Nitric oxide-donating aspirin inhibits colon cancer cell growth via mitogen-activated protein kinase activation

Nitric oxide-donating aspirin (NO-aspirin), representing a new concept in the development of more efficacious nonsteroidal anti-inflammatory drugs, consists of traditional aspirin bearing -ONO2, which releases NO. Conventional aspirin prevents human colon cancer, but its toxicity precludes its application as a chemopreventive agent. NO-aspirin seems safer and in cultured cancer cells it is > 1000-fold more potent than aspirin. To determine the mechanism by which NO-aspirin inhibits cell growth, we studied its effect on mitogen-activated protein kinase (MAPK) signaling in HT-29 human colon cancer cells. NO-aspirin stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 and Akt only marginally. The greatest increases in phosphorylation were seen in cJun NH2-terminal kinase (JNK) and p38 MAP kinases, which were observed as early as 5 min and after 1 h of treatment, averaged more than 10-fold over control. The activation of JNK and p38 was accompanied by large increases in the phosphorylation of the downstream transcription factors cJun and activating transcription factor 2 (ATF-2). We used specific MAPK inhibitors, small interfering (siRNA) gene silencing methods, and dominant-negative cJun to determine the relevance of these phosphorylation events to the ability of NO-aspirin to inhibit colon cancer cell growth. Only the dual inhibitor of p38 and JNK and the use of combined siRNA silencing of p38 and cJun abrogated the ability of NO-aspirin to block cell growth. Our data indicate that NO-aspirin is dependent on both the p38 and the JNK MAP kinase pathways for its ability to inhibit the growth of colon cancer cells.