Nitric Oxide-Mediated Inhibition of NFκB Regulates Hyperthermia-Induced Apoptosis

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 I kappa B alpha and down regulation of NF kappa B-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 NF kappa B-DNA binding activity. The hyperthermia-mediated inhibition of NF kappa B was persistent even after 48 h. Inhibition of NO by L-NAME rescued the NF kappa B-DNA binding activity and inhibits heat-induced apoptosis. Similarly, over-expression of NF kappa B 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 NF kappa B. J. Cell. Biochem. 106: 999-1009, 2009. (c) 2009 Wilcy-Liss, Inc.