Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an 710061, PR China.
Oncol Rep. 2013 Aug;30(2):904-10. doi: 10.3892/or.2013.2534. Epub 2013 Jun 11.
Epidermal growth factor (EGF) signaling and Hedgehog (HH) signaling are both involved in prostate cancer (PCa) progression, yet the mechanisms through which these two pathways are synergistically linked require elucidation. In the present study, we aimed to ascertain how EGF and the HH signaling transcription factor GLI-1 are linked in prostate cancer invasiveness. ARCaP human prostate cancer cells, which included ARCaPE and ARCaPM cells, were used as a model in the present study. The expression of EGF receptor (EGFR) and the HH signaling transcriptional factor GLI-1 were detected in ARCaPE cells by immunofluorescence, and the ARCaPE cells were treated with human recombinant EGF protein (hrEGF) for 4 consecutive days in vitro. Transwell invasion assays were performed in the ARCaPE cells following treatment with DMSO (vehicle control), hrEGF, GATN61 (GLI-1-specific inhibitor), hrEGF plus GANT61 and in the ARCaPM cells. The expression of phosphorylated extracellular signal regulated kinase (p-ERK), total ERK and GLI-1 was detected by western blotting in ARCaPE cells at different time-points following treatment with hrEGF. The expression of EGFR and GLI-1 was detected in ARCaPE cells, which exhibited a cobblestone-like morphology, while after treatment with hrEGF, the cell morphology was altered to a spindle-shaped mesenchymal cell morphology. Transwell invasion assays demonstrated that hrEGF dramatically enhanced the invasive capability of the ARCaPE cells (p<0.05). Additionally, western blot assay demonstrated that the expression levels of p-ERK and GLI-1 in ARCaPE cells increased in a time-dependent manner after treatment with hrEGF (p<0.05); however, the expression levels of total ERK in the cells remained relatively unchanged. It also demonstrated that the GLI-1 inhibitor GANT61 could reverse the enhanced invasive effect induced by EGF in ARCaPE cells (p<0.05). Our preliminary in vitro study showed that EGF signaling may increase the invasive capability of ARCaPE human prostate cancer cells via upregulation of p-ERK and the HH signaling transcriptional factor GLI-1. Additionally, this enhanced cell invasive effect was reversed by a GLI-1-specific inhibitor in vitro. Consequently, it indicates that both EGF and HH signaling are synergistically involved in the progression of human prostate cancer ARCaP cells, and GlI-1 may be one of the important effectors, which is activated by EGF downstream signaling, to promote the invasiveness of ARCaPE prostate cancer cells.
表皮生长因子（EGF）信号和 Hedgehog（HH）信号都参与了前列腺癌（PCa）的进展，但这两种途径协同作用的机制仍需要阐明。在本研究中，我们旨在确定 EGF 和 HH 信号转录因子 GLI-1 如何在前列腺癌侵袭性中相互关联。本研究使用 ARCaP 人前列腺癌细胞（包括 ARCaPE 和 ARCaPM 细胞）作为模型。通过免疫荧光检测 ARCaPE 细胞中 EGF 受体（EGFR）和 HH 信号转录因子 GLI-1 的表达，并在体外连续 4 天用重组人 EGF 蛋白（hrEGF）处理 ARCaPE 细胞。用 DMSO（载体对照）、hrEGF、GATN61（GLI-1 特异性抑制剂）、hrEGF 加 GANT61 处理 ARCaPE 细胞和 ARCaPM 细胞后，进行 Transwell 侵袭实验。用 Western blot 检测不同时间点 hrEGF 处理后 ARCaPE 细胞中磷酸化细胞外信号调节激酶（p-ERK）、总 ERK 和 GLI-1 的表达。用 Western blot 检测 ARCaPE 细胞中 EGFR 和 GLI-1 的表达，ARCaPE 细胞呈鹅卵石样形态，用 hrEGF 处理后，细胞形态变为梭形间充质细胞形态。Transwell 侵袭实验表明，hrEGF 显著增强了 ARCaPE 细胞的侵袭能力（p<0.05）。此外，Western blot 实验表明，hrEGF 处理后 ARCaPE 细胞中 p-ERK 和 GLI-1 的表达水平呈时间依赖性增加（p<0.05）；然而，细胞中总 ERK 的表达水平相对不变。实验还表明，GLI-1 抑制剂 GANT61 可逆转 EGF 诱导的 ARCaPE 细胞侵袭增强效应（p<0.05）。本初步体外研究表明，EGF 信号可能通过上调 p-ERK 和 HH 信号转录因子 GLI-1 增加 ARCaPE 人前列腺癌细胞的侵袭能力。此外，这种增强的细胞侵袭效应可通过 GLI-1 特异性抑制剂在体外逆转。因此，这表明 EGF 和 HH 信号均协同参与人前列腺癌 ARCaP 细胞的进展，而 Gli-1 可能是 EGF 下游信号激活的重要效应子之一，促进 ARCaPE 前列腺癌细胞的侵袭性。
