The effect of phospholipase A2 inhibitors on proliferation and apoptosis of murine intestinal cells.

BACKGROUND

Group II phospholipase A2 (PLA2) enzymes, the rate controlling enzymes in arachidonic acid metabolism, have been well characterized and subdivided into secretory 14-kDa PLA2 (sPLA2) and cytoplasmic 85-kDa PLA2 (cPLA2). Previous research has demonstrated increased PLA2 in colorectal tumors. The present study was performed to determine the effect of specific PLA2 inhibitors on the proliferation and 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-M4) 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 cPLA2 inhibitor, methylarachidonyl fluorophosphate (MAFP), or the sPLA2 inhibitor p-bromophenacyl bromide (BPB). The sPLA2 and cPLA2 protein was estimated by Western blotting. Proliferation of intestinal cells was quantitated by incorporation of [3H]thymidine into DNA and PLA2 activity was evaluated by quantitating arachidonic acid formation and prostaglandin E2 production.

RESULTS

Western blotting of IEC-18 and WB-2054 cell protein demonstrated sPLA2 and cPLA2 enzyme in cells incubated in media containing 10% serum. Spontaneous DNA synthesis was present in both cell lines and serum consistently increased proliferation. In IEC-18 cells [3H]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, produced a dose-dependent increase in apoptotic ratios in both cell lines. Arachidonic acid and PGE2 formation, stimulated by serum, was inhibited by MAFP and BPB.

CONCLUSIONS

A differential effect on intestinal cell mitogenesis was seen with different PLA2 inhibitors. The sPLA2 inhibitor, but not the cPLA2 inhibitor, significantly inhibited [3H]thymidine incorporation in the malignant cell line. This occurred with an induction of apoptosis. sPLA2 inhibitors may be specific inhibitors of growth of malignant cells. The inhibition of arachidonic acid and PGE2 production did not correlate with the inhibition of proliferation, suggesting that the two processes may be unrelated.