Enhancement of radiation and hyperthermia effects for oral cancer by intracellular hydrogen peroxide generator

细胞内过氧化氢发生器增强口腔癌的放射和热疗效果

基本信息

批准号：
16591987
负责人：
WADA Shigehito
金额：
$ 1.6万
依托单位：
University of Toyama (2005)Toyama Medical and Pharmaceutical University (2004)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16591987/
关键词：
hyperthermia radiation therapy intracellular hydrogen peroxide 6-formylpterin oral cancer oxidative stress

项目摘要

The enhancement of heat-induced apoptosis by 6-formylpterin, an intracellular generator of hydrogen peroxide (H_2O_2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 μM (37℃), heat shock (44℃/20min) alone, or a combination of the two, then incubated at 37℃ for 6 h. Assessments of apoptosis, mitochondrial membrane potential, and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation, and changes in the intracellular Ca^<2+> concentration ([Ca^<2+>]i) were examined. Bax,Bcl-2,Bcl-XL,Bid, cytochrome c, and PKCδ were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of … More Bid was noted, although no significant changes in Bax,Bcl-2, and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCδ from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca^<2+>]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca^<2+>]i, the activation of the mitochondria-caspase dependent pathway, and the translocation of PKCδ to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6- formylpterin. These results were confirmed in the cells exposed to X-ray at a dose of 10Gy, too. When cells were exposed to hyperthermia alone (44℃ for 10 min, 15% apoptosis level), 39 up-regulated genes, such as BAG3,DNAJA1,DNAJB1,HSPA1B,HSPA6,HSPH1,SEPW1, and HO-1,and 3 down-regulated gene, such as CCL2,were identified. In combining heat and 6- formylpterin, two up-regulated genes (LOC219962 and NEUROD4) were identified. The expression levels of these genes were confirmed by real-time quantitative polymerase chain reaction. Less

在人脊髓细胞淋巴瘤U937细胞中检查了6-甲基甲基蛋白的热诱导凋亡（H_2O_2）的细胞内发生器（H_2O_2）的增强。单独用单独的6-甲基蛋白的细胞以300μM（37℃），热休克（44℃/20分钟）或两者的组合处理，然后在37℃孵育6小时。通过流式细胞仪进行凋亡，线粒体膜电位和caspase-3激活的评估。此外，检查了caspase-8激活以及细胞内Ca^<2+>浓度的变化（[Ca^<2+>] i）。通过蛋白质印迹检测到BAX，BCL-2，BCL-XL，BID，BID，细胞色素C和PKCδ。通过添加6-甲基甲基蛋白来促进通过形态学观察和DNA碎片评估的热诱导的凋亡的诱导。线粒体膜电位得到了改善，并在结合处理的细胞中增强了caspase -3和-8的激活。尽管在联合治疗后观察到BAX，BCL-2和BCL-XL表达没有明显变化，但提出了更多的出价表达。此外，通过添加6型甲状腺蛋白，可以增强细胞色素C从线粒体到细胞质的释放，以及通过热休克诱导的PKCδ从细胞胞醇转移到线粒体。通过添加6型甲酰基蛋白，增加了[Ca^<2+>]较高的细胞数量。这些发现表明，[Ca^<2+>] I的增加，线粒体 - caspase依赖途径的激活以及PKCδ向线粒体的易位在增强热诱导的凋亡在6-甲基蛋白中的结果中起主要作用。这些结果在暴露于X射线的细胞中也得到了证实，也以10GY的剂量剂量确认。当细胞单独暴露于高温（44℃10分钟，15％凋亡水平）时，39个上调的基因，例如BAG3，DNAJA1，DNAJB1，DNAJB1，HSPA1B，HSPA1B，HSPA6，HSPH1，HSPH1，SEPW1和HO-1和HO-1和HO-1，以及3个下调的基因，例如CCL2。在结合热和6-甲基甲蛋白时，鉴定了两个上调的基因（LOC219962和NeuroD4）。这些基因的表达水平通过实时定量聚合酶链反应证实。较少的