5α-reductase type 3 expression in human benign and malignant tissues: a comparative analysis during prostate cancer progression.

BACKGROUND: A third isozyme of human 5α-steroid reductase, 5α-reductase-3, was identified in prostate tissue at the mRNA level. However, the levels of 5α-reductase-3 protein expression and its cellular localization in human tissues remain unknown. METHODS: A specific monoclonal antibody was developed, validated, and used to characterize for the first time the expression of 5α-reductase-3 protein in 18 benign and 26 malignant human tissue types using immunostaining analyses. RESULTS AND CONCLUSIONS: In benign tissues, 5α-reductase-3 immunostaining was high in conventional androgen-regulated human tissues, such as skeletal muscle and prostate. However, high levels of expression also were observed in non-conventional androgen-regulated tissues, which suggest either multiples target tissues for androgens or different functions of 5α-reductase-3 among human tissues. In malignant tissues, 5α-reductase-3 immunostaining was ubiquitous but particularly over-expressed in some cancers compared to their benign counterparts, which suggests a potential role for 5α-reductase-3 as a biomarker of malignancy. In benign prostate, 5α-reductase-3 immunostaining was localized to basal epithelial cells, with no immunostaining observed in secretory/luminal epithelial cells. In high-grade prostatic intraepithelial neoplasia (HGPIN), 5α-reductase-3 immunostaining was localized in both basal epithelial cells and neoplastic epithelial cells characteristic of HGPIN. In androgen-stimulated and castration-recurrent prostate cancer (CaP), 5α-reductase-3 immunostaining was present in most epithelial cells and at similar levels, and at levels higher than observed in benign prostate. Analyses of expression and functionality of 5α-reductase-3 in human tissues may prove useful for development of treatment for benign prostatic enlargement and prevention and treatment of CaP.

Prostate-specific antigen modulates the expression of genes involved in prostate tumor growth.

Prostate-specific antigen (PSA) is a serine protease that is widely used as a surrogate marker in the early diagnosis and management of prostate cancer. The physiological relevance of tissue PSA levels and their role in prostate tumor growth and metastasis are not known. Free-PSA (f-PSA) was purified to homogeneity from human seminal plasma by column chromatography, eliminating hk2 and all known PSA complexes and retaining its protease activity. Confluent monolayers of prostate cancer cell lines, PC-3M and LNCaP, were treated with f-PSA in a series of in vitro experiments to determine the changes in expression of various genes that are known to regulate tumor growth and metastasis. Gene array, quantitative polymerase chain reaction (QPCR), and enzyme-linked immunosorbent assay (ELISA) results show significant changes in the expression of various cancer-related genes in PC-3M and LNCaP cells treated with f-PSA. In a gene array analysis of PC-3M cells treated with 10 muM f-PSA, 136 genes were upregulated and 137 genes were downregulated. In LNCaP cells treated with an identical concentration of f-PSA, a total of 793 genes was regulated. QPCR analysis reveals that the genes for urokinase-type plasminogen activator (uPA), VEGF, and Pim-1 oncogene, known to promote tumor growth, were significantly downregulated, whereas IFN-gamma, known to be a tumor-suppressor gene, was significantly upregulated in f-PSA-treated PC-3M cells. The effect of f-PSA on VEGF and IFN-gamma gene expression and on protein release in PC-3M cells was distinctly dose-dependent. In vivo studies showed a significant reduction (P = .03) in tumor load when f-PSA was administered in the tumor vicinity of PC-3M tumor-bearing BALB/c nude mice. Our data support the hypothesis that f-PSA plays a significant role in prostate tumor growth by regulating various proangiogenic and antiangiogenic growth factors.

Two step procedure for purification of enzymatically active prostate-specific antigen from seminal plasma.

The role of prostate-specific antigen (PSA) during the onset of prostate cancer and subsequent tumor growth and metastasis is not well understood. We have developed a simple two step procedure, based on principles of hydrophobic charge-induction chromatography and molecular size chromatography to provide pure free-PSA (f-PSA) preparation that is free from all other known PSA complexes as well as human kallikrein 2 (hK2). The overall recovery of f-PSA is 72%. The isolated f-PSA consists of three known isoforms that corresponds to pI of 6.2, 6.4 and 7.2. f-PSA is enzymatically active and its enzymatic activity can be effectively neutralized by a serine protease inhibitor.

Thiophilic interaction chromatography facilitates detection of various molecular complexes of prostate-specific antigen in biological fluids.

BACKGROUND: It is known that total PSA values are often greater than the sum total of free-PSA (f-PSA) and PSA-ACT complex. It is primarily because other PSA complexes are present in the patient serum and currently cannot be quantified. Our studies were aimed in developing methodology that will permit recovery of all molecular forms of PSA from various biological fluids. METHODS: Thiophilic interaction (3S,T-gel) chromatography procedure was used to isolate various PSA molecular forms present in biological fluids. f-PSA and PSA complexes were shown to have strong affinity for T-gel. PSA forms eluted from the column were analyzed by SDS-PAGE/Western blot and were identified by immunostaining with antibody-specific to PSA and to various protease inhibitors. The identity of PSA complexes was also confirmed by Enzyme-linked Immunosorbent-assay (ELISA). RESULTS: One step affinity chromatography procedure (3S,T-gel) was used to isolate different molecular forms of PSA in seminal plasma, patient sera, and in prostate cancer cell culture medium. Seminal plasma was shown to contain f-PSA, PSA-ACT, PSA-A2M, and PSA-PCI. Sera from prostate cancer patients at different stages of the disease contained f-PSA, PSA-ACT, and PSA-A2M. Besides these PSA forms, some patients at late stage of the disease (T3 and T4) also contained two additional PSA-complexes that have not been identified as of yet. Conditioned medium from LNCaP cells was shown to contain f-PSA, PSA-ACT, and PSA-A2M complexes. CONCLUSIONS: Thiophilic gel has strong affinity for all known molecular forms of PSA present in any biological material. Visualization of PSA molecular forms in Western-blot analysis was feasible only after patient serum was processed through T-gel. Our procedure can be used to screen hybridoma and antibodies specific to the individual PSA complex. The PSA molecular forms isolated from patient serum after T-gel procedure may provide useful internal calibrators, and thus may significantly enhance the reliability of PSA measurements.