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

ABSTRACT: 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 (PABG?G) 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.

SUBMITTER: Wu LY

PROVIDER: S-EPMC4237199 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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

Wu Lisa Y LY Johnson Jacqueline M JM Simmons Jessica K JK Mendes Desiree E DE Geruntho Jonathan J JJ Liu Tiancheng T Dirksen Wessel P WP Rosol Thomas J TJ Davis William C WC Berkman Clifford E CE

The Prostate 20140121 5

<h4>Background</h4>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 b ...[more]

PMID: 24449207

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Project description:Objective: To investigate the role of PSMA in the differential diagnosis of adrenocortical carcinoma samples (ACCs) and adrenocortical adenoma samples (ACAs), to validate the prognostic role of PSMA in patients with ACCs, and to explore the possibility that this marker can differentiate localized ACCs from adrenal metastases from other sites. Methods: PSMA protein expression in tissue samples from 50 ACCs, 90 ACAs (including 20 from patients who presented with Cushing's syndrome, 20 aldosterone-producing adenomas and 50 non-functional tumors) and 10 tissues that were metastases from other primary sites was assessed by immunohistochemistry. The clinical and pathological characteristics were compared, the intensity and density were analyzed, and the prognostic role was evaluated. Results: The analysis of clinical and pathological features revealed that the size of ACCs was greater than that of benign tissues and the ACC patients were older than the ACA patients (p < 0.01). The percentage of PSMA-positive vessels, the mean intensity and the degree of staining density were found to be significantly lower in ACAs than in ACCs (p < 0.01). In these 140 samples, 60% of the ACCs were grouped in the positive category. The samples were negative for metastases that were from other primary sites. The ENSAT stage and Ki-67 were correlated with PSMA expression. The survival distribution revealed that high PSMA expression did not show any prognostic relevance in the current ACCs series. Those samples with a score of > 3.5 were 75 times more likely to be malignant (OR = 75). We established a cut-off score of 3.5 (p < 0.05), which had 46% sensitivity and 99% specificity. Paralleling PSMA and Ki-67 maximized the area under the curve, with 72% sensitivity and 100% specificity. Conclusions: Our results strongly confirm that PSMA is helpful for distinguishing benign from malignant tumors and that its high expression levels correlate with a high ENSAT stage and high proliferation. The combination of PSMA and Ki-67 can be particularly useful. Furthermore, PSMA might be a useful tool for the identification of localized adrenal carcinoma and metastatic carcinoma.

Dataset Information

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

Publications

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

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