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

点击翻译按钮获取中文摘要

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