Prostate carcinoma is, at least initially, a hormone-regulated neoplasm, but under therapy eventually escapes and relapses [2, 11–17]. Androgen effects are classically exerted through binding of the hormone to intracellular specific receptors [18, 19], which consequently dimerize, translocate to the nucleus and bind to specific elements of DNA, affecting thus the transcription of androgen-responsive genes . In recent years, evidence has been accumulated, indicating that androgen can alternatively exert on respective target cells short-time non-genomic effects mediated through membrane binding sites and subsequent changing in Ca2+ concentration [4, 5, 20, 21], in cell types presenting [1, 2] or lacking intracellular androgen receptors [3–5]. Quite recently we have described a membrane testosterone binding site in the hormone positive human prostate cancer cell line LNCaP . We have shown that activation of this site initiates within minutes the rearrangement of actin cytoskeleton through a specific signaling cascade (Papakonstanti et al, submitted) and increases the secretion of PSA. This site was selective for androgens, and immunologically different from the intracellular classical testosterone receptor. In addition, no internalization of membrane testosterone sites was obvious after incubation times less than one hour.
The results of the present study indicate that membrane testosterone binding sites can also be identified in freshly prepared human prostate cells. This binding was restrained to epithelial cells and is completed within minutes (Figure 1). Neoplastic cells, morphologically normal prostate epithelium cells and BPH epithelia bind testosterone-BSA analog (Figure 3, Table 1) but with a different intensity. In this respect, as presented in Figure 3, a clear discrimination of malignant and non-malignant cells could be obtained by membrane testosterone binding. This is also evident in touching preparations (Figure 2) and histological preparations of prostate cancer (Figure 4). It is of interest to note that cells underwent prostate intraepithelial neoplasia (PIN) are also intensely stained with testosterone-BSA, indicating that the expression of these sites might correlate with a neoplastic and premalignant (PIN) phenotype. With more sensitive methods however (flow cytometry), some slight staining-reaction of normal and BPH epithelia was also evidenced. In this respect, the difference between neoplastic and non-neoplastic cells is merely a quantitative differential expression. Indeed, two cases of transurethral resection (Table 1) in which high testosterone-BSA-FITC staining was obtained, were revealed to present foci of PIN after a thorough histological examination.
Traditionally the differential diagnosis, grading and staging of prostate carcinoma has been based predominantly on morphological features [9, 22, 23]. A number of immunological markers have been proposed in prostate carcinoma or premalignant conditions (such as PIN), but they lack selectivity and specificity [9, 22–29]. Indeed, a good biomarker can be defined as one that adds independent information to standard prognostic indicators, and which can be reliably reproduced in the laboratory, aiding in diagnostic and/or therapeutic decisions. Our results indicate that membrane testosterone binding sites could fulfill this definition, as they can aid in discrimination between benign and malignant/premalignant prostate conditions. As shown in Figure 3, in only one case of morphologically normal epithelial cells the values overlapped with those obtained in the carcinoma group. It is possible, however, that a number of tumor cells could interfere with the measurement. In addition, two samples, initially considered as BPH, but finally diagnosed as harboring PIN lesions were positively stained for membrane testosterone binding sites, indicating the potential diagnostic usefulness of membrane testosterone binding sites identification and/or measurement. Although further investigations in large series are needed, our results indicate that membrane testosterone binding sites could be a valuable addition in prostate cancer differential diagnosis, and a possible target for novel therapies.