Fig 1.
BC-23 inhibits the binding between β-catenin and Tcf4.
A mixture of 500 nM β-catenin and 20 nM FITC-Tcf4(8–30) was incubated in the absence or presence of the indicated concentrations of BC-23. Fluorescence polarization (FP) values were recorded after 3 h. The relative binding was calculated as (mPT-mPf) /(mPC-mPf)×100. The points represent mean ± S.D. of 3 independent experiments.
Fig 2.
BC-23 inhibits Wnt/β-catenin signaling.
H1299 cells were co-transfected with the reporter TOP-flash and the control Renilla plasmids. Four hours after transfection, cells were treated with various concentrations of BC-23 for 24 h. The TOP luciferase activities were determined and normalized to Renilla luciferase activities. BC-23 exhibited dose-dependent inhibitory activity on TOP luciferase in H1299 cells, with an IC50 value of 2.3 μM. The columns represent mean ± S.D. of 3 independent experiments.
Fig 3.
Predicted binding model for BC-23 and β-catenin.
The binding models were generated by Autodock and PyMol.
Fig 4.
The combination of BC-23 and radiation enhances clonogenic cell death in H1299 cells.
A. H1299 cells were treated with the indicated concentrations of BC-23 alone for 1 (○) and 3 (●) days. Cell viability was determined by the CellTiter Blue assay. B. Sequential exposure of H1299 cells to 2–10 Gy radiation, followed 1 h later by treatment with 3 μM BC-23 (■), dramatically increased the induction of clonogenic cell death when compared to radiation treatment alone (●). Each point represents the mean values of three separate experiments.
Fig 5.
ROS generation is responsible for BC-23 induced radiation enhancement and clonogenic cell death.
A. Combining 3 μM BC-23 with 10 Gy radiation (x) dramatically increased ROS generation, when compared to individual treatments alone: vehicle control (●), 3 μM BC-23 (■), and 10 Gy radiation (▲). Each value is the mean ± SD of three separate experiments. B-D, ROS-dependent cytotoxic effects of BC-23. NAC attenuates the cell death mediated by exposure of H1299 cells to the indicated concentrations of BC-23 in cytotoxicity (B) and colongenic assays (C, representative dishes of clonogenic assays and D, analyses of percentage of surviving cells). *, p<0.05 versus control.
Fig 6.
BC-23 combined with radiation enhances cell cycle arrest in the S phase and relieves radiation-induced G2/M arrest.
H1299 cells were treated with vehicle control (A), 5 μM BC-23 (B), 4 Gy radiation (C), and 4 Gy radiation plus 5 μM BC-23 (D). The percentage of cells in each phase of the cell cycle was determined by propidium iodide (PI) staining and FACS analysis. The results are representative of three independent experiments.
Fig 7.
BC-23 down regulates the mRNA expression of Wnt/β-catenin target genes.
The H1299 cells were treated with the indicated concentrations of BC-23. Total RNA and cDNA were prepared after a 16 h treatment. The cDNA was wuantified by real-time PCR and normalized against a vehicle control. BC-23 treatment significantly inhibited the expression of both Wnt/β-catenin target genes c-Myc and cyclin D1 at the mRNA level. The results are representative of three independent experiments. *, p<0.05 and **, p<0.01 versus control.
Fig 8.
Knockdown of β-catenin by CTNNB1 siRNA significantly blocked the radiosensitizing effect of BC-23.
The H1299 cells were treated with CTNNB1 or with control siRNA. Protein was prepared from a portion of the cells for western blotting and the remaining cells were used for radiosensitivity assays with 6 Gy radiation with/without BC-23. A, CTNNB1 siRNA significantly reduced the β-catenin expression. B, CTNNB1 siRNA significantly blocked the radiosensitizing effect of BC-23. The results are representative of three independent experiments. ****, p<0.001 versus control; ###, p<0.001 versus 6 Gy + control siRNA; &&, p<0.01 versus 6 Gy + control siRNA + BC-23.