Studies On The Treamtment Of Human Glioma Cells By BNCT In Vitro And In Vivo

High-grade glioma is one of the most highly malignant tumors, and is still extremely resistant to all current forms of therapy, including surgery, chemotherapy, radiotherapy, immunotherapy and gene therapy. A new, more effective treatment is therefore needed to improve survival rates in these patients. Among the novel approaches for selective treatment is boron neutron capture therapy (BNCT). Boron neutron capture therapy (BNCT) is based on the nuclear capture and fission reactions that occur when nonradioactive 10B is irradiated with neutrons of the appropriate energy to yield high energy alpha particles (4He) and recoiling lithium-7 (7Li) nuclei. These particles have pathlengths of approximately one cell diameter; their lethality is primarily limited to boron containing cells. Both particles have a high (LET) value. And the high energy transfer from these particles causes severe damage to sensitive structures of the targeted cell causing cell death. It affects both dividing and non-dividing tumor cells. In order for BNCT to be successful, a sufficient amount of 10B must be selectively delivered to the tumor and enough thermal neutrons must be absorbed by them to sustain a lethal 10B (n,a)7Li capture reaction.BNCT has been applied on patients with high-grade gliomas and a much smaller number of patients with other types of brain tumors, however the cellular mechanism of BNCT induced cell death is not well understood. The current study was undertaken to determine if BNCT can induce apoptosis in 3 human glioma cell lines in vitro and if BNCT can improve the survival time of G422 glioma bearing mouse in vivo.1 Effect of boron neutron capture therapy on U87, U251 and SHG44 glioma cells in vitro: This sthudy is design to explore the effect of boron neutron capture therapy(BNCT)on human brain glioma cell lines (U87, U251 and SHG44) in vitro and its mechanisms. U87 cells, U251 cells, SHG44 cells in exponential phase were divided into 6 groups:untreated control,⁶⁰Coγ4 Gy,⁶⁰Coγ8 Gy,nuclear reactor exposure without boronophenylalanine(BPA), BNCT 4 Gy and BNCT 8 Gy. Methyl thiazolyl tetrazolium (MTT) assay was used to test the changes in the proliferation of U87, U251 and SHG44 cells after irradiation. HE staining , Hoechst33342 fluorescence staining , transmission electron microscope(TEM) were applied to observe the changes of cell morphology. The Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) stainings were applied to detect the apoptotisis rate induced by BNCT by flow cytometer(FCM). The anti-tumor effects were analyzed through the apoptotisis rate by FCM, methyl thiazolyl tetrazolium (MTT) assay and tumor colony formation. The expression of P53 protein and BCL-2 protein was studied by immunocytochemistry and the semiquantitative expression of BCL-2 protein and BAX protein was measured by western blot. The results showed that typical morphological changes were observed after BNCT irradiation, and the apoptotisis rates observed 48 h after irradiation were 63.6%, 57.7% and 60.9% for U87 cells, U251 cells and SHG44 cells respectively by BNCT 4 Gy and 85.9%, 80.6% and 88.3% for for U87 cells, U251 cells and SHG44 cells respectively by BNCT 8 Gy. BNCT showed higher apoptotisis rates thanγ–ray control irradiation (P<0.01). The surviving fraction of BNCT was significantly lower than reactor andγ–ray control irradiation (P<0.01). The expression level of P53 protein was negative in control,while it was positive in BNCT 4 Gy and 8 Gy group. On the contrary, the expression level of BCL -2 protein was positive in control,while it was negative in BNCT 4 Gy and 8 Gy group. BNCT promoted expression of BAX protein,at the same time it also inhibited expression of BCL-2 in U87, U251 and SHG44 in a dose- and time-dependent manner. The results suggested that BNCT can inhibit the growth of U87, U251 and SHG44 significantly and can induces U87, U251 and SHG44 apoptosis possibly by the up-regulation of expression of P53 gene and BAX gene and down-regulation of expression of BCL -2.2 Effect of Boron Neutron Capture Therapy for G422 glioma cells in vivo: This study is design to to explore the effect of BNCT on glioma G422 cell line in vivo.Mice transplanted G422 cells in brain were divided into 7 groups: untreated control, ⁶⁰Coγ5 Gy, ⁶⁰Coγ10 Gy, nuclear reactor exposure without BPA 5 Gy and 10 Gy, BNCT 5 Gy and BNCT 10 Gy. The median survival time (MeST) and mean survival time (MST) in days after implantation of the tumor were calculated and Kaplan-Meier survival curve was plotted. The percent increased life span (%ILS) was determined. Analysis of variance was applied to determine levels of significance in survival times between BNCT-treated, irradiated controls and untreated mice. The results showed that the MST of BNCT group was 21.750 d and 27.875 d for 5 Gy and 10 Gy, respectively(P<0.01 vs untreated control). In the same dose, the MST of BNCT was significantly higher than that of reactor (P<0.05) andγ–ray control irradiation (P<0.01). The MST of BNCT 10 Gy was higher than that of BNCT 5 Gy (P<0.01). The percent increased life span(% ILS) of BNCT was 108%(117%)and 321%(300%) for 5 Gy and 10 Gy. The results suggested that BNCT can improve the survival time of G422 glioma bearing mouse significantly in a dose-dependent manner.