Wang et al. BMC Cancer 2010, 10:661 http://www.biomedcentral.com/1471-2407/10/661 Table 5 Survival fractions of glioma cells in different groups Unirradiated control (A) Irradiated in 60Co by 4 Gy (B) Irradiated in 60Co by 8 Gy (C) Irradiated in reactor for the same treatment period as used for Group D (F) Irradiated in reactor for the same treatment period as used for Group E (G) Irradiated in reactor by 4 Gy (H) Irradiated in reactor by 8 Gy (I) Irradiated in reactor with BNCT by 4 Gy (D)* Irradiated in reactor with BNCT by 8 Gy (E)** *P <0.01 vs A, B, F and H **P <0.01 vs A, C, G, I and D able to concentrate in glioma tissues. Therefore, we initially verified uptake of BPA by glioma cells in vitro. We found that the boron concentration in the three glioma cell lines increased constantly over the first 24 hours of incubation. Irradiation was conducted at this time, and the [10B] level attained the theoretical require- ment, in that at least 109 [10B] atoms accumulated in each glioma cell and the thermal neutron fluence could attain 1012-1013n·cm-2, to produce a lethal discharge of high-LET particles [20]. BNCT can inhibit cell proliferation because the RBE is high, as has been shown in animal models. In the pre- sent study, although the three glioma cell lines differed in boron concentration after exposure to BPA, the ther- apeutic efficiency of BNCT was similar in all three lines because the RBE was high. Proliferation of glioma cells was significantly suppressed by BNCT, and the clono- genicity of glioma cells after BNCT treatment was less than 0.01% of control values. Apoptosis, an active process of cellular suicide, is an important means of radiation-induced cell death [21], and may contribute to the therapeutic efficacy of BNCT. Many triggers of apoptosis are known, including cytoge- netic alterations such as micronuclei (MN) formation and chromosomal aberrations (CA) within cellular sys- tems [22]. Furthermore, several studies have sought to correlate the extent of apoptosis after irradiation with Figure 6 The expression of Bcl-2 and Bax proteins in (A) U87 cells, (B) U251 cells, and (C) SHG44 cells, 12 h and 24 h after treatment with 4 Gy or 8 Gy BPA-BNCT. Group Survival fraction (%) Page 6 of 9 SHG44 100 64.5 12.4 2.5 1.6 2.6 1.2 <0.01% <0.01% U87 100 68.1 10.9 3.1 1.6 2.9 1.4 <0.01% <0.01% U251 100 63.7 13.6 2.3 1.4 2.2 0.9 <0.01% <0.01% cellular radiosensitivity [23]. Given that BNCT works mainly through the discharge effects of high-LET Li nuclei and a particles, we investigated whether BNCT could induce apoptosis of glioma cells. Typical apoptotic morphological changes were confirmed in U87, U251, and SHG44 cells after BNCT treatment, using transmis- sion electron microscopy and Hoechst 33342 staining. Furthermore, previous studies have shown that BPA has no toxicity effect on cells at a dosage up to 110 μg [10B]/ml [24]. Few apoptotic changes were apparent in nonirradiated control cells, showing, for the first time, that the pro-apoptotic effect of BNCT plays an impor- tant role in the reduction of cell proliferation. Masunaga et al. described that BNCT induced apoptosis in vivo, the proportion of apoptotic cells increased slightly 6 h after BNCT, and the apoptotic rate was relatively low [17,18]. Fujita et al. stated that apoptosis was a form of cell death induced by BNCT [25]. While Aromando et al. reported that apoptosis did not have a significant role in BNCT-induced hamster cheek pouch tumor control [26], and Kamida et al. described that differences in apoptotic cells pre- and post-BNCT in human oral squamous cell carcinoma xenografts were minimal [27]. Whether BNCT can induce significant apoptosis rate in glioma cells in vivo and whether the pro-apoptotic effect is cell-type-dependant deserved further study. It is well established in mammalian systems that high- LET irradiations have more pronounced effects on bio- logical systems than that of low LET [22], owing to the characteristic mode of energy deposition along a track. Such energy generates complex forms of radiation damage to biological molecules. The transfer of the energy of high-LET radiation to such molecules can break chemical bonds and ionize various cellular components. In the present study, AnnexinV/PI analysis not only confirmed the pro-apoptotic effect of BNCT, but also showed that apoptosis frequencies were higher in groups treated with BNCT compared with g-rays. Moreover, the