The effect of selective cyclooxygenase inhibitors on intestinal epithelial cell mitogenesis

Introduction. Previous research has demonstrated that nonsteroidal anti-inflammatory agents alter the incidence of colorectal cancer. It has been postulated that the response may be due to the effect of these agents on the activities of the cyclooxygenase (COX) enzymes. The COX enzymes catalyze the conversion of arachidonic acid to biologically active prostanoids. Two forms of COX have been identified. COX-1 is a constitutive enzyme, generally involved in cell functions, while COX-2 is commonly an enzyme which is inducible in response to various stimuli, including mitogens. Recently, specific inhibitors of COX-1 and COX-2 enzymes have been developed. Purpose. The present study was undertaken to determine the effects of specific COX-1 and COX-2 inhibitors on the proliferation and the induction of apoptosis of intestinal epithelial cells. Methods. A continuously proliferating rat small intestinal cell line (IEC-18) and a mouse colon cancer cell line (WB-2054) were utilized for these experiments. The cells were placed in microwells with serum-free or serum-supplemented media. The effects of serum on proliferation were then evaluated in the presence of the COX-1 inhibitor, valerylsalicyclic acid (VSA), the COX-2 inhibitor, SC-58125, or indomethacin. The presence of COX-1 and COX-2 protein was evaluated by Western blotting. Proliferation of intestinal cells was quantitated by incorporation of [H-3]thymidine into DNA and cell counting, and apoptosis was determined by evaluating cell attachment. COX activity was evaluated by prostaglandin E-2 production measured by enzyme-linked immunoabsorbent assay (ELISA). Results. Western blotting of IEC-18 and WB-2054 cell protein demonstrated COX-1 enzyme in cells incubated in serum-free media with increased COX-1 expression produced by incubation in media supplemented with 10% serum. COX-2 enzyme was not demonstrated in serum-free media; however, it was present in cells maintained in 10% serum-supplemented media. Spontaneous DNA synthesis was present in both cell lines and serum increased proliferation. In both cell lines [H-3]thymidine incorporation stimulated by serum was inhibited by the COX-2 inhibitor SC-58125, but not by the COX-1 inhibitor VSA. Both indomethacin and SC-58125 produced a dose-dependent increase in apoptotic ratios in both cell lines. PGE(2) formation, stimulated by serum, was inhibited by SC-58125, VSA, and indomethacin. Conclusions. A differential effect on intestinal cell mitogenesis was seen with different COX inhibitors. The COX-2 inhibitor, but not the COX-1 inhibitor, significantly inhibited [H-3]thymidine incorporation in both cell types, suggesting COX-2 inhibitors may be specific inhibitors of normal epithelial cell proliferation and growth of malignant cells. SC-58125, a selective inhibitor of COX-2, has a potent apoptosis inducing effect. The inhibition of PGE(2) production did not correlate with the inhibition of proliferation, suggesting the two processes are unrelated. (C) 1999 Academic Press.