Ubiquitin-proteasome inhibition enhances apoptosis of human pancreatic cancer cells

Background, Tumor necrosis factor (TNF-a)-induced apoptosis is limited by c oactivation of nuclear factor kappa B (NF-kb)-dependent antiapoptotic genes . Nuclear translocation of NF-kappa B 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 hu man pancreatic cancer cells. Methods, Serum-starved BxPC-3 cells (12 hours) were pretreated or not for 5 0 minutes with PST (30 m mol/L), a peptide aldehyde known to inhibit specif ically the chymotrypsin-like activity of the 26S proteasome. Cells were sub sequently stimulated with recombinant human TNF-a (400 units/mL). Western b lots were performed using antibodies to IkB-a and phospho-IkB-a. Level of a poptosis was determined by two methods: enzyme-linked immunosorbent assay d etection of interhistone DNA fragments and flow cytometry with propidium io dide staining. Results. TNF-a-induced degradation of IkB-a was inhibited by PSI. Phospho-I kB-a accumulation was observed 20 minutes after TNF-a stimulation. Apoptosi s relative to constitutive levels was significantly increased after PSI pre treatment, as measured by DNA fragmentation (P less than or equal to .05 by Student t test). Percent apoptosis by flow cytometry confirmed marked incr eases in apoptotic cell fractions from 5.9% (untreated) to 6.8% (TNF-a alon e), 16.4% (PSI alone), and 18.9% (PSI and TNF-a). Conclusions. PSI enhances both constitutive and TNF-a-induced apoptosis thr ough inhibition of IkB-a degradation in BxPC-3 human pancreatic cancer cell s.