Miyoshi Noriyuki, Watanabe Etsuko, Osawa Toshihiko, Okuhira Masashi, Murata Yoshiyuki, Ohshima Hiroshi, Nakamura Yoshimasa
Laboratory of Biochemistry, Graduate School of Nutritional and Environmental Sciences, and Global COE Program, University of Shizuoka, Shizuoka 422-8526, Japan.
Biochim Biophys Acta. 2008 Oct;1782(10):566-73. doi: 10.1016/j.bbadis.2008.07.002. Epub 2008 Jul 14.
Pro-inflammatory death is presumably an undesirable event in cancer prevention process, thus biochemical comprehension and molecular definition of this process could have important clinical implications. In the present study, we examined the cytophysiological conversion of cell death mode by benzyl isothiocyanate (BITC) in human cervical cancer HeLa cells. The detailed studies using flow cytometric and morphological analyses demonstrated that the cells treated with appropriate concentration (25 microM) of BITC showed apoptotic feature, such as chromatin condensation, DNA fragmentation, and preserved plasma membrane integrity, whereas these features were disappeared by treatment with higher concentration (100 microM). The treatment with 2-deoxyglucose, an inhibitor of ATP synthesis, drastically increased in the ratio of necrotic dead cells, while it influences little that of apoptotic cells. Moreover, an analysis using the mitochondrial DNA-deficient HeLa cells demonstrated that the rho degrees cells were more susceptible to the BITC-induced necrosis-like cell death compared to the wild-type (rho(+)) cells, whereas the ROS production was significantly inhibited in the rho degrees cells. It is likely that the BITC-induced ROS is derived from mitochondrial respiratory chain and ruled out the contribution to the mechanism of cell death mode switching. In addition, the BITC treatment resulted in a more rapid depletion of ATP in the rho degrees cells than in the rho(+) cells. Furthermore, a caspase inhibitor, Z-VAD-fmk counteracted not only apoptosis, but also necrosis-like cell death induced by BITC, suggesting that increment in this cell death pattern might be due to the interruption of events downstream of a caspase-dependent pathway. The obtained data suggested that the decline in the intracellular ATP level plays an important role in tuning the mode of cell death by BITC.
促炎性细胞死亡在癌症预防过程中可能是一个不良事件，因此对这一过程的生化理解和分子定义可能具有重要的临床意义。在本研究中，我们检测了异硫氰酸苄酯（BITC）在人宫颈癌HeLa细胞中细胞死亡模式的细胞生理转化。使用流式细胞术和形态学分析的详细研究表明，用适当浓度（25 microM）的BITC处理的细胞表现出凋亡特征，如染色质浓缩、DNA片段化和质膜完整性保留，而用较高浓度（100 microM）处理则这些特征消失。用ATP合成抑制剂2-脱氧葡萄糖处理，坏死死亡细胞的比例急剧增加，而对凋亡细胞的比例影响很小。此外，使用线粒体DNA缺陷型HeLa细胞的分析表明，与野生型（rho(+)）细胞相比，rho度细胞对BITC诱导的坏死样细胞死亡更敏感，而rho度细胞中的ROS产生受到显著抑制。很可能BITC诱导的ROS来源于线粒体呼吸链，排除了其对细胞死亡模式转换机制的贡献。此外，BITC处理导致rho度细胞中的ATP消耗比rho(+)细胞更快。此外，一种半胱天冬酶抑制剂Z-VAD-fmk不仅抵消了凋亡，还抵消了BITC诱导的坏死样细胞死亡，表明这种细胞死亡模式的增加可能是由于半胱天冬酶依赖性途径下游事件的中断。获得的数据表明，细胞内ATP水平的下降在调节BITC诱导的细胞死亡模式中起重要作用。
