The effect of phospholipase A(2) inhibitors on proliferation and apoptosisof murine intestinal cells

Background. Group II phospholipase A(2) (PLA(2)) enzymes, the rate controll ing enzymes in arachidonic acid metabolism, have been well characterized an d subdivided into secretory 14-kDa PLA, (sPLA(2)) and cytoplasmic 85-kDa PL A(2) (cPLA(2)). Previous research has demonstrated increased PLA(2) in colo rectal tumors. The present study was performed to determine the effect of s pecific PLA(2) inhibitors on the proliferation and induction of apoptosis o f intestinal epithelial cells. Methods. A continuously proliferating rat small intestinal cell line (IEC-1 8) and a mouse colon cancer cell line (WB-2054-M-4) 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 evaluat ed in the presence of the cPLA(2) inhibitor, methylarachidonyl fluorophosph ate (MAFP), or the sPLA(2) inhibitor p-bromophenacyl bromide (BPB). The sPL A(2) and cPLA(2) protein was estimated by Western blotting. Proliferation o f intestinal cells was quantitated by incorporation of [H-3]thymidine into DNA and PLA(2), activity was evaluated by quantitating arachidonic acid for mation and prostaglandin E-2 production. Results. Western blotting of IEC-18 and WB-2054 cell protein demonstrated s PLA(2) and cPLA(2) enzyme in cells incubated in media containing 10% serum. Spontaneous DNA synthesis was present in both cell lines and serum consist ently increased proliferation. In IEC-18 cells [H-3]thymidine incorporation stimulated by serum was inhibited by MAFP and BPB, while in the malignant cell line, proliferation was inhibited only by BPB. BPB, but not MAFP, prod uced a dose-dependent increase in apoptotic ratios in both cell lines. Arac hidonic acid and PGE(2) formation, stimulated by serum, was inhibited by MA FP and BPB. Conclusions. A differential effect on intestinal cell mitogenesis was seen with different PLA(2) inhibitors. The sPLA(2), inhibitor, but not the cPLA( 2) inhibitor, significantly inhibited [H-3]thymidine incorporation in the m alignant cell line. This occurred with an induction of apoptosis. sPLA(2) i nhibitors may be specific inhibitors of growth of malignant cells. The inhi bition of arachidonic acid and PGE(2) production did not correlate with the inhibition of proliferation, suggesting that the two processes may be unre lated, (C) 1999 Academic Press.