Department of Chemistry, Washington State University, Pullman, Washington.
Prostate. 2014 May;74(5):451-7. doi: 10.1002/pros.22727. Epub 2014 Jan 21.
Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry.
Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor.
Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1) mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50 = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry.
The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans.
前列腺特异性膜抗原（PSMA）仍然是人类前列腺癌诊断和治疗应用的重要靶标。最近已经开发出模型细胞系来研究犬前列腺癌，但它们的 PSMA 表达和酶活性尚未阐明。本研究集中于确定这些模型犬细胞系中的 PSMA 表达，并使用荧光小分子酶抑制剂通过流式细胞术检测犬 PSMA 表达。
使用 Western blot 和 RT-PCR 确定 Leo 和 Ace-1 犬细胞系中 PSMA 的转录和翻译表达。使用基于终点 HPLC 的测定法监测犬 PSMA 的酶活性和酶抑制剂的效力。使用荧光标记的 PSMA 酶抑制剂通过流式细胞术检测 Leo 和 Ace-1 细胞上表达的 PSMA。
通过 Western blot 和 RT-PCR、酶活性和流式细胞术证实 Leo 细胞系上的犬 PSMA 表达。确定了 PSMA 酶促活性对合成底物（PABGγG）的动力学参数 Km 和 Vmax 分别为 393 nM 和 220 pmol min(-1) mg 蛋白(-1) 。发现抑制剂核心 1 和荧光抑制剂 2 是 PSMA 在 Leo 细胞系上表达的有效可逆抑制剂（IC50 分别为 13.2 和 1.6 nM）。Leo 细胞的荧光标记表明，荧光 PSMA 抑制剂 2 可用于检测 PSMA 阳性犬前列腺肿瘤细胞。流式细胞术未检测到 Ace-1 上 PSMA 的表达。
本文的研究结果表明，PSMA 在犬前列腺肿瘤细胞上表达，并表现出与人类 PSMA 相似的酶学特征。这些发现表明，目前正在研究用于人类前列腺癌诊断和治疗的小分子酶抑制剂也可以扩展到包括犬前列腺癌。重要的是，这些发现表明，在将抑制剂用于人类之前，可以在自然发生前列腺癌的免疫功能正常的动物模型中评估抑制剂用于诊断和治疗的潜力。
