Gutiérrez-Cañas Irene, Juarranz María G, Collado Beatriz, Rodríguez-Henche Nieves, Chiloeches Antonio, Prieto Juan C, Carmena María J
Unidad de Neuroendocrinología Molecular, Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.
Prostate. 2005 Apr 1;63(1):44-55. doi: 10.1002/pros.20173.
Neuroendocrine (NE) differentiation in prostate cancer has been correlated with unfavorable clinical outcome. The mechanisms by which prostate cancer acquires NE properties are poorly understood, but several signaling pathways have been proposed. We have previously observed that vasoactive intestinal peptide (VIP) stimulates cAMP production mainly through VPAC(1) receptor, inducing NE differentiation in LNCaP cells. The aim of this study was to analyze the mechanisms involved in this process.
Reverse transcriptase (RT)-polymerase chain reaction (PCR), quantitative real-time RT-PCR, Western blotting, and immunocytochemistry were performed.
LNCaP cells produce VIP, as demonstrated by RT-PCR and immunocytochemistry. VIP induced NE differentiation of LNCaP cells at a time as short as 1 hr of treatment, and the same occurred with the expression and secretion of neuronal-specific enolase (NSE, a NE differentiation marker). These effects were faster than those exerted by serum-deprivation. VIP induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and NE differentiation by PKA-dependent and independent pathways, since the PKA inhibitor H89 partially blocked VIP-induced NE differentiation and did not affect ERK1/2 phosphorylation. mitogen-activated protein kinase kinase (MEK) and phosphoinositide 3-kinase (PI3K) appear to be also involved since the inhibitors PD98059 and wortmannin abolished ERK1/2 phosphorylation and decreased NE differentiation induced by VIP. Moreover, VIP activated Ras suggesting the involvement of a Ras-dependent pathway.
VIP behaves as autocrine/paracrine factor in LNCaP cells by inducing NE differentiation through PKA, ERK1/2, and PI3K.
前列腺癌中的神经内分泌（NE）分化与不良临床结局相关。前列腺癌获得NE特性的机制尚不清楚，但已提出了几种信号通路。我们之前观察到血管活性肠肽（VIP）主要通过VPAC(1)受体刺激cAMP产生，诱导LNCaP细胞发生NE分化。本研究的目的是分析这一过程中涉及的机制。
进行逆转录（RT）-聚合酶链反应（PCR）、定量实时RT-PCR、蛋白质印迹法和免疫细胞化学。
RT-PCR和免疫细胞化学证明LNCaP细胞产生VIP。VIP在处理1小时时即诱导LNCaP细胞发生NE分化，神经元特异性烯醇化酶（NSE，一种NE分化标志物）的表达和分泌情况也是如此。这些效应比血清剥夺所产生的效应更快。VIP通过PKA依赖和非依赖途径诱导细胞外信号调节激酶1和2（ERK1/2）磷酸化以及NE分化，因为PKA抑制剂H89部分阻断了VIP诱导的NE分化且不影响ERK1/2磷酸化。丝裂原活化蛋白激酶激酶（MEK）和磷脂酰肌醇3激酶（PI3K）似乎也参与其中，因为抑制剂PD98059和渥曼青霉素消除了ERK1/2磷酸化并降低了VIP诱导的NE分化。此外，VIP激活了Ras，提示存在Ras依赖途径。
VIP在LNCaP细胞中作为自分泌/旁分泌因子，通过PKA、ERK1/2和PI3K诱导NE分化。
