Li F J, Kondo T, Zhao Q L, Tanabe K, Ogawa R, Li M, Arai Y
Department of Radiological Sciences, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, 930-0194, Japan.
Free Radic Res. 2001 Sep;35(3):281-99. doi: 10.1080/10715760100300821.
To elucidate the mechanism how a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0 degrees C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca2+ concentration ([Ca2+]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The effects of hyperthermia on LPO and [Ca2+]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (delta psi m) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca2+ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamin E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca2+]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca2+]i arising from increased expression of IP3R1 and LPO. Additional increase in [Ca2+]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.
为阐明自由基引发剂2,2'-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)在高温下诱导细胞死亡的机制，我们研究了人组织细胞淋巴瘤细胞系U937中的细胞凋亡情况。通过用5,5-二甲基-1-吡咯啉-N-氧化物(DMPO)进行自旋捕获，检测了AAPH热分解产生的自由基形成。采用DNA片段化检测、细胞核形态变化观察以及磷脂酰丝氨酸(PS)外化的流式细胞术来检测细胞凋亡，并揭示了AAPH产生的自由基增强了44.0℃高温诱导的细胞凋亡。然而，单独由AAPH产生的自由基并不诱导细胞凋亡。高温诱导了脂质过氧化(LPO)的产生、细胞内Ca2+浓度([Ca2+]i)的增加以及1型肌醇1,4,5-三磷酸受体(IP3R1)表达的增强。与AAPH联合使用时，高温对LPO和[Ca2+]i的影响显著增强。仅在高温和AAPH处理的细胞中发现Bcl-2表达显著降低、Bax表达增加、线粒体膜电位(Δψm)丧失以及细胞色素c表达显著增加。虽然细胞内Ca2+离子螯合剂BAPTA-AM完全抑制了DNA片段化，但水溶性维生素E生育酚(Trolox)仅部分抑制了DNA片段化和[Ca2+]i的增加。相反，生育酚完全抑制了LPO，但未发现BAPTA-AM对其有抑制作用。这些结果表明，单独高温诱导的细胞凋亡是由于IP3R1表达增加和LPO导致的[Ca2+]i增加所致。LPO增加和线粒体-半胱天冬酶依赖性途径激活导致的[Ca2+]i额外增加在高温与AAPH联合使用增强细胞凋亡中起主要作用。
