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 IκBα and down regulation of NFκB-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°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°C. Apoptosis measured by Annexin V expression and cell survival by clonogenic assay showed a 20% increase in apoptosis after 43°C treatments. EMSA analysis showed a dose-dependent inhibition of NFκB-DNA binding activity. The hyperthermia-mediated inhibition of NFκB was persistent even after 48 h. Inhibition of NO by L-NAME rescued the NFκB-DNA binding activity and inhibits heat-induced apoptosis. Similarly, over-expression of NFκB 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 NFκB.