Wada S, Cui Z-G, Kondo T, Zhao Q-L, Ogawa R, Shoji M, Arai T, Makino K, Furuta I
Department of Oral and Maxillofacial Surgery, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.
Int J Hyperthermia. 2005 May;21(3):231-46. doi: 10.1080/02656730400025404.
The enhancement of heat-induced apoptosis by 6-formylpterin, an intra-cellular generator of hydrogen peroxide (H2O2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 microM (37 degrees C), heat shock (44 degrees C per 20 min) alone or a combination of the two, then incubated at 37 degrees C 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 intra-cellular Ca2+ concentration ([Ca2+]i) were examined. Bax, Bcl-2, Bcl-XL, Bid, cytochrome c and PKCd 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 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 PKCd 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 [Ca2+]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca2+]i, the activation of the mitochondria-caspase dependent pathway and the translocation of PKCd to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6-FP.
在人骨髓单核细胞淋巴瘤U937细胞中，研究了细胞内过氧化氢（H2O2）生成剂6-甲酰基蝶呤对热诱导凋亡的增强作用。细胞分别用无毒浓度300 microM（37℃）的6-甲酰基蝶呤单独处理、单独热休克（44℃，每20分钟一次）或二者联合处理，然后在37℃孵育6小时。通过流式细胞术评估凋亡、线粒体膜电位和半胱天冬酶-3激活情况。此外，还检测了半胱天冬酶-8激活和细胞内Ca2+浓度（[Ca2+]i）的变化。通过蛋白质免疫印迹法检测Bax、Bcl-2、Bcl-XL、Bid、细胞色素c和蛋白激酶Cδ（PKCd）。通过形态学观察和DNA片段化评估的热诱导凋亡的诱导作用，在添加6-甲酰基蝶呤后得到促进。联合处理的细胞中线粒体膜电位降低，半胱天冬酶-3和-8的激活增强。虽然联合处理后Bax、Bcl-2和Bcl-XL的表达没有显著变化，但Bid的表达降低。此外，热休克诱导的细胞色素c从线粒体释放到细胞质以及PKCd从细胞质转移到线粒体的过程，在添加6-甲酰基蝶呤后均增强。添加6-甲酰基蝶呤后，[Ca2+]i较高的细胞数量也增加。这些发现表明，[Ca2+]i的增加、线粒体-半胱天冬酶依赖性途径的激活以及PKCd向线粒体的转移在6-甲酰基蝶呤增强热诱导凋亡中起主要作用。
