Albrecht Martin, Mittler Anja, Wilhelm Beate, Lundwall Ake, Lilja Hans, Aumüller Gerhard, Bjartell Anders
Department of Anatomy and Cell Biology, Philipps-University, Marburg, Germany.
Eur Urol. 2003 Oct;44(4):415-22. doi: 10.1016/s0302-2838(03)00322-1.
Neutral Endopeptidase (NEP) is a cell surface enzyme that cleaves and inactivates neuropeptides. When present on androgen-dependent prostate cancer (PC) cells, NEP inactivates growth stimulatory neuropeptides. After androgen ablation NEP expression decreases and neuropeptides can enhance cell growth, leading to the development of androgen-independent, neuropeptide stimulated PC. Aim of the study was to analyse the expression, localisation and distribution of NEP in benign and malignant prostatic tissues and its relation to the cytoskeleton.
Immunohistochemistry (IHC) was performed to localise NEP in fixed specimens from normal prostatic tissue, benign prostate hyperplasia (BPH) and PC of Gleason grade 2-5. In situ hybridisation and Western blotting experiments were carried out to confirm NEP gene expression and translation to mature protein in BPH and PC tissue. Confocal laser scanning microscopy was utilised to investigate whether development of high grade prostate tumours was accompanied by changes in intracellular actin/NEP colocalisation patterns. Finally, the proliferative activity in relation to loss of NEP expression was investigated by dual staining of NEP and Ki-67 in prostatic tumours.
In situ hybridisation studies revealed preserved expression of NEP mRNA in epithelial cells of PC. NEP was by IHC shown to be located in the apical plasma membrane of normal epithelial cells and BPH tissue. In PC a Gleason grade dependent shift of the NEP distribution pattern towards a heterogeneous, partly cytoplasmic allocation of the protein was found. Compared to BPH tissue, specimens derived from PC showed very low IHC-staining intensity for NEP protein. In high grade PC the typical apical colocalisation of actin and NEP was lost and a strong granular cytoplasmic NEP staining was found. PC areas with a high expression of NEP displayed diminished proliferative activity i.e. low staining intensity for Ki-67.
NEP is differentially expressed in the normal and the pathologically altered prostate with a clear shift from a membrane bound to a cytoplasmic distribution pattern in high-grade tumours and loss of NEP expression in areas of high proliferative activity. The data presented support an active involvement of NEP in the progression of androgen-independent PC. Further studies are needed to unravel the mechanisms underlying the cytoplasmic NEP distribution in PC.
中性内肽酶（NEP）是一种细胞表面酶，可裂解并使神经肽失活。当存在于雄激素依赖性前列腺癌（PC）细胞上时，NEP会使生长刺激神经肽失活。雄激素去除后，NEP表达降低，神经肽可促进细胞生长，导致雄激素非依赖性、神经肽刺激的PC发生。本研究的目的是分析NEP在良性和恶性前列腺组织中的表达、定位和分布及其与细胞骨架的关系。
采用免疫组织化学（IHC）方法对正常前列腺组织、良性前列腺增生（BPH）和Gleason分级为2-5级的PC固定标本中的NEP进行定位。进行原位杂交和蛋白质印迹实验以证实BPH和PC组织中NEP基因的表达及向成熟蛋白的翻译。利用共聚焦激光扫描显微镜研究高级别前列腺肿瘤的发生是否伴随着细胞内肌动蛋白/NEP共定位模式的变化。最后，通过对前列腺肿瘤中的NEP和Ki-67进行双重染色，研究与NEP表达缺失相关的增殖活性。
原位杂交研究显示PC上皮细胞中NEP mRNA表达保留。免疫组织化学显示NEP位于正常上皮细胞和BPH组织的顶端质膜。在PC中，发现NEP分布模式随Gleason分级向异质性、部分蛋白胞质分配转变。与BPH组织相比，PC来源的标本中NEP蛋白的免疫组织化学染色强度非常低。在高级别PC中，肌动蛋白和NEP典型的顶端共定位消失，发现强烈的颗粒状胞质NEP染色。NEP高表达的PC区域增殖活性降低，即Ki-67染色强度低。
NEP在正常和病理改变的前列腺中差异表达，在高级别肿瘤中明显从膜结合分布模式转变为胞质分布模式，且在高增殖活性区域NEP表达缺失。所提供的数据支持NEP积极参与雄激素非依赖性PC的进展。需要进一步研究以阐明PC中胞质NEP分布的潜在机制。
