Novel integrative gene therapy for hormone refractory prostate cancer

前列腺特异性抗原（PSA）被广泛用作前列腺癌的诊断血清标志物，PSA启动子也被用作前列腺特异性载体，用于传递治疗基因。PSA启动子受雄激素受体（AR）以及其他转录因子的调控。我们实验室最近的一项研究表明，NF-κB通过结合其顺式元件XBE下调PSA的反活化和表达。在本研究中，我们研究了糖皮质激素受体（GR）在雄激素非依赖性DU145或PC-3前列腺癌细胞中调控PSA启动子活性的作用。首先，我们证明了gr特异性mRNA和蛋白在DU145或PC-3细胞中表达，而不是在雄激素依赖性LNCaP细胞中表达，并且合成糖皮质激素地米松（DEX）在DU145和PC-3细胞中以剂量依赖性的方式显著诱导由糖皮质激素应答元件（GREs）的连续重复驱动的启动子活性。其次，转录因子数据库搜索和凝胶转移分析的结果确定了PSA启动子远端增强子中的两个GREs （GREI和II）。第三，全长和嵌合启动子报告子中的GREs突变证实了GR可以通过与GREs结合激活PSA启动子。此外，嵌合PSA启动子构建中XBE的缺失进一步增强了GR在AI DU145或PC-3细胞中的转录活性，其中NF-κB具有组成性活性，并且与此观察一致，NF-κB的p65亚基抑制GR功能。总的来说，我们的数据是第一个物理证据，证明DEX通过内在GR调节AI PC-3和DU145细胞中的PSA启动子。这些发现表明GR介导的PSA启动子激活可能适用于AI前列腺癌治疗基因的表达。

Prostate specific antigen (PSA) is widely used as a diagnostic serum marker of prostate cancer, and the PSA promoter is also utilized as a prostate-specific vector for the delivery of therapeutic genes. The PSA promoter is regulated by androgen receptor (AR), as well as the other transcription factors. A recent study from our laboratory has demonstrated that NF-κB down-regulates PSA transactivation and expression by binding to its cis-element, named XBE. In this study, we investigated the role of glucocorticoid receptor (GR) in regulating PSA promoter activity in androgen-independent DU145 or PC-3 prostate cancer cell lines. First, we demonstrated that GR-specific mRNA and protein were expressed in DU145 or PC-3 cells, not in androgen dependent LNCaP cells, and that dexamethasone (DEX), a synthetic glucocorticoid, induced markedly the promoter activity driven by tandem repeat of glucocorticoid responsive elements (GREs) in DU145 and PC-3 cells in a dose-dependent manner. Second, the results of transcription factor database search and gel shift assays identified two GREs (GREI and II) within the distal enhancer of the PSA promoter. Third, the mutation of GREs in the full-length and chimeric promoter reporters confirmed that GR could activate the PSA promoter by binding to GREs. Moreover, the deletion of XBE in the chimeric PSA promoter constructs further enhanced the transcriptional activity of GR in AI DU145 or PC-3 cells, where NF-κB constitutively active, and consistent with this observation, p65 subunit of NF-κB inhibited GR function. Collectively, our data are the first physical evidence that DEX regulates the PSA promoter in AI PC-3 and DU145 cells through the intrinsic GR. These findings suggest that GR mediated-PSA promoter activation may be applicable for the expression of therapeutic genes in AI prostate cancers.