Nitric oxide-mediated inhibition of NFkappaB 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 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.
Aravindan
Natarajan
N
Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. naravind@ouhsc.edu
Mohan
Sumathy
S
Herman
Terence S
TS
Natarajan
Mohan
M
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms / Q000628:therapy*; D045744:Cell Line, Tumor; D003131:Combined Modality Therapy; D004247:DNA / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005260:Female; D006801:Humans; D006979:Hyperthermia, Induced; D020672:I-kappa B Proteins / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D009569:Nitric Oxide; D004247:DNA; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D019331:NG-Nitroarginine Methyl Ester