smo receptor inhibitor vismodegib  (Genentech inc)

Journal: Frontiers in immunology

Article Title: A Critical Function for the Transcription Factors GLI1 and GLI2 in the Proliferation and Survival of Human Mast Cells.

doi: 10.3389/fimmu.2022.841045

Figure Lengend Snippet: FIGURE 1 | Modulation of GLI1/2 expression in human mast cells. (A) Simplified illustration of the canonical hedgehog signaling pathway and some of the small molecules used for its activation or inhibition. (B) Relative GLI1 mRNA levels (2-DDCq) in human mast cell lines (HMC-1.1, HMC-1.2, LAD2) and primary human mast cells (hMC) after 20 h incubation with the SMO agonist SAG (200 nM) or the PTCH1 ligand SHH (500 ng/mL). (C) Relative GLI1 mRNA levels (2-DDCq) in mast cells transduced with lentiviral particles containing two separate shRNA constructs to knockdown SUFU (see Supplementary Figure 2). (D) Relative GLI1 and GLI2 mRNA levels (2-DDCq) after 5 h culture with the GLI1/2 inhibitor HPI-1 (20 or 40 µM) or with the SMO inhibitor vismodegib (40 µM). Results are expressed as Mean ± SD of three independent experiments. GAPDH and ACTB were used for normalization. Each individual experiment was done in triplicate. Two-way ANOVA followed by Dunnet multiple comparisons test was used for statistical analysis. **p < 0.01; ****p < 0.0001.

Article Snippet: The tyrosine kinase inhibitor (TKI) dasatinib, SMO agonist (SAG)- hydrochloride, GLI1/2 inhibitor GANT61, and SMO antagonist vismodegib (GDC-0449) were purchased from Selleckchem (Houston, TX).

Techniques: Expressing, Activation Assay, Inhibition, Incubation, Transduction, shRNA, Construct, Knockdown

Journal: Frontiers in immunology

Article Title: A Critical Function for the Transcription Factors GLI1 and GLI2 in the Proliferation and Survival of Human Mast Cells.

doi: 10.3389/fimmu.2022.841045

Figure Lengend Snippet: FIGURE 3 | GLI1/2 inhibitors reduce human mast cell growth by decreasing viability and proliferation. (A) HMC-1.1 (left panel), HMC-1.2 (middle panel), and LAD2 (right panel) cells were cultured for 72 h (HMC-1.1 and HMC-1.2 cells) or 7 days (LAD2 cells) with increasing concentrations of the indicated inhibitors of GLI1/2 (HPI- 1, GANT61 and ATO) or SMO (vismodegib). Cell growth was assessed by measuring relative units of fluorescence (RFU) that represent viable cells using a Cyquant assay; or counting viable cells with a cell counter (LAD2 cells). Results were normalized to vehicle control (represented as concentration 0 µM) and are expressed as Mean ± SD of three independent experiments. Two-way ANOVA followed by Dunnet multiple comparisons test (compared to vehicle) were used for statistical analysis. Approximated IC50 values, estimated using a non-linear fit between the inhibitor concentration and the normalized response (variable slope) with GraphPad Prism 9, were for HPI-1: 25 µM in HMC-1.1 and 16 µM in HMC1.2; and for GANT61: 15 µM in HMC-1.1 and 14 µM in HMC1.2. (B) Representative dot-plots of three independent experiments in which HMC-1.2 and LAD2 were cultured for 72 h or 7 days, respectively, in the presence of the indicated inhibitors (20 µM). Cells were stained with Cell Trace Violet (CTV) before the incubation and at the end of the experiment stained with green dead cell stain. The percentage of dead cells and median fluorescence intensity (MFI) for CTV is shown in each dot-plot. (C) Representative histograms of primary human mast cells (hMC; 1 healthy donor out of 2) cultured for 5 days with the indicated inhibitors (20 µM) and stained with green dead cell stain. Averages of CTV MFI and percentages of dead cells in three independent experiments are shown in Supplementary Figure 4. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The tyrosine kinase inhibitor (TKI) dasatinib, SMO agonist (SAG)- hydrochloride, GLI1/2 inhibitor GANT61, and SMO antagonist vismodegib (GDC-0449) were purchased from Selleckchem (Houston, TX).

Techniques: Cell Culture, CyQUANT Assay, Control, Concentration Assay, Staining, Incubation

Journal: Frontiers in immunology

Article Title: A Critical Function for the Transcription Factors GLI1 and GLI2 in the Proliferation and Survival of Human Mast Cells.

doi: 10.3389/fimmu.2022.841045

Figure Lengend Snippet: FIGURE 5 | GLI1/2 inhibition causes apoptosis in human mast cells. (A) HMC-1.1, HMC-1.2, LAD2, and primary human mast cells (hMC) were cultured in the presence of HPI-1, GANT61, or vismodegib (20 µM) for 48 h (HMC-1.1 and HMC-1.2 cells), 6 days (LAD2 cells) and 4 days (hMC). The percentages of annexin V positive and dead cells were quantified by flow cytometry. Representative dot-plots from one of three independent experiments are shown. The bar graph represents the average percentage of apoptotic cells (annexinV+/dead cell stain-) in three independent experiments using the indicated cells and treatments. (B) Caspase 3/7 activity was quantified by measuring relative units of fluorescence (RFU) of a caspase 3/7 substrate in HMC-1.1 and HMC 1.2 after 48h incubation with increasing concentrations of inhibitors, vehicle control is represented as concentration 0 µM. Results expressed as Mean ± SD of three independent experiments. Two-way ANOVA followed by Dunnet multiple comparisons test (compared to vehicle) were used for statistical analysis. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Article Snippet: The tyrosine kinase inhibitor (TKI) dasatinib, SMO agonist (SAG)- hydrochloride, GLI1/2 inhibitor GANT61, and SMO antagonist vismodegib (GDC-0449) were purchased from Selleckchem (Houston, TX).

Techniques: Inhibition, Cell Culture, Cytometry, Staining, Activity Assay, Incubation, Control, Concentration Assay

Journal: Cellular signalling

Article Title: Coordinated D -cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

doi: 10.1016/j.cellsig.2017.12.007

Figure Lengend Snippet: Canonical Shh signaling drives proliferation in iMEFs. (A) Wild-type (WT) and Gli2−/−3−/− iMEFs were cultured ± SHH ligand and ± the SMO antagonist vismodegib (Vis). Cell number is shown relative to vehicle-treated cells. SHH increased proliferation in WT iMEFs, but not in Gli2−/−3−/− iMEFs. This increase in proliferation was blocked by the addition of vismodegib (n = 5). (B) Histogram of fluorescence intensity of cells treated with the cell tracing reagent CellTrace™ Far Red and analyzed by flow cytometry. SHH treatment resulted in a leftward shift in fluorescence compared to vehicle-treated cells, indicating an increase in cellular divisions (n = 1). Significance: * = p < 0.05. Error bars show SEM.

Article Snippet: The SMO inhibitor, vismodegib, was purchased from LC Laboratories (CAS No 879085–55–9) and dissolved in DMSO.

Techniques: Cell Culture, Fluorescence, Flow Cytometry

Journal: Cellular signalling

Article Title: Coordinated D -cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

doi: 10.1016/j.cellsig.2017.12.007

Figure Lengend Snippet: Foxd1 is a target of canonical Sonic Hedgehog signaling. (A) Gene expression was determined for WT iMEFs cultured ± SHH ligand and ± the SMO antagonist vismodegib (Vis). Expression changes are shown as a fold change from vehicle-treated cells. SHH treatment resulted in an increase in Gli1 and Foxd1 expression, which was blocked by the addition of vismodegib (n = 6). (B) Expression of Gli1 and Foxd1 is increased in Ptch1−/− iMEFs relative to wild-type (inset) and significantly reduced by vismodegib treatment. (C) Expression of Gli1 and Foxd1 is increased in iMEFs overexpressing a constitutively active form of human Smoothened (SMOM2). Expression changes are shown as a fold change from cell overexpressing GFP (n = 5). Insert shows SMO expression. (D) Expression of Gli1 and Foxd1 is not significantly changed in Gli2−/−3−/− iMEFs treated with SHH ligand (n = 4). Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Error bars show SEM.

Article Snippet: The SMO inhibitor, vismodegib, was purchased from LC Laboratories (CAS No 879085–55–9) and dissolved in DMSO.

Techniques: Gene Expression, Cell Culture, Expressing

Journal: Cellular signalling

Article Title: Coordinated D -cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

doi: 10.1016/j.cellsig.2017.12.007

Figure Lengend Snippet: SHH suppresses Cdkn1c expression. (A) Gene expression was determined for wild-type (WT) iMEFs cultured ± SHH ligand and ± the SMO antagonist vismodegib (Vis). Expression changes are shown as a fold change from vehicle-treated cells. SHH treatment resulted in an increase in Ccnd1 and a decrease in Cdkn1c expression, both of which were blocked by the addition of vismodegib. Expression changes are shown as a fold change from vehicle-treated cells. (n = 9). (B) Expression of Ccnd1 is increased in Ptch1−/− iMEFs relative to WT (inset). Vismodegib treatment Ptch1−/− iMEFs results in decreased Ccnd1 expression and increased Cdkn1c expression (C) Expression of Gli1 and Foxd1 is not significantly changed in Gli2−/− 3−/− iMEFs treated with SHH ligand (n = 4). (C) In iMEFs overexpressing a constitutively active form of human Smoothened (SMOM2), expression of Ccnd1 was increased while Cdkn1c expression was decreased. Expression changes are shown as a fold change from cells overexpressing GFP (n = 5). (D) To determine the temporal sequence of gene expression changes, WT cells were harvested at 6, 12, 24, 48, and 72 h after treatment ± SHH ligand. Gli1 expression (white arrow) is significantly increased by 6 h followed by increased Foxd1 expression (black arrow) by 24 h, and Cdkn1c expression (gray arrow) is not significantly decreased until 48 h post-treatment. Expression changes are shown as a fold change from vehicle-treated cells at respective time points (n = 5). Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Error bars show SEM.

Article Snippet: The SMO inhibitor, vismodegib, was purchased from LC Laboratories (CAS No 879085–55–9) and dissolved in DMSO.

Techniques: Expressing, Gene Expression, Cell Culture, Sequencing

Journal: Cancer treatment reviews

Article Title: Targeting the Wnt/beta-catenin Pathway in Cancer: Update on Effectors and Inhibitors

doi: 10.1016/j.ctrv.2017.11.002

Figure Lengend Snippet: Examples of drugs/agents that inhibit Wnt/β-Catenin Signaling

Article Snippet: Vismodegib , SMO inhibitor , Roche/Genentech , Advanced pancreatic cancer, prostate cancer, gastric cancers( NCT01195415 , NCT01088815 , NCT00878163 ) , 62.

Techniques: Binding Assay