P21WAF1/cip1 is a cyclin dependent kinase inhibitor that plays a critical role in regulating intestinal homeostasis. Our previous studies demonstrated that sulindac, a non-steroidal anti-inflammatory drug, induced p21 expression in vivo and in vitro, and that the induction of p21 suppressed intestinal tumor formation in Apc1638 mice by inhibiting cell proliferation and increasing cell differentiation and apoptosis in the intestine. Our current studies showed that sulindac also induced JNK1, that JNK1 was bound to p21, and that JNK1 and p21 were synergistic in regulating cell differentiation and apoptosis in vitro and in vivo. JNK1 is a member of the mitogen-activated protein kinase (MAPK) family and plays an important role in coordinating the cellular stress response, including apoptosis, cell proliferation and transformation. First, we found that JNK1 expression was increased with spontaneous differentiation of the colonic carcinoma cells Caco-2, HT29-CL19A and CL16E , and that the expression of JNK1 was well correlated with p21 expres sion. Both p21 and JNK1 were significantly induced by sulindac in Caco2 and HCT116 cells, resulting in increased apoptosis and cell differentiation. Moreover, immunoprecipitation/western blot experiments showed that p21 was bound to JNK1. To determine the importance of JNK1 in response to sulindac, JNK1 and -/- murine embryonic fibroblasts were treated with sulindac. Sulindac increased apoptosis in both cell lines, but, the increase of apoptosis in JNK1-/- cells was less than that in JNK1 wild-type cells. Further studies revealed that JNK1 was dramatically induced by sulindac in the JNK1 cells; no JNK1 was detectable in JNK1-/- cells regardless of treatment. Sulindac also induced p21 expression in the fibroblasts, but the induction of p21 in JNK1-/- cell was less than that in JNK1 cells. Again, the induction of JNK1 was well correlated with p21 induction. However, c-myc was suppressed by sulindac both in JNK1-/- or cells. Finally, we determined the expression of JNK1 in the intestinal mucosa of Apc,p21 or Apc, p21-/- mice. We have reported that inactivation of p21 enhances Apc-initiated tumor formation in mice by decreasing cell differentiation and apoptosis and increasing cell proliferation. Compared to p21 mice, JNK1 expression was lower in the normal mucosa of p21-/- mice. When fed a sulindac supplemented (0.2%) diet, there was no alteration of JNK1 in p21-/- mice. However, more than a three-fold increase of JNK1 was induced by sulindac in the p21 mice. This indicate that tumor inhibition by sulindac is not only p21-dependent, but also might require JNK1.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]