Ubiquitin-proteasome inhibition enhances apoptosis of human pancreatic cancer cells.
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.
McDade
T P
TP
Department of Surgery, University of Massachusetts Medical School, Worcester 01655, USA.
Perugini
R A
RA
Vittimberga
F J
FJ
Callery
M P
MP
D017209:Apoptosis / Q000187:drug effects*; D003546:Cysteine Endopeptidases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes / Q000502:physiology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000473:pathology*; D046988:Proteasome Endopeptidase Complex; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014452:Ubiquitins / Q000037:antagonists & inhibitors*
D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex