Salicylates inhibit NF-kappaB activation and enhance TNF-alpha-induced apoptosis in human pancreatic cancer cells.
BACKGROUND
Tumor necrosis factor (TNF-alpha)-induced apoptosis is limited by its coactivation of nuclear factor kappa B (NF-kappaB)-dependent anti-apoptotic genes. Sodium salicylate (NaSal) inhibits NF-kappaB activation by limiting phosphorylation and degradation of its bound inhibitor protein, IkappaB-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. PANC-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 demonstrate IkappaB-alpha phosphorylation and degradation. Western blots of nuclear extracts were performed to assess nuclear translocation of NF-kappaB. In separate cultures, apoptosis was measured 4.5 h after TNF-alpha stimulation by both ELISA detection of interhistone DNA fragments and flow cytometry with propidium iodide staining.


RESULTS
TNF-alpha induced IkappaB-alpha phosphorylation and degradation, which was inhibited by NaSal in both cell lines. TNF-alpha-induced apoptosis (DNA fragmentation) increased significantly when BxPC-3 cells were pretreated 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 in apoptosis were measured in the PANC-1 cell line among the various treatment groups.


CONCLUSIONS
NaSal enhances TNF-alpha-induced apoptosis while inhibiting IkappaB-alpha phosphorylation and degradation in BxPC-3 human pancreatic cancer cells.
McDade
T P
TP
Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Perugini
R A
RA
Vittimberga
F J
FJ
Carrigan
R C
RC
Callery
M P
MP
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D012980:Sodium Salicylate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D012980:Sodium Salicylate