Significance of NE-κB activation and therapeutic strategies for targeting its pathway in androgen-independent prostate cancer

前言：虽然雄激素治疗是雄激素依赖型(AD)前列腺癌最有效的治疗方法，但随着时间的推移，它最终会失效，然后部分癌症会复发为雄激素非依赖性(AI)前列腺癌。最近，研究表明，NF-κB的结构性激活对包括前列腺癌在内的许多人类恶性肿瘤都是有害的。一些报道表明，NE-κB在AI、PC-3和DU145细胞中被结构性激活，但在AD LNCaP细胞系中不被激活。由于已经建立了衍生的LNCaP AI亚系，LNCaP细胞模型是研究AI进展的一个很好的选择。为了阐明AI进展早期的机制，我们测定了短暂雄激素剥夺后LNCaP细胞中NE-κB的活性状态。材料和方法：采用瞬时转染法和荧光素酶活性测定法比较雄激素κ类似物R1881对LNCaP细胞中NE-…B启动子活性的影响更多。对NE-κB亚基及其抑制物I-κBα蛋白进行Western印迹分析，对NE-κB依赖的细胞因子进行ELISA法检测，并对NF-κB亚基p65进行免疫细胞化学定位。为验证NE-κB活化对LNCaP细胞生长的促进作用，构建了携带IκBα超抑制子的重组腺病毒载体(Ad-SR-IκBα)。结果：剥夺R1881后，NE-κB启动子活性显著增强(5~6倍)，磷酸化I-κBα表达增加约10倍；p65、p50和I-κBα蛋白表达无明显变化，直到R1881停用后4d，IL-6、β-α和IL-1的分泌也未见明显变化。免疫组织化学染色显示，R1881的去除促进了p65从胞浆到胞核的移位，尽管在R1881存在的情况下，几乎所有的p65都只定位于胞浆。在未加R1881的情况下，用Ad-SR-IκBκBα阻断去甲肾上腺素受体B的活性可促进细胞的凋亡，并抑制细胞的生长。结论：我们的数据表明去雄激素后培养的人前列腺癌细胞中NE-κB迅速激活。NE-κB的激活可能是AI进展的早期事件。我们认为，IκBα磷酸化增加和伴随的NE-KB激活是AI进展的关键步骤。用Ad-SR-IκBκBα阻断核转录因子B的激活，为诱导AI进展中的前列腺癌细胞死亡提供了一种新的治疗方法。较少

Introduction: Although androgen ablation is the most effective therapy for androgen-dependent (AD) prostate cancer, it eventually fails with time and then a portion of the cancer relapses to androgen-independent (AI) prostate cancer. Recently it has become clear that constitutive activation of NF-κB is detrimental to a number of human malignancies including prostate cancer. Several reports have showed that NE-κB is constitutively activated in AI PC-3 and DU145 but not in AD LNCaP cell lines. LNCaP cell model is an excellent choice for the study of AI progression for the reason that derivative LNCaP AI sublines have been established. To elucidate the mechanisms responsible for an early step of AI progression, we determined the status of NE-κB activity in LNCaP cells after a brief androgen deprivation. Materials and Method: Transient transfections and luciferase assay were performed to compare NE-κB promoter activity in LNCaP cells with and without R1881, a synthetic analogue of androgen … More . Western blot analysis of NE-κB subunits and their inhibitor IκBα proteins, ELISA assay of NE-κB-dependent cytokines, and immunocytochemical localization of NF-κB subunit p65 in LNCaP cells were also performed. To verify that NE-κB activation can promote the growth of LNCaP cells, an adenoviral vector bearing superrepressor of IκBα (Ad-SR- IκBα), a dominant negative inhibitor of NF-κB, was assessed. Results: After R1881 deprivation, NE-κB promoter activity was markedly elevated (5 to 6-fold), and phosphorylated IκBα expression increased approximately 10-fold with time There was no significant change in the expression of p65, p50, or IκBα protein, and no evidence of altered IL-6, TNF-a, or IL-1β secretion up to 4 days after R1881 withdrawal. Immunostaining revealed that R1881 removal promoted the translocation of p65 from cytoplasm to cell nucleus, even though almost all p65 localized only to cytoplasm in the presence of R1881. Without R1881, blockade of NE-κB activity by Ad-SR- IκBα accelerated apoptosis and decreased cell growth as demonstrated by TUNEL and MTT assay, respectively. Conclusions: Our data suggest that NE-κB activation occurs rapidly after androgen deprivation in cultured human prostate cancer cells. NE-κB activation could arise as an early event for AI progression. We propose that increased IκBα phosphorylation and concomitant NE-KB activation account for the critical step of AI progression. Blocking NF-κB activation with Ad-SR- IκBα offers a novel therapeutic approach to induce the death of prostate cancer cells undergoing AI progression. Less