Wang et al. BMC Cancer 2010, 10:661 http://www.biomedcentral.com/1471-2407/10/661 Page 5 of 9 Figure 3 Morphologic changes in glioma cells following BPA- BNCT treatment. (A) Hoechst staining. White arrows indicate apoptotic cells in U87 cells, U251 cells, and SHG44 cells, respectively. Original magnification×400; bar: 10 μm. (B) Phase-contrast microscopy. White arrows indicate apoptotic cells in U87 cells, U251 cells, and SHG44 cells, respectively. Original magnification×400; bar: 10 μm. (C) Transmission electron microscopy. Left, U87 cells; original magnification×6,000; bar: 1 μm; middle, U251 cells; original magnification×5,000; bar: 1 μm; right, SHG44 cells; original magnification×6,000; bar: 1 μm. Figure 4 Apoptosis of glioma cells induced by BPA-BNCT. (A) U87 cells; a-1, nonirradiated control; a-2, 48 h after treatment with 4 Gy BPA-BNCT; a-3, 48 h after treatment with 8 Gy BPA-BNCT. (B) SHG44 cells; b-1, nonirradiated control; b-2, 48 h after treatment with 4 Gy BPA-BNCT; b-3, 48 h after treatment with 8 Gy BPA-BNCT. (C) U251 cells; c-1, nonirradiated control. c-2, 48 h after treatment with 4 Gy BPA-BNCT; c-3, 48 h after treatment with 8 Gy BPA-BNCT. Figure 5 At 48 h after irradiation the apoptotic frequencies of glioma cells in the nine 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. BPA-BNCT was higher than that seen after treatment with 4 Gy BPA-BNCT (P < 0.01). Also, the apoptotic frequen- cies of U87 cells after BPA-BNCT treatment were greater than those noted after delivery of equivalent reactor doses of irradiation (P < 0.05). Similar effects were observed in SHG44 and U251 cells (Figure 4, 5). BNCT inhibits colony formation The survival rates of nonirradiated control cells were set at 100%. Cell survival proportions were shown in Table 5. Survival analysis of U87, U251, and SHG44 cells showed that all three cell lines were inhibited more potently by BNCT than by irradiation with [60Co] g-rays (P < 0.01) or by general reactor neutron irradiation (P < 0.01). Altered expression of Bcl-2 and Bax after BNCT In all of U87, U251, and SHG44 cells, Bcl-2 was initially abundantly expressed, whereas Bax expression was low. After treatment with 4 Gy or 8 Gy of BPA-BNCT, Bcl-2 expression was down-regulated, while Bax expression increased simultaneously. Furthermore, 24 h after treat- ment with 4 Gy BPA-BNCT, Bcl-2 expression was lower than that seen 12 h after treatment with 4 Gy BPA- BNCT, and the level of Bax expression was higher. The levels of Bcl-2 and Bax expression were similar 12 h and 24 h after treatment with 8 Gy BPA-BNCT (Figure 6). Discussion Two criteria must be fulfilled when BNCT is to be applied: an appropriate source of high-flux thermal neu- trons is necessary, along with a boron carrier that is