Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

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 (PABGgG) 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一个重要靶点。最近已经开发出一些模型细胞系来研究犬前列腺癌，但其PSMA表达和酶活性尚未阐明。本研究重点在于确定这些犬模型细胞系中的PSMA表达，以及利用荧光小分子酶抑制剂通过流式细胞术检测犬PSMA表达。 采用蛋白质印迹法和逆转录聚合酶链反应（RT - PCR）来确定PSMA在犬Leo和Ace - 1细胞系中的转录和翻译表达。采用基于高效液相色谱（HPLC）的终点检测法来监测犬PSMA的酶活性以及酶抑制剂的效力。利用流式细胞术，采用一种荧光标记的PSMA酶抑制剂来检测Leo和Ace - 1细胞上表达的PSMA。 通过蛋白质印迹法、RT - PCR、酶活性检测以及流式细胞术证实了Leo细胞系上存在犬PSMA表达。合成底物（PABGgG）的PSMA酶活性的动力学参数Km和Vmax分别确定为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Leo细胞系上表达的PSMA的强效可逆抑制剂（半数抑制浓度IC₅₀分别为13.2 nM和1.6 nM）。对Leo细胞的荧光标记表明，荧光PSMA抑制剂2可用于检测PSMA阳性的犬前列腺肿瘤细胞。Ace - 1细胞上的PSMA表达量低，无法通过流式细胞术检测到。 本文所述结果表明，PSMA在犬前列腺肿瘤细胞上有表达，并且表现出与人类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扩展到犬前列腺癌。重要的是，这些发现表明，在用于人类之前，可以在一种自然发生前列腺癌的免疫健全动物模型中评估这些抑制剂用于诊断和治疗的潜力。