Thermal neutron (Gy) Epithermal neutron (Gy) Fast neutron (Gy) 0.013 0.013 0.013 5.778 5.350 5.992 Wang et al. BMC Cancer 2010, 10:661 http://www.biomedcentral.com/1471-2407/10/661 Table 4 The dose components of BNCT 8 Gy U87 U251 SHG44 0.056 0.052 0.058 Thereafter, membranes were incubated for 1 h with HRP-labeled secondary antibodies (Amersham Pharma- cia Biotech, Uppsala, Sweden), either sheep anti-mouse (diluted 1:2,500) or donkey anti-rabbit (1:5,000), and the blots were finally developed using an ECL system, according to the manufacturer’s instructions (Amersham Bioscience, Buckinghamshire, UK). Statistical analysis Outcome variables are expressed as means ± standard deviations (SDs). Statistical analysis was performed using SPSS Version 13.0 (SPSS, Chicago, IL). ANOVA was used for data analysis. All statistical tests were two- sided, and P values < 0.05 were considered to be statisti- cally significant. Results Boron uptake As expected, [10B] was taken up by U87, U251, and SHG44 cells during incubation for 24 hours in medium containing 50 μg [10B]/ml. The boron concentrations in cells even- tually reached 2.72 ± 0.25 μg/107 cells, 9.78 ± 0.49 μg/107 cells, and 2.48 ± 0.34 μg/107 cells for the U87, U251, and SHG44 cell lines, respectively, at 24 h (Figure 1). Tumor cell numbers fall after BPA-BNCT treatment The inhibitory effect of BPA-BNCT on proliferation of U87 cells was much more significant than that afforded by g-rays, as confirmed by the MTT assay (P < 0.01, Figure 2). Any inhibitory effect was marginal in cells Figure 1 Time course of boron accumulation. Data represent the mean ± SD of triplicate experiments. Page 4 of 9 g-ray (Gy) 0.846 0.748 0.878 10B(n,a)7Li (Gy) 1.306 1.800 1.058 receiving 4 Gy or 8 Gy of g-rays, whereas significant inhi- bition was observed in cells treated with 4 Gy or 8 Gy of BPA-BNCT. The number of viable U87 cells decreased in 48 h after treatment in all of Groups D, E, F, G, H, and I, and the most potent inhibition was observed in the group receiving 8 Gy of BPA-BNCT. Similar effects were observed in SHG44 and U251 cells (data not shown). BNCT induces apoptosis Typical apoptotic morphological changes were found in all of the U87, U251, and SHG44 cell lines 12 h after treatment with BPA-BNCT; the changes included shrinkage, deformation, and detachment from culture dishes. Nuclear condensation and chromatin margina- tion were evident by Hoechst 33342 staining (Figure 3A, 3B), and chromatin margination, nuclear condensation, and segmentation were noted on transmission electron microscopy (Figure 3C). The apoptotic frequency of U87 cells treated with 4 Gy or 8 Gy of BPA-BNCT was significantly higher than that of cells treated with equivalent doses of g-rays (P < 0.01), as shown by annexin V/PI staining (Figure 4, 5). Furthermore, the apoptotic frequency of cells treated with 8 Gy Figure 2 Growth curves of U87 cells in different groups. Group A; control nonirradiated cells; Group B: cells treated with 4 Gy of [60Co] g-rays; Group C: cells treated with 8 Gy of [60Co] g-rays; Group D: cells treated with 4 Gy BPA-BNCT; Group E: cells treated with 8 Gy BPA-BNCT; Group F: cells irradiated in the reactor for the same treatment period as used for Group D; Group G: cells irradiated in the reactor for the same treatment period as used for Group E; Group H: cells irradiated with 4 Gy in the reactor; Group I: cells irradiated with 8 Gy in the reactor. Data represent the mean ± SD of triplicate experiments.