Studies about Application of BNCT for Head and Neck Malignancies

我们最近报道，硼中子俘获疗法(BNCT)代表了一种新的、有前途的治疗方法，即使是对巨大或非常晚期的头颈部恶性肿瘤也是如此。但BNCT细胞死亡的机制尚不清楚。特别是针对P53蛋白的表达，我们研究了BNCT对细胞生长、细胞凋亡和细胞周期相关P53蛋白的影响。我们使用野生型P53的SAS/neo细胞和突变型P53的SAS/mP53细胞，两者都是相同的遗传背景，除了P53的表达。癌细胞与p-硼丙氨酸孵育2小时后，在京都大学研究反应堆中进行中子束照射。用结肠法测定细胞存活率，用四甲基偶氮唑盐比色法测定细胞生长。流式细胞仪分析细胞周期。用Hoechst33342染色检测BNCT诱导的细胞凋亡小体。(1)集落形成实验显示，SAS/neo细胞集落形成受到抑制，呈物理剂量依赖性。(2)用2GY和6GY(物理剂量)照射SAS/neo细胞48h，细胞生长抑制率分别为80%和70%左右。(3)流式细胞仪和Hoechst染色显示，SAS/neo细胞的凋亡率呈时间依赖性地高于SAS/mp53细胞，这表明SAS/mp53细胞对BNCT的耐药性高于SAS/neo细胞，且呈剂量依赖关系。头颈部肿瘤的主要抑癌基因、P53状态可能影响BNCT的预后。

We recently reported that boron neutron capture therapy (BNCT) represents a new and promising treatment approach even for huge or far advanced head and neck malignancies. But the mechanism of cell death from BNCT has not clarified yet. Especially focused on p53 status, we investigated effects of BNCT on cell growth, apoptosis and cell cycle related p53. We used SAS/neo cell with wild type p53 and SAS/mp53 cell with mutant type p53, both are identical genetic backgrounds except for the p53 expression.The cancer cells were irradiated with neutron beam at Kyoto University Research Reactor after incubation with p-boronopheylalanine for 2 hours. Surviving fraction of each cell was measured by colonogenic assay.Cell growth was measured by MTT assay. Cell cycles were analyzed by flow cytometric assay. BNCT-induced apoptotic bodies were detected by Hoechst33342 stain.Results are as follows. (1) Colony formation assay revealed the number of SAS/neo cell-colony formation were suppressed, physical dose-dependently. (2) When SAS/neo cell were irradiated at 2Gy and 6Gy (physical dose), MTT assay showed the growth suppression rate of the cell, compared with that of SAS/mp53 cell were 80% and about 70% respectively for 48 hours. (3) Flow cytometric assay and Hoechst stain revealed that apoptosis rates were increased in SAS/neo cell compared with that in SAS/mp53 time-dependently.These data suggests that SAS/mp53 cell was more resistant to BNCT than SAS/neo cell physical dose-dependently. Major tumor suppression gene, p53 status of head and neck cancer might influence on the outcomes of BNCT.