Article Figures & Data
Figures
- Figure 1.
Identification of a small-molecule SMO inhibitor. A, structure of lead compound AZD8542. B, treatment of HeLa cells expressing SMO with AZD8542 (0.1 and 1000 nmol/L) was able to compete away BODIPY-labeled cyclopamine in a dose-dependent fashion. C, in the BODIPY-labeled cyclopamine competition assay, affinity of AZD8542 for mouse and human SMO showed similar binding Kd values for both species (mouse, 5 nmol/L; human, 20 nmol/L).
- Figure 2.
Hedgehog pathway gene expression. RNA from various pancreas cell lines was analyzed for expression of SMO (A), SHH (B), PTCH1 (C), and GLI1 (D) by qPCR. Expression was normalized to that of the HPRT gene.
- Figure 3.
Stimulation of HPSCs by rSHH induces proliferation and GLI1 expression. A, rSHH (μg/mL) was added to HPSCs grown in 1% DMEM. A, proliferation was measured by MTS assay at 72 hours. *, P ≤ 0.05, **, P ≤ 0.005 vs. 0 μg/mL rSHH control. B, HPSCs were treated with rSHH (2 μg/mL) with or without AZD8542. GLI1 expression was measured by qPCR and normalized to HPRT. *, P ≤ 0.05 vs. 2 μg/mL rSHH, 0 nmol/L AZD8542 sample.
- Figure 4.
Effect of AZD8542 in an orthotopic model of pancreatic cancer with luciferase-labeled Bxpc3 cells coinjected with HPSCs. Mice were treated daily with 80 mg/kg AZD8542 (black) or vehicle alone (white). A, luciferase signal, indicating tumor growth, was measured weekly. B, final pancreatic tumor volume was measured after 32 days of drug treatment. C, D, expression of downstream targets GLI1 and PTCH1 was analyzed in tumors (T/S ratio 1:3) using qPCR. *, P ≤ 0.05 and **, P ≤ 0.005 vs. vehicle controls.
- Figure 5.
Hedgehog pathway signaling in PDAC in vitro and in vivo. A, quantitative PCR for Gli1 expression. HPSC-GFP cells were cultured with or without Bxpc3 for 96 hours and sorted by flow cytometry; **, P < 0.0001. Immunohistochemistry for Ki67 (B) and CD31 (C) was done on mouse pancreatic tumors with tumor/stroma ratio 1:3 treated with AZD8542 or control. Total magnification 100×. Data are shown as mean ± SEM; *, P < 0.005 and **, P < 0.0001.
- Figure 6.
Effect of AZD8542 on liver metastases in an orthotopic model of pancreatic cancer with luciferase-labeled Bxpc3 cells coinjected with HPSCs. Mice were treated daily with 80 mg/kg AZD8542 or vehicle alone. A, liver metastases were measured by luciferase signal. B, histology was confirmed by H&E staining in mice from the T/S 1:3 group (total magnification 200×); *. P = 0.06.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods, Figures Legends 1-7 - PDF file - 78K
- Supplementary Figures 1-7 - PDF file - 705K, S1. Identification of potent Hh pathway inhibitors. S2. SHH and IHH expression and effects on HPSCs. S3. Effect of AZD8542 in an orthotopic model of pancreatic cancer with luciferase-labeled MPanc96 cells co-injected with HPSCs. S4. AZD8542 is a potent in vivo inhibitor of the Hh pathway. S5. In vivo efficacy of AZD8542 in an HT29/MEF co-implant model. S6. Effect of AZD8542 in an orthotopic model of pancreatic cancer with luciferase-labeled Bxpc3 cells coinjected with HPSCs. S7. IVIS imaging of luciferase in an orthotopic model of PDAC
Volume 10, Issue 9
