Overexpression of p65 attenuates celecoxib-induced cell death in MDA-MB-231 human breast cancer cell line

The effects of p65 overexpression on celecoxib-inhibited NF-kappaB transcriptional activity were examined by western blotting, electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay. Cell viability and cell death were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, and the levels of cleaved poly(ADP-ribose) polymerase (PARP) and caspase. Anti-apoptotic NF-kappaB target genes and cell cycle regulators were examined by western blotting to screen for the expression of target genes under direct regulation by p65.Overexpression of p65 increased NF-kappaB transcriptional activity and interfered with celecoxib-mediated apoptosis as assessed by MTT assay and caspase-3, caspase-9, and PARP expressions. Exogenously overexpressed p65 upregulated NF-kappaB-responsive genes, including anti-apoptotic genes such as survivin and XIAP, and the cell cycle regulatory gene cyclin D1. However, p65 overexpression did not affect celecoxib-induced p-Akt inactivation, suggesting that celecoxib might have separate molecular mechanisms for regulating Akt signaling independently of its inhibition of NF-kappaB transcriptional activity.p65 is a pivotal anti-apoptotic factor that can reverse celecoxib-induced growth inhibition in MDA-MB-231 cells.