NO-donating aspirin induces apoptosis via the intrinsic pathway, dissociates adherens junctions and inhibits Wnt signaling in SW480 colon cancer cells

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Preclinical studies have shown that nitric oxide donating aspirin (NO-ASA) is more potent than traditional ASA in colon cancer chemoprevention, but the mechanism by which it prevents colon cancer is not well understood. We have examined the mechanism of NO-ASA-induced cell death in SW480 human colon cancer cells. NO-ASA induced apoptosis by activating the intrinsic apoptosis pathway as evidenced by the release of cytochrome c into the cytosol and activation of caspase 9 but not caspase 8; caspase 3 was also activated with its catalytic activity increased up to 6-fold. During apoptosis NO-ASA dissociated adherens junctions through the time- and concentration-dependent cleavage of β- and γ-catenin, but not α-catenin or E-cadherin. These cleavages were inhibited by the caspase-3 inhibitor Z-DEVD-FMK and the pan-caspase inhibitor Z-VAD-FMK, but not by the proteasome inhibitor MG-132, suggesting that they were caspase 3-dependent. Treatment of these cells with NO-ASA also inhibited Wnt signaling via a dual mechanism. During the early stages of treatment or at low concentrations of NO-ASA, Wnt signaling was inhibited though a steric hindrance effect on β-catenin and the transcription factor Tcf. At higher NO-ASA concentrations and after apoptosis was underway Wnt signaling inhibition was compounded through the caspase-3 dependent cleavage of β-catenin. Both mechanisms decreased the expression of cyclin D, a gene dependent for its transcription on Wnt signaling. A central early event was the NO-ASA induced change in the cells redox status, assayed directly with the use of appropriate reporter molecules; N-acetylcysteine significantly reversed all of the above effects. A structure-activity study revealed that these events were dependent on the presence of the -ONO₂ moiety on NO-ASA, which releases NO. Taken together, our findings suggest that NO-ASA induced the intrinsic apoptosis pathway, impaired cell-cell adhesion and inhibited Wnt signaling resulting in reduced cyclin D1 expression. These findings may explain, at least in part its chemoprevention activity. Grant support: NIH CA92423