The novel chemopreventive nitric oxide-donating aspirin (NO-ASA) prevents nearly 90% of ductal adenocarcinomas in a animal tumor model. To decipher the mechanism of this effect, we studied in BxPC-3 human pancreatic cancer cells the sequence of signaling events leading from NO-ASA treatment to cell growth inhibition. NO-ASA inhibited the growth of BxPC-3 cells (IC(50) =13 microM), by inhibiting proliferation modestly and inducing apoptosis, necrosis and G(1)/S cell cycle block. At 15 min of treatment with NO-ASA, the intracellular levels of reactive oxygen species (ROS) began increasing (peak at 8h, baseline levels by 24h). ROS activated almost immediately in a time- and concentration-dependent manner the MAPK pathways p38, ERK and JNK (their activation was abrogated by the antioxidant N-acetylcysteine). MAPK activation induced p21(cip-1), which suppressed the levels of cyclin D1 that controls the G(1)/S cell cycle transition. NO-ASA induced COX-2 expression starting 90 min after p21(cip-1) was induced. When COX-2 expression was knocked down using siRNA against cox-2, the expression of p21(cip-1) was induced by NO-ASA, regardless of the level of expression of COX-2, suggesting a marginal, if any, role for COX-2 in the growth inhibitory effect of NO-ASA. These findings along with the temporal sequence of individual changes indicate a signaling sequence that involves ROS-->MAPKs-->p21(cip-1)-->cyclin D1-->cell death. Our findings establish the critical role of ROS as proximal signaling molecules in the action of anticancer compounds and may be useful in designing mechanism-driven approaches to cancer control.