Salicylates inhibit NF-kappa B activation and enhance TNF-alpha-induced apoptosis in human pancreatic cancer cells

Introduction. Tumor necrosis factor (TNF-alpha)-induced apoptosis is limite d by its coactivation of nuclear factor kappa B (NF-kappa B)-dependent anti -apoptotic genes. Sodium salicylate (NaSal) inhibits NF-KB activation by li miting phosphorylation and degradation of its bound inhibitor protein, I ka ppa B-alpha.We examined whether NaSal enhances TNF-alpha-induced apoptosis in cultured human pancreatic cancer cell lines. Methods. Two cultured human pancreatic cancer cell lines were studied. PANG -1 and BxPC-3 cells were serum-starved for 12 h, pretreated or not for 1 h with NaSal (5-20 mM), and then stimulated with recombinant human TNF-alpha (400 units/ml). Western blots of cytoplasmic lysates were performed to demo nstrate I kappa B-alpha phosphorylation and degradation. Western blots of n uclear extracts were performed to assess nuclear translocation of NF-kappa B. In separate cultures, apoptosis was measured 4.5 h after TNF-alpha stimu lation by both ELISA detection of interhistone DNA fragments and flow cytom etry with propidium iodide staining. Results. TNF-alpha induced I kappa B-alpha phosphorylation and degradation, which was inhibited by NaSal in both cell lines. TNF-alpha-induced apoptos is (DNA fragmentation) increased significantly when BxPC-3 cells were pretr eated with NaSal. Flow cytometry confirmed this, demonstrating increases in apoptotic cell fractions: 8.5% (untreated), 9.3% (TNF-alpha alone), 14.9% (15 mM NaSal), and 22.9% (NaSal and TNF-alpha). In contrast, no increases i n apoptosis were measured in the PANG-1 cell line among the various treatme nt groups. Conclusions. NaSal enhances TNF-alpha-induced apoptosis while inhibiting I kappa B-alpha phosphorylation and degradation in BxPC-3 human pancreatic ca ncer cells. (C) 1999 Academic Press.