Muenchen H J, Lin D L, Walsh M A, Keller E T, Pienta K J
Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0946, USA.
Clin Cancer Res. 2000 May;6(5):1969-77.
Prostate cancer patients experiencing a relapse in disease often express high serum tumor necrosis factor-alpha (TNF-alpha) levels. Many androgen-insensitive prostate cancer cells are TNF-alpha insensitive because of the expression of antiapoptotic genes as part of the nuclear factor-kappaB (NF-kappaB) family of transcription factors. NF-kappaB stimulates gene transcription when expressed in the nucleus; however, in resting cells, this nuclear import is prevented by association with the cytoplasmic inhibitor IkappaBalpha. This cytoplasmic retention of NF-kappaB is uncoupled by many extracellular signals including low levels of TNF-alpha. During normal cell activation, nuclear translocation of NF-kappaB is preceded by phosphorylation and degradation of IkappaBalpha. When phosphorylation is blocked, IkappaBalpha remains intact, thereby blocking NF-kappaB translocation to the nucleus and subsequent activation of antiapoptotic genes that cause TNF-alpha insensitivity. We tested whether a "super-repressor" of NF-kappaB activity could be transfected into prostate cancer cells and make them TNF-alpha sensitive. PC-3 and LNCaP cells were stimulated with TNF-alpha (10 ng/ml) for 24 h in the presence or absence of the IkappaBalpha "super-repressor" (p6R-IkappaB(S32A + S36A)). NF-kappaB activity was measured by electrophoretic mobility shift assay and the steady state levels of the cytoplasmic IkappaBalpha protein were measured by Western blot. Secretory IL-6 and IL-6 mRNA were measured by ELISA. p6R-IkappaB(S32A + S36A) blocked the stimulation of NF-kappaB activity by TNF-alpha in prostate cancer cells. It also subsequently decreased IL-6 production by TNF-alpha. We conclude that these data demonstrate that inhibition of NF-kappaB selectively sensitizes previously insensitive prostate cancer cells to TNF-alpha.
经历疾病复发的前列腺癌患者通常血清肿瘤坏死因子-α（TNF-α）水平较高。许多雄激素不敏感的前列腺癌细胞对TNF-α不敏感，这是因为作为核因子-κB（NF-κB）转录因子家族一部分的抗凋亡基因的表达。NF-κB在细胞核中表达时会刺激基因转录；然而，在静息细胞中，这种核输入会因与细胞质抑制剂IκBα结合而被阻止。NF-κB的这种细胞质滞留会被包括低水平TNF-α在内的许多细胞外信号解除。在正常细胞激活过程中，NF-κB的核转位之前会发生IκBα的磷酸化和降解。当磷酸化被阻断时，IκBα保持完整，从而阻断NF-κB向细胞核的转位以及随后导致TNF-α不敏感的抗凋亡基因的激活。我们测试了是否可以将NF-κB活性的“超级抑制剂”转染到前列腺癌细胞中并使其对TNF-α敏感。在存在或不存在IκBα“超级抑制剂”（p6R-IκB(S32A + S36A)）的情况下，用TNF-α（10 ng/ml）刺激PC-3和LNCaP细胞24小时。通过电泳迁移率变动分析测量NF-κB活性，通过蛋白质印迹法测量细胞质IκBα蛋白的稳态水平。通过酶联免疫吸附测定法测量分泌的白细胞介素-6（IL-6）和IL-6信使核糖核酸（mRNA）。p6R-IκB(S32A + S36A)阻断了TNF-α对前列腺癌细胞中NF-κB活性的刺激。它随后还降低了TNF-α诱导的IL-6产生。我们得出结论，这些数据表明抑制NF-κB可选择性地使先前不敏感的前列腺癌细胞对TNF-α敏感。
