Ubiquitin-proteasome inhibition enhances apoptosis of human pancreatic cancer cells

Surgery. 1999 Aug;126(2):371-7.


Background: Tumor necrosis factor (TNF-a)-induced apoptosis is limited by coactivation of nuclear factor kappa B (NF-kb)-dependent antiapoptotic genes. Nuclear translocation of NF-kB requires degradation of ubiquitinated phospho-IkB-a by the 26S proteasome. We examined whether inhibition of the ubiquitin-proteasome pathway enhances TNF-a-induced apoptosis in BxPC-3 human pancreatic cancer cells.

Methods: Serum-starved BxPC-3 cells (12 hours) were pretreated or not for 50 minutes with PSI (30 m mol/L), a peptide aldehyde known to inhibit specifically the chymotrypsin-like activity of the 26S proteasome. Cells were subsequently stimulated with recombinant human TNF-a (400 units/mL). Western blots were performed using antibodies to IkB-a and phospho-IkB-a. Level of apoptosis was determined by two methods: enzyme-linked immunosorbent assay detection of interhistone DNA fragments and flow cytometry with propidium iodide staining.

Results: TNF-a-induced degradation of IkB-a was inhibited by PSI. Phospho-IkB-a accumulation was observed 20 minutes after TNF-a stimulation. Apoptosis relative to constitutive levels was significantly increased after PSI pretreatment, as measured by DNA fragmentation (P < or = .05 by Student t test). Percent apoptosis by flow cytometry confirmed marked increases in apoptotic cell fractions from 5.9% (untreated) to 6.8% (TNF-a alone), 16.4% (PSI alone), and 18.9% (PSI and TNF-a).

Conclusions: PSI enhances both constitutive and TNF-a-induced apoptosis through inhibition of IkB-a degradation in BxPC-3 human pancreatic cancer cells.

MeSH terms

  • Apoptosis / drug effects*
  • Cysteine Endopeptidases / physiology*
  • Cysteine Proteinase Inhibitors / pharmacology*
  • DNA-Binding Proteins / metabolism
  • Humans
  • I-kappa B Proteins*
  • Multienzyme Complexes / physiology*
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / metabolism
  • Pancreatic Neoplasms / pathology*
  • Proteasome Endopeptidase Complex
  • Tumor Cells, Cultured
  • Tumor Necrosis Factor-alpha / pharmacology
  • Ubiquitins / antagonists & inhibitors*


  • Cysteine Proteinase Inhibitors
  • DNA-Binding Proteins
  • I-kappa B Proteins
  • Multienzyme Complexes
  • NF-kappa B
  • NFKBIA protein, human
  • Tumor Necrosis Factor-alpha
  • Ubiquitins
  • NF-KappaB Inhibitor alpha
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex