Preparation and characterization of the antibody recognizing AMACR inside its catalytic center

Abstract

Alpha-methylacyl-CoA racemase (AMACR) catalyzes the β-oxidation of fatty acids and is overexpressed in carcinomas in various organs, while its inactivation results in the inhibition of cancer growth. In the present study, we prepared and characterized 20 different mouse monoclonal antibodies against human AMACR. In the course of biopanning of a phage peptide commercial library against in-house prepared 6H9 and 2A5, and commercial 13H4 antibodies, 10 phage mimotopes recognized by each type of the antibody were selected. Using the program Pepitope and the crystal structure of AMACR from Mycobacterium tuberculosis, we reveal for the first time, at least to the best of our knowledge, that the epitopes recognizing the antibody against AMACR are composed of conformation sequences localized inside the AMACR catalytic center. When delivered into live HeLa cells using cationic lipid-based PULSin reagent, the specific antibodies against AMACR were co-localized with peroxisomes. The in-house made 6H9 antibody exhibited a low level of this co-localization compared to the commercially available 63340 antibody, and did not inhibit the growth rate of HeLa and T98G cells. The results obtained suggest that antibody against AMACR may possess anti-AMACR catalytic activity and needs to be further investigated as a potential drug for use in anticancer therapy. On the whole, in this study, we generated several clones of AMACR antibodies and demonstrated that these antibodies can be colonized into live cells. Currently, we are testing the growth inhibitory properties of these antibodies against AMACR.

Early Cell Cultures from Prostate Cancer Tissue Express Tissue Specific Epithelial and Cancer Markers

“…First, we evaluated cancerous and normal prostate tissue samples for the presence of cancer markers AMACR and EZH2. PCaT samples were positive for these markers (similar to the T98G glioblastoma cells that served as a positive control [31]), while normal prostate tissue did not express them (Figure 2a). ilar to the T98G glioblastoma cells that served as a positive control [31]), while normal prostate tissue did not express them (Figure 2a).…”

“…PCaT samples were positive for these markers (similar to the T98G glioblastoma cells that served as a positive control [31]), while normal prostate tissue did not express them (Figure 2a). ilar to the T98G glioblastoma cells that served as a positive control [31]), while normal prostate tissue did not express them (Figure 2a). Protein extracts (30 µg of total protein per lane) were loaded onto 10% polyacrylamide gel.…”

“…Literature data show that the level of EZH2 is stably elevated in aggressive PCa but not in localized PCa [30]. AMACR is commonly used as a marker of all PCa stages [31]. TMPRSS2-ERG is a specific PCa marker, although its expression has been found in less than 50% of patients with localized PCa [32].…”

“…AMACR regulates fatty acids β-oxidation and energy metabolism, which is activated in PCa and other malignancies. However, the mechanisms of AMACR hyper-production in cancer are still unknown [31]. Evaluation of the expression of these cancer markers may facilitate the use of PCCs as a preclinical model of PCa.…”

Organoid and Primary Epithelial Cultures of Human Prostate Show the Key Role of the Epithelial-to-Mesenchymal Transition in Generation of Tissue-Specific Stromal Cells

“…The following antibodies were used for immunofluorescence: rabbit anti-PanCK (Thermo Fisher Scientific, United States), mouse anti-vimentin (Sigma, United States), and mouse monoclonal antibodies against AMACR (Popov et al, 2018). Goat antibodies against mouse IgG (H + L) conjugated with Cy3 cyanine dye and goat antibodies against rabbit IgG conjugated with Cy5 (Molecular Probes, Thermo Fisher Scientific, United States) served as second antibodies for immunofluorescence.…”