Wang et al. BMC Cancer 2010, 10:661 http://www.biomedcentral.com/1471-2407/10/661 primarily limited to boron-containing cells [3]. BNCT has been carried out in numerous studies using syn- geneic rat gliomas [3-6]. Approaches such as administer- ing [10B] by intra-arterial injection with or without blood-brain barrier (BBB) disruption [7-9], or intracer- ebral delivery of high molecular weight agents by means of convection enhanced delivery following neutron beam irradiation [2,10], confirm that BNCT is highly effective for treatment glioma xenograft models. In clinic, BNCT has been used in various countries to treat patients with high-grade glioma, melanoma, liver metastasis of colon adenocarcioma, malignant melanomas, oral cancer, and therapeutically refractory, recurrent tumors of the head and neck [3,6,11-15]; while the mechanism of BNCT- induced cell death remains unclear. We treated U87, U251, and SHG44 glioma cells with BNCT in vitro, using L-p-borono-phenylalanine (L-BPA) as the boron carrier and the thermal neutron source of the Xi’an Pulsed Reactor (XAPR) in China as the irra- diator. The relative biological effect (RBE) of BNCT on tested cells, especially with respect to apoptosis induc- tion, was assessed, and a possible mechanism of BNCT action was presented. Methods Glioma cell culture The human glioblastoma multiforme cell lines U87 and U251 were obtained from the American Type Culture Collection (ATCC; Manassas, VA). The human anaplas- tic astrocytoma cell line SHG44 was purchased from the Institute of Biochemistry and Cell Biology (IBCB; Shang- hai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) with 10% (v/v) fetal bovine serum (Gibco) and 0.5% (w/v) penicillin-streptomycin solution (Gibco), at 37°C under 5% CO2 and 95% air (both v/v). Boron uptake by glioma cells in vitro [10B]-enriched L-BPA (Ryscor Inc., Raleigh, NC) was used in the study. L-BPA was added to the culture media of glioma cells to 50 μg [10B]/ml. Cells in the exponential phase of growth were cultured in boron- containing medium for 3, 6, 12, or 24 h before harvest- ing, and boron concentrations were determined by inductively coupled plasma atomic emission spectro- scopy (ICP-AES) (VISTA-MPX; Varian Co., Walnut Creek, CA) according to a previously described method [16]. In vitro irradiation In vitro irradiation of the three cell lines was performed using the neutron beam at the XAPR whereas in vitro g-ray irradiation by the [60Co] g source was performed in the Fourth Military Medical University (Xi’an, China). Page 2 of 9 Cells were divided into nine groups. Group A was con- trol nonirradiated cells; Group B cells were irradiated with 4 Gy of [60Co] g-rays; Group C cells were irradiated with 8 Gy of [60Co] g-rays; Group D cells were irra- diated with 4 Gy BPA-BNCT; Group E cells were irra- diated with 8 Gy BPA-BNCT; Group F cells were irradiated in the XAPR for the same treatment period as used for Group D; Group G cells were irradiated in the XAPR for the same treatment period as used for Group E; Group H cells were irradiated with 4 Gy in the XAPR; and Group I cells were irradiated with 8 Gy in the XAPR. In Group D and Group E with BPA-BNCT, cells were incubated for 24 h in medium with BPA and washed three times with phosphate-buffered saline (PBS); the medium was next replaced with boron-free medium; cells were exposed to the XAPR with irradia- tion doses of 4 Gy and 8 Gy, respectively. In the g-ray- treated Group B and Group C, cells incubated in growth medium without BPA were exposed to the g-rays with irradiation doses of 4 Gy and 8 Gy, respectively. In Group F and Group G, cells incubated in medium with- out BPA were exposed to the XAPR with identical treat- ment durations of Group D and Group E, respectively. In Group H and Group I, cells incubated in growth medium without BPA were exposed to the XAPR with irradiation doses of 4 Gy and 8 Gy, respectively. Irra- diated and nonirradiated cells were placed at room tem- perature and under a normal atmosphere during irradiation. At the beginning of the experiment, all cells were in the exponential phase of growth and at a den- sity of 3×105/ml. The components of the reactor neu- tron source and the [60Co] g source were shown in Table 1; the irradiation periods of various groups were listed in Table 2; the dose components of BNCT were listed in Table 3 and Table 4. The irradiation doses were calculated according to previous studies [17,18]. Proliferation assays Cell survival was determined using the 3-(4,5- dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT) (Sigma, St. Louis, MO) assay. After irradiation, cells were trypsinized and washed, and cell concentrations were adjusted to 104 cells/ml. Cells were seeded into the Table 1 Dose rates for reactor neutron source and [60Co] g source Components Dose rate (Gy/min) Thermal neutron 1.04×10-3 Dose rates for the reactor Epithermal neutron 2.48×10-4 neutron source Fast neutron 1.07×10-1 g-ray 1.57×10-2 10B(n,a)7Li 5.09×10-4/ppm10B Dose rate for the [60Co] g source 1.40×101