Prostate specific antigen (psa): from the catalytic activity to the clinical value

Abstract

Prostate-specific antigen (PSA) is a serine protease belonging to the kallikrein family and it is also known as kallikrein-related peptidase 3 (KLK3). The major physiologic substrates for PSA appear to be the gel-forming proteins in freshly ejaculated semen, semenogelin I (SgI) and semenogelin II (SgII) which are synthesized and secreted by the seminal vesicles. PSA can also cleave a number of growth regulatory proteins that are important in cancer development and survival as IGFBP, PTH-related protein, latent TGF-β2, and extracellular matrix components fibronectin and laminin. Part of this thesis is dedicated to deepening the catalytic activity of PSA in order to clarify the hypothetical role of this enzyme in prostate cancer (PCa) progression and metastasization. In this light, the steady state and pre-steady state kinetics of the PSA-catalyzed hydrolysis of a fluorogenic substrate (Mu-His-Ser-Ser-Lys-Leu-Gln-AMC) has been determined between pH 6.5 and 9.0 at 37°C temperature. The obtained kinetic pattern was characterized by the presence of an initial burst phase which precedes the insurgence of the steady-state phase. This feature could be referred to a mechanism where the acylation and deacylation steps of the PSA-catalyzed cleavage of the fluorescent substrate displayed different rate constants (k2 and k3, respectively). The possibility of a quantitatively satisfactory description of the two processes by parameters which are mutually consistent (i.e., kcat, k2, k3, Km and Ks) gave a great support to the fact that the mechanism described was suitable to account for the observed behavior. Fitting the obtained data the pH-dependence of the pre-steady-state and steady-state parameters for the PSA-catalyzed hydrolysis of the fluorescent substrate was demonstrated. The overall description of the proton linkage for the different parameters required the protonation / deprotonation of (at least) two groups. The global fitting of the pH-dependence of all parameters has allowed to define a set of six pKa values (i.e., pKU1, pKU2, pKES1, pKES2, pKL1, and pKL2) which satisfactorily describe all proton linkages modulating the enzymatic activity of PSA. Consequently to a prostate tissue damage, large amount of PSA could be released in bloodstream and this explain the extensive clinical application of PSA in clinical practice as a PCa-related marker. Although the routine use of serum PSA testing has undoubtedly increased PCa detection, one of its main drawbacks is represented by its low specificity. High levels of PSA, in fact, can be found not only in malignant but also in benign prostatic pathologies, such as prostatitis and, first of all, in benign prostatic hyperplasia (BPH). In this view several new PCa biomarkers have been intensively scrutinized over the last decade, but despite new findings and good performance characteristics, they are currently not uniformly accepted in clinical practice. In this thesis the diagnostic performances of one of this marker, the prostate cancer gene 3 (PCA3), are discussed in both term of its diagnostic and prognostic potential. Through a prospective study, first of all, the different distribution in PSA, expressed as total PSA (tPSA) and free PSA (fPSA) to tPSA ratio (f/tPSA), and PCA3 score in two groups of subjects with negative (n=212) and positive biopsy (n=195) was evaluated. tPSA values overlapped between the two groups indicating that this parameter cannot be used to discriminate PCa from non-PCa patients. Considering instead the f/tPSA ratio, a most powerful marker was found, as it resulted significantly lower in subjects with PCa. However, the best result was obtained from the PCA3 urine test, that shows the highest difference between the two groups, demonstrating to be a more sensitive test, resulting strongly associated to the prostate oncologic pathology. Conversely to the PSA, the PCA3 test showed also a good prognostic performance, since the percentage of patients with more aggressive PCa (Gleason score ≥ 7) had significantly higher PCA3 score values respect to patients with lower grade PCa (Gleason score ≤ 6). Attempting to improve the specificity of the PSA serum test for the early diagnosis of PCa, the clinical utility of other PSA forms were evaluated. With a second retrospective study, the clinical utility of PSA, [-2]proPSA (p2PSA) and Galectin 3 (Gal3) serum tests was investigated, comparing the different distribution of these markers along two populations, one with PCa and a second with BPH. The obtained results showed that none of these markers was able to discriminate the malignant from the benign prostatic pathology. However, things deeply changed when these parameters were combined together to calculate different indices, such as the PSA ratio (fPSA/tPSA), the percentage of p2PSA (p2PSA/fPSA), the prostate health index (p2PSA/fPSA*tPSA1/2) and the Galphi (Gal3*tPSA/fPSA), a novel index of our creation here investigated for the first time. In this case, interestingly, all the considered indices were able to predict PCa in a good manner. In particular, the p2PSA derivatives (i.e. the percentage of p2PSA and the prostate health index) showed a diagnostic accuracy greater than tPSA and f/tPSA tests, with prostate health index resulting the most accurate. At the same time, the Galphi resulted able to reach good diagnostic performances, in some respects comparable to that obtained from p2PSA related indices, showing, for the first time, that a Gal3 related index may be used for the same purpose of other quoted PCa biomarkers. Once that a PCa is diagnosed and characterized in its histopathological features, a treatment approach must be planned. The first line treatment of organ-confined PCa involves, generally, a surgical resection of the prostate. In 1997, Schuessler described the first laparoscopic radical prostatectomy (LRP) and since that time numerous European and US centers tried to improve and refine technical aspects of the laparoscopic approach, introducing, for example, several robotic systems such as the “da Vinci” system. The radical prostatectomy (RP) surgery, when applicable, is generally resolutive; however, in some cases, PCa tends to generate metastases after a few years, even if they are not detected during the surgery. Accordingly, some investigations have suggested that a number of factors in the perioperative period could promote metastasization. These include the surgery approach and its associated stress response, the anaesthetic regimen, the acute pain, and the administration of opioid analgesics. The hypothesis is that different anesthetic protocols and surgery techniques can differently activate the clotting system, thereby generating thrombin, or stimulate mononuclear cells, platelets and endothelial cells. The consequent formation of a fibrin matrix, together with cell activation, appear to promote tumor growth and neo-angiogenic processes. In this view part of this PhD thesis was dedicated to investigate whether two group of PCa patients, undergoing conventional LRP or robot assisted laparoscopic prostatectomy (RALP), with two different intra-operative anaesthetic regimens, total intravenous anesthesia with target-controlled infusion (TIVA-TCI) and balanced inhalation anaesthesia (BAL), showed different changes in coagulation, cell activation and angiogenesis activation markers during the perioperative period. Both TIVA-TCI and BAL patients showed a marked and significant increase in pro-coagulant factors, with consequent reduction in haemostatic system inhibitors, in the early post-operative period, while the RALP approach showed a significant increase in pro-thrombotic markers as compared to LRP. In TIVA patients undergoing LRP, instead, a lower variation of p-selectin levels, compared to BAL, was observed. Considering the variations of pro-angiogenesis and endothelial activation markers a significant increment in vascular endothelial growth factor (VEGF) and von Willebrand factor (vWf), in the perioperative period, was found for all conditions. At the same time, patients undergoing LRP showed a significantly higher variation in VEGF levels. Interestingly, in all the analyzed groups, an opposite trend in VEGF and vWf variations was always observed, manifesting, for the first time in vivo, the possible negative regulation of angiogenic processes by vWf. The obtained results fundamentally support the hypothesis that the administration of anti- thrombotic and/or anti-angiogenic agents could be helpful in preventing the pro-metastasization events derived from some of the perioperative manipulations received from PCa patients during the radical prostatectomy.