title: Inhibition of NF-kappaB sensitizes A431 cells to epidermal growth factor-induced apoptosis, whereas its activation by ectopic expression of RelA confers resistance.
abstract: Epidermal growth factor (EGF) is a well known mitogen, but it paradoxically induces apoptosis in cells that overexpress its receptor. We demonstrate for the first time that the EGF-induced apoptosis is accelerated if NF-kappaB is inactivated. To inactivate NF-kappaB, human epidermoid carcinoma cells (A431) that overexpress EGF receptor were stably transfected with an IkappaB-alpha double mutant construct. Under the NF-kappaB-inactivated condition, A431 cells were more sensitive to EGF with decreased cell viability and increased externalization of phosphatidylserine on the cell surface, DNA fragmentation, and activation of caspases (3 and 8 but not 9), typical features of apoptosis. These results were further supported by the potentiation of the growth inhibitory effects of EGF by chemical inhibitors of NF-kappaB (curcumin and sodium salicylate) and the protective role of RelA evidenced by the resistance of A431-RelA cells (stably transfected with RelA) to EGF-induced apoptosis. EGF treatment or ectopic expression of RelA in A431 cells induced DNA binding activity of NF-kappaB (p50 and RelA) and the expression of c-IAP1, a downstream target of NF-kappaB. A431-RelA cells exhibited spontaneous phosphorylation of Akt (a downstream target of phosphatidylinositol 3-kinase and regulator of NF-kappaB) and EGF treatment stimulated it further. Blocking this basal Akt phosphorylation with LY294002, an inhibitor of phosphatidylinositol 3-kinase, did not affect their viability but blocking of EGF-induced phosphorylation of Akt sensitized the otherwise resistant A431-RelA cells to EGF-mediated growth inhibition. Our results favor an anti-apoptotic role for NF-kappaB in the regulation of EGF-induced apoptosis.
authors.affiliation: Division of Cancer Biology, Rajiv Gandhi Center for Biotechnology, Thiruvananthapuram, Kerala-695014, India.
authors.forename: Ruby John
authors.initials: RJ
authors.lastname: Anto
authors.forename: Manickam
authors.initials: M
authors.lastname: Venkatraman
authors.forename: Devarajan
authors.initials: D
authors.lastname: Karunagaran
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002455:Cell Division; D002470:Cell Survival; D002867:Chromones / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D020552:Comet Assay; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D051996:Transcription Factor RelA; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D002867:Chromones; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D009025:Morpholines; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D010718:Phosphatidylserines; D013778:Tetrazolium Salts; D013844:Thiazoles; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004815:Epidermal Growth Factor; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C022616:thiazolyl blue; D013936:Thymidine