Article Figures & Data
Figures
- Figure 1.
BV6 enhances γ-irradiation–induced apoptosis in glioblastoma cells. In A–C, glioblastoma cells were treated for 168 hours (A172, U87MG) or 144 hours (T98G) with indicated doses of γ-irradiation and/or 2 μmol/L BV6 (A172, T98G) or 3 μmol/L BV6 (U87MG). In D, A172 cells were treated with indicated doses of γ-irradiation and/or indicated concentrations of BV6 for 168 hours. In D, A172 cells were treated with indicated doses of γ-irradiation and/or indicated concentrations of BV6 for 168 hours. In E, A172 cells were treated with 8 Gy γ-irradiation and/or 2 μmol/L BV6 for indicated times. In A–C, E, apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide–stained nuclei. In D, cell viability was determined by MTT assay and is expressed as percentage of untreated controls. Data are mean + SEM of 3 independent experiments conducted in triplicate; **, P < 0.001. In E, combined treatment with BV6 and radiation was compared with radiation treatment alone. In F–H, clonogenic colony formation assay was done as described in Materials and Methods, and the surviving fraction after 11 to 14 days is shown. Data are mean + SD of at least 3 independent experiments; +, P < 0.05; *, P < 0.01.
- Figure 2.
BV6-mediated sensitization to γ-irradiation–induced apoptosis is caspase dependent. A172 cells were treated for 168 hours with 8 Gy γ-irradiation and/or 2 μmol/L BV6 in the presence or absence of 20 μmol/L zVAD.fmk or 100 μg/mL Enbrel. Treatment with 10 ng/mL TNFα and 2 μmol/L BV6 for 48 hours served as a positive control for Enbrel. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide–stained nuclei. Data are mean + SEM of 3 (A, B) or 1 (C) independent experiments conducted in triplicate; **, P < 0.001 comparing BV6 with solvent; n.s., not significant.
- Figure 3.
NF-κB activation is required for BV6-mediated sensitization to γ-irradiation–induced apoptosis. In A, A172 cells were left untreated (Co) or were treated with 8 Gy γ-irradiation and/or 2 μmol/L BV6 for indicated times. Stimulation with 10 ng/mL TNFα for 1 hour served as positive control. NF-κB activation was assessed by the analysis of NF-κB DNA binding by EMSA. One representative out of 3 experiments is shown. In B, NF-κB complex composition following treatment of A172 cells with 8 Gy γ-irradiation and/or 2 μmol/L BV6 for 12 hours was assessed by subjecting nuclear extracts to EMSA analysis with or without preincubation with specific antibodies against p50, p65, RelB, cRel or p52 or with IgG. In C–E, A172 (C), U87MG (D) or T98G (E) cells stably transduced with a vector containing IκBα-SR or empty control vector were treated with indicated doses of γ-irradiation and/or 2 μmol/L BV6 (A172, T98G) or 3 μmol/L BV6 (U87MG) for 168 hours (C, D) or 144 hours (E). Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide–stained nuclei. Data are mean + SEM of 3 independent experiments conducted in triplicate; *, P < 0.005; **, P < 0.001 comparing IκBα-SR overexpressing to control vector cells.
- Figure 4.
NF-κB inhibition by IκBα-SR prevents BV6-mediated sensitization to γ-irradiation–induced caspase activation and mitochondrial outer membrane permeabilization. A172 cells stably transduced with a vector containing IκBα-SR or empty control vector were treated with 2 μmol/L BV6 and/or 8 Gy γ-irradiation for indicated times. In A, cleavage of caspase-8 and -3 was assessed by Western blotting; cleavage fragments are indicated by arrowhead. GAPDH served as loading control. One representative of 2 experiments is shown. In B and C, mitochondrial transmembrane potential (B) and cytochrome c release (C) were assessed by FACS analysis. Data are mean + SEM of 3 independent experiments conducted in triplicate; **, P < 0.001 comparing IκBα-SR overexpressing with control vector cells following γ-irradiation.
- Figure 5.
NF-κB inhibition by kinase dead IKKβ inhibits BV6-mediated radiosensitization. A, A172 (left) and U87MG (right) cells were stably transduced with a vector containing kinase dead IKKβ (IKKβ-KD) or empty control vector (co). Expression of IKKα/β was determined by Western blotting. In B, NF-κB activation in control and IKKβ-KD overexpressing cells was determined by EMSA after stimulation with 10 ng/mL TNFα for 1 hour. In C, IKKβ-KD overexpressing and control cells were treated with 2 μmol/L BV6 and/or 8 Gy (A172) or 6 Gy (U87MG) γ-irradiation for 168 hours. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide–stained nuclei. Data are mean + SEM of 3 independent experiments conducted in triplicate; **, P < 0.001 comparing IKKβ-KD overexpressing with control vector cells.
- Figure 6.
BV6 enhances γ-irradiation–induced apoptosis in primary cultured glioblastoma and glioblastoma-initiating cancer stem cells. In A, primary cultured glioblastoma cells were treated with 8 Gy γ-irradiation and/or 2 μmol/L BV6 for 168 hours. In B–D, glioblastoma-initiating cancer stem cells (GBSC1-3) were seeded at 0.05 × 105/cm2 and treated with 6 Gy γ-irradiation and/or 6 μmol/L BV6 for 168 hours. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide–stained nuclei. Data are mean + SEM of 3 independent experiments conducted in triplicate; **, P < 0.001 comparing BV6 with solvent.
Tables
- Table 1.
Radiation response of glioblastoma cells upon treatment with BV6 and radiation
Cell line treatment Plating efficiency [%] α [Gy−1] β [Gy−2] LD50 [Gy] Radiation enhancement ratio LD10 [Gy] Radiation enhancement ratio A172 Mock treated 32.4 0.2274 0.0178 2.54 6.66 DMSO treated 28.6 0.1813 0.0232 2.81 6.79 BV6 [1.5 μmol/L] 1.1 0.0000 1.1488 0.78 3.62 1.42 4.80 T98G Mock treated 15.7 0.2142 0.0029 3.11 9.53 DMSO treated 16.0 0.1984 0.0050 3.23 9.39 BV6 [1.5 μmol/L] 6.2 0.2738 0.0045 2.43 1.33 7.49 1.25 U87MG Mock treated 14.2 0.2371 0.0067 2.72 7.94 DMSO treated 13.9 0.2224 0.0080 2.83 8.03 BV6 [1.5 μmol/L] 8.0 0.1905 0.0146 2.96 0.95 7.63 1.05 NOTE: Radiation enhancement ratios at 50% (LD50) and 10% survival (LD10) were calculated by transforming the linear quadratic equation (SF = exp [−α × D − β × D2]) using α and β values of the individual survival curves.
Supplementary Data
Files in this Data Supplement:
- Supplementary Table 1 - PDF file-54KB
- Supplementary Figures 1-4 - PDF file-589KB
- Supplementary Figure Legends and Supplementary Methods Section - PDF file-82KB
Volume 10, Issue 10
