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

本文研究了细胞内过氧化氢（H_2O_2）产生剂6-甲酰蝶呤（6-formylpterin）对人骨髓单核细胞淋巴瘤U937细胞热诱导凋亡的增强作用。将细胞单独用无毒浓度300 μM（37℃）的6-甲酰蝶呤处理，单独用热休克（44℃/20 min）处理，或两者联合处理，然后在37℃孵育6 h。通过流式细胞术评估细胞凋亡、线粒体膜电位和caspase-3活化。此外，还检测了caspase-8的激活和细胞内Ca^2+浓度（[Ca^2+]i）的变化。Western blotting检测Bax、Bcl-2、Bcl-XL、Bid、细胞色素c和PKCδ的表达。通过形态学观察和DNA片段化评估，6-甲酰蝶呤的加入促进了热诱导凋亡的诱导。线粒体膜电位降低，caspase-3和-8的活化增强，在细胞与联合处理。一个减少的表达 ...更多信息 注意到BID，尽管在联合治疗后未观察到Bax、Bcl-2和Bcl-XL表达的显著变化。此外，6-甲酰蝶呤还能增强热休克诱导的细胞色素c从线粒体向胞浆的释放和PKCδ从胞浆向线粒体的转位。加入6-甲酰蝶呤后，[Ca^<2+>]i较高的细胞数量也增加。这些结果表明，[Ca^<2+>]i的增加，caspase依赖途径的激活，以及PKCδ向线粒体的移位在6-甲酰蝶呤增强热诱导的细胞凋亡中起主要作用。这些结果在10 Gy剂量X射线照射的细胞中也得到了证实。在单纯高温（44℃，10 min，15%凋亡水平）条件下，共检测到BAG 3、DNAJA 1、DNAJB 1、HSPA 1B、HSPA 6、HSPH 1、SEPW 1、HO-1等39个基因表达上调，CCL 2等3个基因表达下调。在结合热和6-甲酰蝶呤，两个上调基因（LOC 219962和NEUROD 4）被确定。通过实时定量聚合酶链反应证实这些基因的表达水平。少

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