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

导读：虽然雄激素消融术是雄激素依赖型（AD）前列腺癌最有效的治疗方法，但它最终会随着时间的推移而失效，然后一部分癌症复发为雄激素非依赖型（AI）前列腺癌。近年来，NF-κB的组成性激活对包括前列腺癌在内的许多人类恶性肿瘤是有害的。一些报道表明，NE-κB在AI PC-3和DU145中被组成性激活，而在AD LNCaP细胞系中不被激活。LNCaP细胞模型是研究人工智能进展的一个很好的选择，因为衍生的LNCaP人工智能分支已经建立。为了阐明AI早期发展的机制，我们测定了短暂雄激素剥夺后LNCaP细胞中NE-κB活性的状态。材料和方法：通过瞬时转染和荧光素酶测定，比较了LNCaP细胞中含有和不含R1881（一种雄性激素的合成类似物）的NE-κB启动子活性。Western blot分析NE-κB亚基及其抑制剂i -κB α蛋白，ELISA检测NE-κB依赖性细胞因子，以及LNCaP细胞中NF-κB亚基p65的免疫细胞化学定位。为了验证NE-κ b激活能促进LNCaP细胞的生长，我们对一种携带NF-κ b的显性负抑制因子i -κ b α超抑制因子（Ad-SR- i -κ b α）的腺病毒载体进行了评估。结果：剥夺R1881后，NE-κB启动子活性显著升高（5 ~ 6倍），磷酸化的i -κB α表达随时间增加约10倍，p65、p50和i -κB α蛋白表达无显著变化，IL-6、TNF-a和IL-1β分泌无明显改变。免疫染色显示R1881的去除促进了p65从细胞质向细胞核的易位，尽管在R1881存在的情况下，几乎所有的p65只定位在细胞质上。TUNEL和MTT实验分别表明，在不添加R1881的情况下，Ad-SR- i -κ b α阻断NE-κ b活性可加速细胞凋亡，降低细胞生长。结论：我们的数据表明，在体外培养的人前列腺癌细胞中，雄激素剥夺后NE-κB激活迅速发生。NE-κB激活可能作为AI进展的早期事件出现。我们认为，增加的i - b α磷酸化和伴随的NE-KB激活是AI进展的关键步骤。用Ad-SR- i -κ b α阻断NF-κ b活化为诱导AI进展中的前列腺癌细胞死亡提供了一种新的治疗方法。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