McDade T P, Perugini R A, Vittimberga F J, Carrigan R C, Callery M P
Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
J Surg Res. 1999 May 1;83(1):56-61. doi: 10.1006/jsre.1998.5560.
Tumor necrosis factor (TNF-alpha)-induced apoptosis is limited by its coactivation of nuclear factor kappa B (NF-kappaB)-dependent anti-apoptotic genes. Sodium salicylate (NaSal) inhibits NF-kappaB activation by limiting phosphorylation and degradation of its bound inhibitor protein, IkappaB-alpha. We examined whether NaSal enhances TNF-alpha-induced apoptosis in cultured human pancreatic cancer cell lines.
Two cultured human pancreatic cancer cell lines were studied. PANC-1 and BxPC-3 cells were serum-starved for 12 h, pretreated or not for 1 h with NaSal (5-20 mM), and then stimulated with recombinant human TNF-alpha (400 units/ml). Western blots of cytoplasmic lysates were performed to demonstrate IkappaB-alpha phosphorylation and degradation. Western blots of nuclear extracts were performed to assess nuclear translocation of NF-kappaB. In separate cultures, apoptosis was measured 4.5 h after TNF-alpha stimulation by both ELISA detection of interhistone DNA fragments and flow cytometry with propidium iodide staining.
TNF-alpha induced IkappaB-alpha phosphorylation and degradation, which was inhibited by NaSal in both cell lines. TNF-alpha-induced apoptosis (DNA fragmentation) increased significantly when BxPC-3 cells were pretreated with NaSal. Flow cytometry confirmed this, demonstrating increases in apoptotic cell fractions: 8.5% (untreated), 9.3% (TNF-alpha alone), 14.9% (15 mM NaSal), and 22.9% (NaSal and TNF-alpha). In contrast, no increases in apoptosis were measured in the PANC-1 cell line among the various treatment groups.
NaSal enhances TNF-alpha-induced apoptosis while inhibiting IkappaB-alpha phosphorylation and degradation in BxPC-3 human pancreatic cancer cells.
肿瘤坏死因子（TNF-α）诱导的细胞凋亡受其对核因子κB（NF-κB）依赖性抗凋亡基因的共激活作用限制。水杨酸钠（NaSal）通过限制其结合的抑制蛋白IκB-α的磷酸化和降解来抑制NF-κB的激活。我们研究了NaSal是否能增强培养的人胰腺癌细胞系中TNF-α诱导的细胞凋亡。
研究了两种培养的人胰腺癌细胞系。将PANC-1和BxPC-3细胞血清饥饿12小时，用NaSal（5-20 mM）预处理或不预处理1小时，然后用重组人TNF-α（400单位/毫升）刺激。对细胞质裂解物进行蛋白质免疫印迹以证明IκB-α的磷酸化和降解。对核提取物进行蛋白质免疫印迹以评估NF-κB的核转位。在单独的培养物中，在TNF-α刺激4.5小时后，通过ELISA检测组蛋白间DNA片段和用碘化丙啶染色的流式细胞术测量细胞凋亡。
TNF-α诱导IκB-α的磷酸化和降解，在两种细胞系中均被NaSal抑制。当BxPC-3细胞用NaSal预处理时，TNF-α诱导的细胞凋亡（DNA片段化）显著增加。流式细胞术证实了这一点，显示凋亡细胞分数增加：8.5%（未处理）、9.3%（单独TNF-α）、14.9%（15 mM NaSal）和22.9%（NaSal和TNF-α）。相比之下，在PANC-1细胞系中各治疗组之间未检测到细胞凋亡增加。
NaSal在抑制BxPC-3人胰腺癌细胞中IκB-α磷酸化和降解的同时增强TNF-α诱导的细胞凋亡。
