| Autor: |
McDade, T.P., Perugini, R.A., Vittimberga, F.J., Callery, M.P. |
| Zdroj: |
Surgery; August 1999, Vol. 126 Issue: 2 p371-377, 7p |
| Abstrakt: |
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 @mmol/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 I@kB-@a and phospho-I@kB-@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 I@kB-@a was inhibited by PSI. Phospho-I@kB-@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 @? .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 I@kB-@a degradation in BxPC-3 human pancreatic cancer cells. (Surgery 1999;371-7.) |
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