Aravindan Natarajan, Mohan Sumathy, Herman Terence S, Natarajan Mohan
Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
J Cell Biochem. 2009 Apr 15;106(6):999-1009. doi: 10.1002/jcb.22079.
Ascertaining the upstream regulatory mechanisms of hyperthermia-induced apoptosis is important to understand the role of hyperthermia in combined modality cancer therapy. Accordingly, we investigated whether (i) hyperthermia-induced apoptosis is mediated through the nitric oxide (NO) signaling pathway and (ii) inhibition of post-translational modification of IkappaBalpha and down regulation of NFkappaB-DNA binding activity is an intermediate step in NO-dependent apoptosis in MCF-7 breast cancer cells. For hyperthermia treatment, the cells were exposed to 43 degrees C. Intracellular NO levels measured by the fluorescent intensity of DAF-2A and iNOS expression by immunobloting revealed an increased level of iNOS dependent NO production after 43 degrees C. Apoptosis measured by Annexin V expression and cell survival by clonogenic assay showed a 20% increase in apoptosis after 43 degrees C treatments. EMSA analysis showed a dose-dependent inhibition of NFkappaB-DNA binding activity. The hyperthermia-mediated inhibition of NFkappaB was persistent even after 48 h. Inhibition of NO by L-NAME rescued the NFkappaB-DNA binding activity and inhibits heat-induced apoptosis. Similarly, over-expression of NFkappaB by transient transfection inhibits heat-induced apoptosis. These results demonstrate that apoptosis upon hyperthermia exposure of MCF-7 cells is regulated by NO-mediated suppression of NFkappaB.
确定热疗诱导细胞凋亡的上游调控机制对于理解热疗在联合癌症治疗中的作用至关重要。因此，我们研究了：（i）热疗诱导的细胞凋亡是否通过一氧化氮（NO）信号通路介导；（ii）抑制IkappaBalpha的翻译后修饰以及下调NFkappaB-DNA结合活性是否是MCF-7乳腺癌细胞中NO依赖性细胞凋亡的中间步骤。对于热疗处理，将细胞暴露于43℃。通过DAF-2A的荧光强度测量细胞内NO水平，并通过免疫印迹检测诱导型一氧化氮合酶（iNOS）表达，结果显示43℃处理后iNOS依赖性NO生成水平增加。通过膜联蛋白V表达测量细胞凋亡，通过克隆形成试验检测细胞存活，结果显示43℃处理后细胞凋亡增加了20%。电泳迁移率变动分析（EMSA）显示NFkappaB-DNA结合活性呈剂量依赖性抑制。即使在48小时后，热疗介导的NFkappaB抑制作用仍然持续。L-NAME抑制NO可恢复NFkappaB-DNA结合活性并抑制热诱导的细胞凋亡。同样，通过瞬时转染过表达NFkappaB可抑制热诱导的细胞凋亡。这些结果表明，MCF-7细胞热疗暴露后的细胞凋亡是由NO介导的NFkappaB抑制所调控的。
