, Flow Cytometry, In Vitro, Injection, Vaccines