McDade T P, Perugini R A, Vittimberga F J, Callery M P
Department of Surgery, University of Massachusetts Medical School, Worcester 01655, USA.
Surgery. 1999 Aug;126(2):371-7.
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.
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.
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).
PSI enhances both constitutive and TNF-a-induced apoptosis through inhibition of IkB-a degradation in BxPC-3 human pancreatic cancer cells.
肿瘤坏死因子（TNF-α）诱导的细胞凋亡受到核因子κB（NF-κB）依赖性抗凋亡基因共激活的限制。NF-κB的核转位需要26S蛋白酶体降解泛素化的磷酸化IκB-α。我们研究了泛素-蛋白酶体途径的抑制是否能增强TNF-α诱导的BxPC-3人胰腺癌细胞凋亡。
血清饥饿的BxPC-3细胞（12小时）用PSI（30μmol/L）预处理或不预处理50分钟，PSI是一种已知能特异性抑制26S蛋白酶体胰凝乳蛋白酶样活性的肽醛。随后用重组人TNF-α（400单位/mL）刺激细胞。使用针对IκB-α和磷酸化IκB-α的抗体进行蛋白质印迹分析。通过两种方法测定细胞凋亡水平：酶联免疫吸附测定法检测组蛋白间DNA片段，以及碘化丙啶染色的流式细胞术。
PSI抑制了TNF-α诱导的IκB-α降解。TNF-α刺激20分钟后观察到磷酸化IκB-α的积累。通过DNA片段化测量，PSI预处理后相对于组成水平的细胞凋亡显著增加（学生t检验，P≤0.05）。流式细胞术检测的细胞凋亡百分比证实凋亡细胞分数显著增加，从5.9%（未处理）增加到6.8%（单独使用TNF-α）、16.4%（单独使用PSI）和18.9%（PSI和TNF-α）。
PSI通过抑制BxPC-3人胰腺癌细胞中IκB-α的降解来增强组成性和TNF-α诱导的细胞凋亡。
