Enzymatic Activity Of Prostate-specific Antigen Research Articles

Powerful Chemiluminescence Probe for Rapid Detection of Prostate Specific Antigen Proteolytic Activity: Forensic Identification of Human Semen.

The prostate specific antigen (PSA), a serine protease with chymotrypsin-like activity, is predominantly expressed in the prostate and is considered as the most common marker in use to identify and follow the progress of prostate cancer. In addition, it is also now accepted as a marker for detecting semen in criminal cases. Here, we describe the design, synthesis, and evaluation of the first chemiluminescence probe for detection of PSA enzymatic activity. The probe activation mechanism is based on a catalytic cleavage of a specific peptidyl substrate, followed by a release of a phenoxy-dioxetane luminophore, that then undergoes efficient chemiexcitation to emit a green photon. The probe exhibits a significant turn-on response upon reaction with PSA and produces strong light emission signal with an extremely high signal-to-noise ratio. Comparison of the chemiluminescence probe with an analogous fluorescence probe showed superior detection capability in terms of response time and sensitivity. In addition, the probe was able to efficiently detect and image human semen traces on fabric, even after 3 days from sample preparation. The advantageous sensitivity and simplicity of a chemiluminescence assay to detect seminal fluid was effectively demonstrated by on-site measurements using a small portable luminometer. It is expected that the new chemiluminescence probe would be broadly useful for numerous applications in which PSA detection or imaging is required.

NEW MONOCLONAL ANTIBODIES TO THE PROSTATE-SPECIFIC ANTIGEN: OBTAINING AND STUDYING BIOLOGICAL PROPERTIES

Original set of 15 clones of hybridomas producers of monoclonal antibodies (mAbs) to prostate-specific antigen (PSA) (using as a source of lymphocytes of mice of inbred strains NZB and Balb/c) was obtained. The activity in ELISA, affinity constant and titer in the culture fluid of the mAbs obtained from NZB mice are higher compared to the mAbs obtained from the Balb/c mice splenocytes. Conjugates of obtained mAbs with horseradish peroxidase were synthesized, and it allowed performing comparative epitope characterization of the resulting set of mAbs. Studied mAbs are directed to 4 epitopes of PSA molecule: 2 mAbs interacting with one of the epitopes showed cross-activity with PSA-related protein – human kallikrein 2; 2 mAbs of the same epitope specificity blocked the enzymatic activity of PSA; mAbs of the remaining two epitopes either did not affect PSA chymotrypsin activity at all, or this inhibitory effect was not significant. The obtained mAbs recognize antigenic determinants that are not screened when interacting with α1-antichymotrypsin, as well as those that are localized in the places where α1-antichymotrypsin interacts with PSA. Within each of the given two mAbs groups, there are antibodies belonging to different epitopes, which give prospects for the further use of the obtained mAbs in immunoassay and immunobiotechnology. Two alternative methods for determining of the mAbs constants of affinity (by Friguet and Scatchard) are comparable (for determination of the affinity constants of anti-PSA antibodies): coefficient of linear correlation between the affinity constants defined by different methods was 0.90.

PSA enzymatic activity: A new biomarker for assessing prostate cancer aggressiveness

With the advent of widespread prostate-specific antigen (PSA) testing in recent decades, prostate cancer (PCa) has emerged as the most frequently diagnosed non-skin cancer among men in the U.S. and Europe. Greater screening rates coupled with improved detection methods have caused a controversial upsurge in the number of men undergoing prostate biopsy and subsequent treatment. However, current diagnostic techniques generally suffer from limited ability to identify which seemingly indolent cancers are biologically aggressive. We collected prostatic fluid from 778 post-radical prostatectomy specimens and randomly selected samples from both the clinically confirmed aggressive (n = 50) and non-aggressive (n = 50) prostate cancer populations. We measured the level of proteolytic enzyme activity of PSA (aPSA) in each sample and used receiver operating characteristic (ROC) analysis to correlate aPSA levels with prostate cancer aggressiveness. We found aPSA in prostatic fluid to be inversely proportional to disease stage, such that patients with the most aggressive PCa have on average significantly reduced aPSA compared to those with less aggressive disease. Significantly, our results suggest that many (22% in our study population) of the diagnosed patients with non-aggressive PCa could have averted or delayed radical prostatectomy. Given the high level of debate surrounding PSA screening effectiveness [3-5] and the recent U.S. Preventative Services Task Force recommendation to discontinue PSA screening [6], our results provide renewed hope that a clinical monitoring tool may emerge that truly refines PCa treatment decision-making.

Detection and quantification of enzymatically active prostate-specific antigenin vivo

Assays for blood levels of prostate-specific antigen (PSA), performed in prostate cancer detection, measure mostly inactive/complexed PSA and do not provide information regarding enzymatically active PSA, which is biologically more relevant. Thus, we designed and synthesized an enzymatically cleavable peptide sequence labeled with near-infrared (NIR) fluorophores (ex/em 740/770 nm) and coupled it to a pharmacokinetic modifier designed to improve its plasma kinetics. In its native state, the agent, PSA750 FAST™ (PSA750), is optically quenched (>95%) and only becomes fluorescent upon cleavage by active PSA, yielding a significant increase in signal. This activation is highly selective for PSA relative to a large panel of disease-relevant enzymes. Active PSA was detected in tumor frozen sections using PSA750 and this activity was abolished in the presence of the inhibitor, alpha-1 anti-chymotrypsin. In vivo imaging of tumor-bearing mice using fluorescence molecular tomography demonstrated a significantly higher fluorescent signal in PSA+ LNCaP tumors as compared to PSA- prostate cancer 3 tumors (13.0±3.7 versus 2.8±0.8 pmol, p=0.023). Ex vivo imaging of tumor sections confirms PSA750-derived NIR signal localization in nonvascular tissue. This is the first report that demonstrates the feasibility and effectiveness of noninvasive, real time, fluorescence molecular imaging of PSA enzymatic activity in prostate cancer.

Abstract 5306: Tissue-specific prostate specific antigen (PSA).

Abstract We attempted to isolate and characterize PSA from prostate tissue. The aim was to isolate tissue-specific PSA and its initial characterization. This included: (i) determination of PSA molecular forms (free and complexed PSA), (ii) enzymatic activity, and its inhibition by metal ions, (iii) and their relative anti-angiogenic activity. Prostate, tumor and non-tumor, tissue was acquired under IRB approved protocol. PSA was isolated from prostate non-tumor (NT-PSA) and tumor tissue (T-PSA) and characterized using various chromatographic techniques including ligands such as Thiophilic gel, Concanavalin-A agarose, Protein-A agarose, and other lectins. The average NT-PSA concentration was 25±6.3ng PSA/μg of DNA (p=0.0001) and the average T-PSA concentration was 13±5.7ng PSA/μg of DNA (p=0.0001). On an average, tumor PSA (EA=29.64±11.34 p=0.0001)) had twice as much enzymatic activity as compared to non-tumor PSA (EA=13.29±3.300 p=0.001). The inhibition of enzymatic activity of PSA by metal ion (Zn2+)showed that seminal plasma-PSA (SP-PSA) and NT-PSA were inhibited to the same degree [85.3% and 87.0% respectively (p<0.05)]. However, the level of EA of T-PSA was significantly lower (52.4%) as compared to T-PSA. In regards to its molecular complexes, a higher ratio of free to complex-PSA (ACT and A2M) was obtained from non-tumor prostate tissue-PSA (1.51 p=0.0001) as compared to tumor prostate tissue-PSA (1.15 p=0.0001). Initial studies on the effect of SP-PSA on in vitro angiogenesis revealed a dose-dependent inhibitory effect (1μM, 5μM, 10μM concentrations) on the formation of tubular structures in HUVEC-Matrigel assay. The inhibitory effect of T-PSA & NT-PSA was examined and compared to the effect obtained from SP-PSA. Concentrations of 0.1μM, 1μM and 5μM gave similar levels of inhibition for SP-PSA, NT-PSA, and T-PSA (NT-PSA p-values for 1μM and 5μM: p=0.0057, p=0.0006, respectively; T-PSA p-values for 1μM and 5μM: p=0.0038, p=0.0002, respectively). At 0.1 μM concentration, both T-PSA and NT-PSA were not statistically different from control (p=0.3561 and p=0.2532, respectively). A 10μM concentration of both SP-PSA and NT-PSA displayed similar results (p=0.0001). When comparing the effects between NT-PSA and T-PSA, only 0.1μM concentration produced statistically significant results (p=0.0149; 1μM and 5μM: p=0.2301 and p=0.0752, respectively). We found that both NT-PSA and T-PSA also produced significant dose-dependent inhibition (1μM and 5μM NT-PSA and T-PSA (% inhibition): 76.21, 72.58, and 55.07, 54.98, respectively (p<0.05)). A larger sample size is needed to further assess the differences between the two groups. Our results provide some insight into the role of PSA in pathobiology of prostate cancer. PSA levels in prostate cancer tissue are lower as disease progresses suggesting that PSA has a role in prostate pathology. Citation Format: Srikant S. Chakravarthi, Kailash C. Chadha, Gary J. Smith. Tissue-specific prostate specific antigen (PSA). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5306. doi:10.1158/1538-7445.AM2013-5306

Does Prostate-Specific Antigen Contribute to Bone Metastases?

Advanced prostate cancers consistently display common features, i.e., progression to androgen resistance, rising prostate-specific antigen (PSA), and metastases to the skeleton. In this issue of Clinical Cancer Research , Nadiminty et al. report a provocative mechanistic link between PSA and bone

Inhibition of the enzymatic activity of prostate-specific antigen by boric acid and 3-nitrophenyl boronic acid.

Prostate specific antigen (PSA) is a well-established marker of prostate cancer, but it can also degrade extracellular matrix proteins such as fibronectin and could be involved in tumor progression and metastasis. In this study, we have addressed the use of boric acid and 3-nitrophenyl boronic acid (NPBA) as PSA inhibitors in vitro. The inhibition of PSA by boric acid was studied by using specific fluorogenic substrates. Fibronectin, a biologically relevant substrate for PSA, was used as a substrate in a zymographic assay, and the degradation of fibronectin by PSA in the presence of boric acid and NPBA was followed by Western Blot. Low concentrations of boric acid partially inhibited the proteolytic activity of PSA toward a synthetic fluorogenic substrate. Also, by Western blot, we have found significant inhibition in the proteolysis of fibronectin by PSA in the presence of boric acid as well as NPBA. Results indicate that the boronated compounds used in this study can be used for the modulation of PSA activity. PSA activity is inhibited in vitro by boric acid and NPBA. If degradation of fibronectin by PSA were, in fact, an important step in the progression of prostate cancer, then borate-induced inhibition of PSA activity should help reduce the development and proliferation of prostate carcinomas.

A 5-fluorodeoxyuridine prodrug as targeted therapy for prostate cancer

A method for targeted delivery of the cytotoxic agent 5-fluorodeoxyuridine (FudR) ( 1) to sites of metastatic prostate cancer is described. The prodrug was synthesized by coupling the active drug (FudR) to the PSA-peptide via a self-cleaving diamino acid linker to produce HSSKLQ-Leu-Aib-FudR. This prodrug serves as a substrate for prostate specific antigen (PSA). This approach permitted efficient conversion of the inactive prodrug back to the active cytotoxic state by the enzymatic activity of PSA which is highly expressed by prostate cells.

ANALYSIS OF PROSTATE SPECIFIC ANTIGEN AND α1-ANTICHYMOTRYPSIN INTERACTION USING ANTIPEPTIDE MONOCLONAL ANTIBODIES

The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs). Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane. Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA. The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.

Growth regulation of prostatic stromal cells by prostate-specific antigen.

Prostate-specific antigen (PSA) is a serine protease that can cleave insulin-like growth factor-binding protein-3 (IGFBP3), thereby decreasing its affinity for insulin-like growth factor-I (IGF-I). Dissociation of the IGF-I-IGFBP3 complex renders IGF-I available to bind to its receptor and stimulates cellular proliferation. We evaluated the potential for PSA to modulate the effects of IGF-I and IGFBP3 on the proliferation of human benign prostatic hyperplasia (BPH)-derived fibromuscular stromal cells in primary cultures. We cultured BPH-derived stromal cells for 48 hours in serum-free RPMI-1640 medium supplemented with 0.2% bovine serum albumin and studied the effects of IGF-I, IGFBP3, PSA, and ZnCl(2) at varying concentrations. Differences in cell growth between control and treated cultures were evaluated by use of Dunnett's test. Concentration-related trends were evaluated by linear regression of log-transformed concentrations of test reagents on BPH-derived stromal cell number responses. Statistical tests were two-sided. We observed a concentration-dependent proliferative response of BPH-derived stromal cells to IGF-I. IGFBP3 inhibited this response in a concentration-dependent fashion. IGFBP3 alone had no effect on stromal cell proliferation. When stromal cells were incubated with PSA alone or with PSA, IGF-I, and IGFBP3, an increase in stromal cell numbers that was dependent on PSA concentration was evident in both instances. Zinc, an endogenous inhibitor of PSA enzymatic activity, was able to attenuate the stimulatory effect of PSA at intraprostatic physiologic concentrations. These results are consistent with the idea that PSA can modulate in vitro interactions between IGF-I and IGFBP3 and suggest that PSA may play a role in the regulation of human prostatic fibromuscular cell growth.

Anti-Free Prostate-specific Antigen Monoclonal Antibody Epitopes Defined by Mimotopes and Molecular Modeling

Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer, and the free PSA/total PSA ratio has been shown to be efficient for distinguishing prostate cancer from benign prostatic hyperplasia. We report here the characterization of seven mouse monoclonal antibodies (mAbs) and the partial localization of two conformational epitopes identified by anti-free PSA mAbs. The mAbs were studied by competition and sandwich assays, and the epitope localization of the two anti-free PSA mAbs (6C8D8 and 5D3D11) was performed using phage displayed peptide libraries and molecular modeling. The seven mAbs were classified into three groups according to their recognition specificities and their ability to inhibit the enzymatic activity of PSA and the formation of PSA-alpha1-antichymotrypsin (ACT) complex. Among the anti-free PSA mAb group, 6C8D8 recognized the phage displayed peptide RKLRPHWLHFHPVAV, two parts of which presented similarities with two regions distant on the PSA sequence but joined in the tridimensional structure. mAb 5D3D11 recognized the peptide DTPYPWGWLLDEGYD, which is similar to a PSA region located on the board of the groove containing the PSA enzymatic site. Both epitopes were located in the theoretical ACT binding site described previously. Moreover, these mAbs were able to inhibit the enzymatic activity of PSA. These epitope localizations are in agreement with the ability of both mAbs to inhibit enzymatic activity and ACT fixation. The results presented here could bring information for the generation of clinically relevant PSA assays.

Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies

To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the association targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also examined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with alpha 1-antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited > 90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT association by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA.

Prostate Specific Antigen Cleaves Parathyroid Hormone-Related Protein in the PTH-like Domain

To determine whether parathyroid hormone-related protein (PTHrP) is a substrate of prostate-specific antigen (PSA) and how the biological activity of PTHrP may be altered by cleavage with PSA. Prostate-specific antigen cleavage of recombinant human PTHrP 1-141 was conducted in vitro at 37C and analyzed by SDS-PAGE. Five rounds of automated amino-terminal amino acid sequence analysis were performed on blotted PSA-cleaved PTHrP peptide fragments to determine the PSA cleavage sites. The mouse osteoblast cell line MC3T3-E1 was used to test whether PSA cleavage of PTHrP 1-141 altered its ability to stimulate cAMP production. Prostate-specific antigen was found to specifically cleave PTHrP 1-141 in a time- and dose-dependent manner. Cleavage of PTHrP 1-141 by PSA generated fragments on Coomassie-stained acrylamide gels that migrated with mobilities that corresponded to 19.5, 17, 15 and < 7 kd. The preferred PSA cleavage site of PTHrP 1-141 was determined to be at the carboxyl-terminus of phenylalanine 23, consistent with chymotryptic-like enzymatic activity of PSA. Cleavage of PTHrP by PSA completely abolished the ability of PTHrP to stimulate cAMP production. Cleavage of PTHrP 1-141 by PSA carboxyl-terminal to phenylalanine 23 represents a unique pattern of PTHrP processing that may be specific to the prostate. Prostate-specific antigen inactivation of the cAMP-inducing activity of PTHrP 1-141 demonstrates that PSA cleavage regulates the biological activity of PTHrP. These results have implications for the role of PTHrP in prostate cancer metastasis to bone and its subsequent regulation of bone remodeling. Study of the biological activities of the PSA-generated PTHrP peptides identified in this study merits further investigation.

Presence and enzymatic activity of prostate-specific antigen in archival prostate cancer samples

Patients with advanced prostate cancer frequently have a poor prognosis as a result of metastasis. The serum prostate-specific antigen (PSA) test is widely used for the diagnosis of prostate cancer. The enzymatic activity of PSA may be involved in the invasion of prostate cancer. We set out to determine the prevalent form of PSA in human prostate adenocarcinoma samples by ELISA and Western blot analysis and its enzymatic activity using a synthetic substrate S-2586 and fibronectin. Our results show that in serum from prostate cancer patients and in tumour homogenates, the prevalent form was PSA bound to alpha1-antichymotrypsin. All homogenates showed enzymatic activity towards a synthetic PSA substrate, whereas only five samples showed activity at 28 kDa towards fibronectin as determined by enzymography, which is most likely due to active PSA. Human prostate cancer LNCaP cells produced largely inactive PSA. In comparison, 22Rv1 cells produced 29-fold less PSA, but with high specific activity. Similarly, our results from the human prostate cancer tissue samples also show that free PSA appears to exist in diverse forms of very different specific activity. Since PSA, as a serine protease, may be involved in the invasion of prostate cancer, our results suggest that prostate cancers have potentially diverse invasive capacity due to differences in specific enzymatic activity of PSA.

Enzymatic activity of prostate‐specific antigen and its reactions with extracellular serine proteinase inhibitors

Prostate-specific antigen (PSA) is one of the three most abundant prostatic-secreted proteins in human semen. It is a serine proteinase that, in its primary structure, manifests extensive similarities with that of the Arg-restricted glandular kallikrein-like proteinases. When isolated from semen by the addition of chromatography on aprotinin-Sepharose to a previously described procedure, PSA displayed chymotrypsin-like activity and cleaved semenogelin and the semenogelin-related proteins in a rapid and characteristic pattern, but had no trypsin-like activity. About one third of the purified protein was found to be enzymatically inactive, due to cleavage carboxy-terminal of Lys145. Active PSA formed SDS-stable complexes with alpha 1-antichymotrypsin, alpha 2-macroglobulin-analogue pregnancy zone protein. PSA formed inhibitory complexes with alpha 1-antichymotrypsin at a molar ratio of 1:1, a reaction in which PSA cleaved the inhibitor in a position identical to that reported from the reaction between chymotrypsin and alpha 1-antichymotrypsin. The formation of stable complexes between PSA and alpha 1-antichymotrypsin occurred at a much slower rate than that between chymotrypsin and alpha 1-antichymotrypsin, and at a similar or slightly slower rate than that between PSA and alpha 2-macroglobulin. When added to normal blood plasma in vitro, active PSA formed stable complexes both with alpha 2-macroglobulin and alpha 1-antichymotrypsin. This complex formation may be a crucial determinant of the turnover of active PSA in intercellular fluid or blood plasma in vivo.