Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used drugs for the treatment of inflammatory disease and have a chemopreventive effect on colorectal cancer. NSAIDs inhibit cyclooxygenase (COX)-1 and/or COX-2 activity, but the chemopreventive effect may be, in part, independent of prostaglandin inhibition. NSAID-activated gene (NAG-1) was previously identified as a gene induced by some NSAIDs in cells devoid of COX activity. NAG-1 has proapoptotic and antitumorigenic activity in vitro and in vivo. To determine whether the induction of NAG-1 by NSAIDs is influenced by COX expression, we developed COX-1- and COX-2-overexpressing HCT-116 cells. COX expression did not affect NSAID-induced NAG-1 expression as assessed by transient and stable transfection. Also, NAG-1 expression was not affected by PGE(2) and arachidonic acid, suggesting that NAG-1 induction by NSAIDs occurs by a prostanoid-independent manner. We also report that indomethacin increased NAG-1 expression in a number of cells from tissues other than colorectal. In conclusion, NSAIDs have dual function, induction of NAG-1 expression and inhibition of COX activity that occurs in a variety of cell lines.