Prostatic Acid Phosphatase Research Articles

Therapeutic Potential of 5α-Reductase Inhibitor 17-oxo-17a-aza-D-homo-5-androsten-3β-yl phenoxyacetate (17a-aza Steroid) in the Management of Prostate Cancer

Background: The present study highlights the comparative therapeutic efficacy of the newly synthesized drug 17-Oximino-5-androsten-3β-yl 4-aminobenzoate (17a-aza steroid) and finasteride for the treatment of experimentally induced prostate carcinogenesis in rats. Considering the inhibitory activity of the oximes to 5α-reductase and the role of the ester group in augmenting the anti- androgenic property, we decided to synthesize a compound with a lactam moiety in ring D and esters at 3β position of androsterone nucleus as a more potent 5α-reductase inhibitor. Methods: In this study, 30 rats were divided into six groups, viz. Group I as normal controls, Group II was treated with carcinogen N-methyl-N-nitrosourea (MNU) and hormone testosterone propionate (TP) for inducing prostate cancer, Group III rats were treated with finasteride (10mg/Kg of body weight for 8 weeks through gavage), Group IV was treated with 17a-aza steroid (10mg/Kg of body weight for 8 weeks through gavage), whereas, Group V and Group VI were given finasteride and 17a-aza steroid, respectively, with similar doses as in Group III and IV after pretreatment with a carcinogen (MNU) and hormone (TP). Results: The induction of carcinogenesis by treating animals with MNU+TP revealed a significant reduction in weight loss as compared to the normal control group, which, however, increased following 17a-aza steroid treatment. Furthermore, the study witnessed a significant improvement in prostatic biomarker, viz prostatic acid phosphatase (PAcP) in serum following finasteride and 17a-aza steroid treatment to MNU+TP treated rats. Histological investigation of prostate tissue sections of 17aaza steroid-treated prostate cancer rats showed milder hyperplastic glandular epithelium and exhibited signs of recovery as compared to carcinogen-treated rats. Conclusion: The present findings are preliminary and suggest that the 17a-aza steroid has therapeutic efficacy for the management of experimentally induced prostate cancer, as evidenced by changes in prostate weight, PAcP levels, and histology. In comparison to finasteride, further exploration is needed to assertively conclude its comparative therapeutic efficacy.

7323 Prostatic Acid Phosphatase Is Negatively Regulated by Androgens and Persists in Castration-resistant Prostate Cancer

Abstract Disclosure: A. Kirschenbaum: None. P. Cheung: None. S. Yao: None. P. Cheung: None. Background: Prostate cancer (PCa) is the most common cancer and second leading cause of cancer death in American men. Most patients with metastatic PCa respond initially to androgen deprivation therapy (ADT) but the majority will progress to lethal, metastatic, castrate-resistant PCa (mCRPC). The identification of markers of mCRPC that are not regulated or negatively regulated by androgens can serve as markers and therapeutic targets in mCRPC. We hypothesized that prostatic acid phosphatase (PAP), a protein phosphatase and 5’ectonucleotidase that is expressed in both the cytoplasm and transmembrane (TM-PAP) of PCa cells, is negatively regulated by androgen signaling and can serve as a therapeutic target. Methods: PAP protein expression was assessed by immunohistochemistry (IHC) in PCa cell line spheroids (VCaP, 22Rv1, LNCaP, C42B) +/- androgen addition as well as in LNCaP s.c. xenografts +/- castration. Protein expression of PAP was assessed with western blotting in PCa cell lines after treatment with androgens (DHT) and anti-androgen (enzalutamide). Results: In spheroids derived from PCa cell lines, there is some IHC expression of PAP with the strongest staining in VCaP and 22Rv1 spheroids. DHT decreased PAP IHC expression in all spheroids. LNCaP and VCaP s.c. xenografts demonstrated that castration decreased tumor growth rates but that in castrate as well as control tumors PAP expression persisted. All forms of PAP including TM-PAP are expressed in VCaP cell line and were negatively regulated by androgen addition. Conclusions: The identification of markers of mCRPC that are not regulated or are negatively regulated by androgen could help identify recalcitrant tumor cells and mount targeted delivery of therapeutic agents. In several androgen sensitive PCa cell lines, DHT addition decreased PAP expression in spheroids. In vivo, PAP expression persists after castration in spite of decreases in tumor growth rates. Finally, DHT decreases and enzalutamide restores PAP expression in vitro. These data demonstrate that PAP is a more progenitor cell marker in PCa that persists after castration and may be targeted in castration-resistant disease. Presentation: 6/3/2024

7323 Prostatic Acid Phosphatase Is Negatively Regulated By Androgens And Persists In Castration-Resistant Prostate Cancer

7323 Prostatic Acid Phosphatase Is Negatively Regulated By Androgens And Persists In Castration-Resistant Prostate Cancer

Irreversible electroporation of localised prostate cancerdownregulates immune suppression andinduces systemic anti-tumour T-cell activation - IRE-IMMUNO study.

To prospectively compare systemic anti-tumour immune responses induced by irreversible electroporation (IRE) and robot-assisted radical prostatectomy (RARP) in patients with localised intermediate-risk prostate cancer (PCa). Between February 2021 and June 2022, before and after treatment (at 5, 14 and 30 days) peripheral blood samples of 30 patients with localised PCa were prospectively collected. Patient inclusion criteria were: International Society of Urological Pathologists Grade 2-3, clinical cancer stage ≤T2c, prostate-specific antigen level <20 ng/mL). Patients were treated with IRE (n = 20) or RARP (n = 10). Frequency and activation status of lymphocytic and myeloid immune cell subsets were determined using flow cytometry. PCa-specific T-cell responses to prostatic acid phosphatase(PSAP) and cancer testis antigen (New York oesophageal squamous cell carcinoma 1 [NY-ESO-1]) were determined by interferon-γ enzyme-linked immunospot assay (ELISpot). Repeated-measures analysis of variance and two-sided Student's t-tests were used to compare immune responses over time and between treatment cohorts. Patient and tumour characteristics were similar between the cohorts except for age (median 68 years [IRE] and 62 years [RARP], P = 0.01). IRE induced depletion of systemic regulatory Tcells (P = 0.0001) and a simultaneous increase in activated cytotoxic T-lymphocyte antigen 4 (CTLA-4)+ cluster of differentiation (CD)4+ (P < 0.001) and CD8+ (P = 0.032) Tcells, consistent with reduction of systemic immune suppression allowing for effector T-cell activation, peaking 14 days after IRE. Effects were positively correlated with tumour volume/ablation size. Accordingly, IRE induced expansion of PSAP and/or NY-ESO-1 specific T-cell responses in four of the eight immune competent patients. Temporarily increased activated myeloid derived suppressor cell frequencies (P = 0.047) were consistent with transient immunosuppression after RARP. Irreversible electroporation induces a PCa-specific systemic immune response in patients with localised PCa, aiding conversion of the tumour microenvironment into a more immune permissive state. Therapeutic efficacy might be further enhanced by combination with CTLA-4 checkpoint inhibition, potentially opening up a new synergistic treatment paradigm for high-risk localised or (oligo)metastatic disease.

Race-Related Differences in Sipuleucel-T Response among Men with Metastatic Castrate-Resistant Prostate Cancer.

Our novel findings of higher expression of co-stimulatory receptor ICOS on CD4+ and CD8+ T cells in African American patients with metastatic castrate-resistant prostate cancer (mCRPC) prior and post-sipuleucel-T suggest activation of CD4+ and CD8+ T cells. The data indicate that racial differences observed in these and other immune correlates before and after sipuleucel-T warrant additional investigation to further our understanding of the immune system in African American men and other men with mCRPC.

Distribution of prostatic markers in glands of the female urethra and anterior vaginal wall-a rapid autopsy study.

There are varying reports of immunohistochemically detected prostatic marker protein distribution in glands associated with the female urethra that may be related to tissue integrity at the time of fixation. In this study we used tissue derived from rapid autopsies of female patients to determine the distribution of glandular structures expressing prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) along the female urethra and in surrounding tissues, including the anterior vaginal wall (AVW). Tissue blocks from 7 donors that contained the entire urethra and adjacent AVW were analyzed. These tissue samples were fixed within 4-12hours of death and divided into 5-mm transverse slices that were paraffin embedded. Sections cut from each slice were immunolabeled for PSA or PSAP and a neighboring section was stained with hematoxylin and eosin. The sections were reviewed by light microscopy and analyzed using QuPath software. In tissue from all donors, glandular structures expressing PSA and/or PSAP were located within the wall of the urethra and were present along its whole length. In the proximal half of the urethra from all donors, small glands expressing PSAP, but not PSA, were observed adjacent to the and emptying into the lumen. In the distal half of the urethra from 5 of the 7 donors, tubuloacinar structures lined by a glandular epithelium expressed both PSA and PSAP. In addition, columnar cells at the surface of structures with a multilayered transitional epithelium in the distal half of the urethra from all donors expressed PSAP. No glands expressing PSA or PSAP were found in tissues surrounding the urethra, including the AVW. Greater understanding of the distribution of urethral glands expressing prostatic proteins in female patients is important because these glands are reported to contribute to the female sexual response and to urethral pathology, including urethral cysts, diverticula, and adenocarcinoma. Strengths of the present study include the use of rapid autopsy to minimize protein degradation and autolysis, and the preparation of large tissue sections to demonstrate precise anatomical relations within all the tissues surrounding the urethral lumen. Limitations include the sample size and that all donors had advanced malignancy and had undergone previous therapy which may have had unknown tissue effects. Proximal and distal glands expressing prostate-specific proteins were observed in tissue from all donors, and these glands were located only within the wall of the urethra.

CD8+ T cell targeting of tumor antigens presented by HLA-E.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8+ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8+ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E+ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8+ T cell-based immunotherapies.

Evaluation of t-PSA, f-PSA and prostatic acid phosphatase in two immunoassays from Snibe and Abbott

Evaluation of t-PSA, f-PSA and prostatic acid phosphatase in two immunoassays from Snibe and Abbott

Androgenic Properties and Subchronic Toxicity of the Aqueous Extract of Pycnanthus angolensis (Welw.) Warb. Wood (Myristicaceae)

Aims: Pycnanthus angolensis (Welw.) Warb. also known as “Etengué” in the Baka language, is a medicinal plant used by the Baka Pygmies of Cameroon to treat erectile dysfunction. This study aimed to evaluate the androgenic activity and subchronic toxicity of the aqueous extract of Pycnanthus angolensis wood.&#x0D; Study Design: Experimental design.&#x0D; Place and Duration of Study: Laboratory of Biochemistry, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, between November 2022 and October 2023.&#x0D; Methodology: Twenty-five male Wistar strain rats were divided into 5 groups of 5 rats each including a positive control group that received testosterone enanthate (5 mg/kg) intramuscularly once a week; a normal control group that received distilled water (10 ml/kg) and three test groups which received 134 and 267 and 533 mg/kg of the aqueous extract of Pycnanthus angolensis daily per os. On the 29th day, the animals were killed, and several biochemical parameters were assessed for androgenic properties.&#x0D; Results: At a dose of 134 mg/kg, the extract significantly increased (p&lt;0.05) the levels of testicular cholesterol, testosterone and serum prostatic acid phosphatase activity compared to those of the normal control. A toxicity study showed that at doses of 134 and 267 mg/kg, the extract did not induce any significant variation (p&gt;0.05) in the creatinine level, but a significant reduction (p&lt;0.05) in of aspartate and alanine aminotransferase activities was recorded. Histopathology revealed destruction of testicular spermatozoa at a dose of 533 mg/kg compared to that of the controls.&#x0D; Conclusion: These results showed that at a dose of 134 mg/kg, the aqueous extract of Pycnanthus angolensis (Welw.) Warb. wood is capable of stimulating the production of androgens and is tolerated by the rat's body. Our results justified the traditional use of this plant for the treatment of male infertility and erectile dysfunction.

Abstract 3417: Dissecting tumor microenvironment to predict clinical outcome in prostate cancer

Abstract Prostate cancer is one of the major causes of cancer death in males, and its prevalence is particularly high in developed countries such as the United States and Western Europe. However, death rates in developing and underdeveloped countries are greater, presumably, due to a lack of routine clinical check-ups, poor health awareness, inadequate screening resources, and clinical intervention. Much progress has already been made using cutting-edge technologies like whole genome sequencing, RNAseq, phosphoproteomics, and targeted transcriptomic Nanostring profiling, etc. However, understanding of the prostate tumor microenvironment as a whole in clinical samples remains very limited. Here we use the latest genomic, proteomics, and metabolomic tools not only to understand the biology of prostate cancers but also their correlation with patient demography and clinical outcome. We recruited 80 patients from both tertiary research hospitals and clinical centers in the eastern region of India. All of their demographic information is collected along with their clinical outcome. Pathological evaluation revealed only 50 patients with positive Prostate Carcinoma. We performed a detailed analysis using experimental data (from genomic, and proteomic) in a cohort of patients' routine biomarkers (PSA and PSMA), histopathology, and clinical outcome. Our data demonstrate no linear correlation of any of the individual markers or parameters like AR status, PSA, Prostate Specific Acid Phosphatase, Prostate-Specific Membrane Antigen (PSMA), Alpha-methylacyl-CoA racemase (AMACR), Mesothelin, PD-L1, and other potential genomic and proteomic alterations or any histopathological readouts. However, a clear understanding of the relationship between these multiparameter data, disease pathology, and clinical outcomes in a group of patients has been established. The heterogeneity of these individual data for tumor microenvironments will lead us to identify novel biomarkers for the clinical response of targeted drugs, accelerating personalized treatment strategies. Citation Format: Aritri Bhattacharjee, Manjusha Biswas, Amjad Husain, Asim Kr Das, Sourav Karmakar Karmakar, Tapan Mondal, Pinaki Roy, Manas Kr Mondal, Badal Kumar Sahu, Nabendu Murmu, Partho Nath, Chandra C. Ghosh, Pradip Kumar Majumder. Dissecting tumor microenvironment to predict clinical outcome in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3417.

Catechol-Siderophore Mimics Convey Nucleic Acid Therapeutics into Bacteria.

Antibacterial resistance is a major threat for human health. There is a need for new antibacterials to stay ahead of constantly-evolving resistant bacteria. Nucleic acid therapeutics hold promise as powerful antibiotics, but issues with their delivery hamper their applicability. Here, we exploit the siderophore-mediated iron uptake pathway to efficiently transport antisense oligomers into bacteria. We appended a synthetic siderophore to antisense oligomers targeting the essential acpP gene in Escherichia coli. Siderophore-conjugated PNA and PMO antisense oligomers displayed potent antibacterial properties. Conjugates bearing a minimal siderophore consisting of a mono-catechol group showed equally effective. Targeting the lacZ transcript resulted in dose-dependent decreased β-galactosidase production, demonstrating selective protein downregulation. Applying this concept to Acinetobacter baumannii also showed concentration-dependent growth inhibition. Whole-genome sequencing of resistant mutants and competition experiments with the endogenous siderophore verified selective uptake through the siderophore-mediated iron uptake pathway. Lastly, no toxicity towards mammalian cells was found. Collectively, we demonstrate for the first time that large nucleic acid therapeutics can be efficiently transported into bacteria using synthetic siderophore mimics.

Catechol‐Siderophore Mimics Convey Nucleic Acid Therapeutics into Bacteria

AbstractAntibacterial resistance is a major threat for human health. There is a need for new antibacterials to stay ahead of constantly‐evolving resistant bacteria. Nucleic acid therapeutics hold promise as powerful antibiotics, but issues with their delivery hamper their applicability. Here, we exploit the siderophore‐mediated iron uptake pathway to efficiently transport antisense oligomers into bacteria. We appended a synthetic siderophore to antisense oligomers targeting the essential acpP gene in Escherichia coli. Siderophore‐conjugated PNA and PMO antisense oligomers displayed potent antibacterial properties. Conjugates bearing a minimal siderophore consisting of a mono‐catechol group showed equally effective. Targeting the lacZ transcript resulted in dose‐dependent decreased β‐galactosidase production, demonstrating selective protein downregulation. Applying this concept to Acinetobacter baumannii also showed concentration‐dependent growth inhibition. Whole‐genome sequencing of resistant mutants and competition experiments with the endogenous siderophore verified selective uptake through the siderophore‐mediated iron uptake pathway. Lastly, no toxicity towards mammalian cells was found. Collectively, we demonstrate for the first time that large nucleic acid therapeutics can be efficiently transported into bacteria using synthetic siderophore mimics.

CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner

BackgroundClinical and experimental studies have shown that the myocardial inflammatory response during pathological events varies between males and females. However, the cellular and molecular mechanisms of these sex differences remain elusive. CD73/adenosine axis has been linked to anti-inflammatory responses, but its sex-specific cardioprotective role is unclear. The present study aimed to investigate whether the CD73/adenosine axis elicits sex-dependent cardioprotection during metabolic changes and myocarditis induced by hypobaric hypoxia.MethodsFor 7 days, male and female mice received daily injections of the CD73 inhibitor adenosine 5′- (α, β-methylene) diphosphate (APCP) 10 mg/kg/day while they were kept under normobaric normoxic and hypobaric hypoxic conditions. We evaluated the effects of hypobaric hypoxia on the CD73/adenosine axis, myocardial hypertrophy, and cardiac electrical activity and function. In addition, metabolic homeostasis and immunoregulation were investigated to clarify the sex-dependent cardioprotection of the CD73/adenosine axis.ResultsHypobaric hypoxia-induced cardiac dysfunction and adverse remodeling were more pronounced in male mice. Also, male mice had hyperactivity of the CD73/adenosine axis, which aggravated myocarditis and metabolic shift compared to female mice. In addition, CD73 inhibition triggered prostatic acid phosphatase ectonucleotidase enzymatic activity to sustain adenosine overproduction in male mice but not in female mice. Moreover, dual inhibition prostatic acid phosphatase and CD73 enzymatic activities in male mice moderated adenosine content, alleviating glycolytic shift and proinflammatory response.ConclusionThe CD73/adenosine axis confers a sex-dependent cardioprotection. In addition, extracellular adenosine production in the hearts of male mice is influenced by prostatic acid phosphatase and tissue nonspecific alkaline phosphatase.

Purification, identification and Cryo-EM structure of prostatic acid phosphatase in human semen

Prostatic acid phosphatase (PAP) is a glycoprotein that plays a crucial role in the hydrolysis of phosphate ester present in prostatic exudates. It is a well-established indicator for prostate cancer due to its elevated serum levels in disease progression. Despite its abundance in semen, PAP's influence on male fertility has not been extensively studied. In our study, we report a significantly optimized method for purifying human endogenous PAP, achieving remarkably high efficiency and active protein recovery rate. This achievement allowed us to better analyze and understand the PAP protein. We determined the cryo-electron microscopic (Cryo-EM) structure of prostatic acid phosphatase in its physiological state for the first time. Our structural and gel filtration analysis confirmed the formation of a tight homodimer structure of human PAP. This functional homodimer displayed an elongated conformation in the cryo-EM structure compared to the previously reported crystal structure. Additionally, there was a notable 5-degree rotation in the angle between the α domain and α/β domain of each monomer. Through structural analysis, we revealed three potential glycosylation sites: Asn94, Asn220, and Asn333. These sites contained varying numbers and forms of glycosyl units, suggesting sugar moieties influence PAP function. Furthermore, we found that the active sites of PAP, His44 and Asp290, are located between the two protein domains. Overall, our study not only provide an optimized approach for PAP purification, but also offer crucial insights into its structural characteristics. These findings lay the groundwork for further investigations into the physiological function and potential therapeutic applications of this important protein.

Tumor epitope spreading by a novel multivalent therapeutic cellular vaccine targeting cancer antigens to invariant NKT-triggered dendritic cells in situ.

Cancer is categorized into two types based on the microenvironment: cold and hot tumors. The former is challenging to stimulate through immunity. The immunogenicity of cancer relies on the quality and quantity of cancer antigens, whether recognized by T cells or not. Successful cancer immunotherapy hinges on the cancer cell type, antigenicity and subsequent immune reactions. The T cell response is particularly crucial for secondary epitope spreading, although the factors affecting these mechanisms remain unknown. Prostate cancer often becomes resistant to standard therapy despite identifying several antigens, placing it among immunologically cold tumors. We aim to leverage prostate cancer antigens to investigate the potential induction of epitope spreading in cold tumors. This study specifically focuses on identifying factors involved in secondary epitope spreading based on artificial adjuvant vector cell (aAVC) therapy, a method established as invariant natural killer T (iNKT) -licensed DC therapy. We concentrated on three prostate cancer antigens (prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP)). By introducing allogeneic cells with the antigen and murine CD1d mRNA, followed by α-galactosylceramide (α-GalCer) loading, we generated five types of aAVCs, i.e, monovalent, divalent and trivalent antigen-expressing aAVCs and four types of prostate antigen-expressing cold tumors. We evaluated iNKT activation and antigen-specific CD8+ T cell responses against tumor cells prompted by the aAVCs. Our study revealed that monovalent aAVCs, expressing a single prostate antigen, primed T cells for primary tumor antigens and also induced T cells targeting additional tumor antigens by triggering a tumor antigen-spreading response. When we investigated the immune response by trivalent aAVC (aAVC-PROS), aAVC-PROS therapy elicited multiple antigen-specific CD8+ T cells simultaneously. These CD8+ T cells exhibited both preventive and therapeutic effects against tumor progression. The findings from this study highlight the promising role of tumor antigen-expressing aAVCs, in inducing efficient epitope spreading and generating robust immune responses against cancer. Our results also propose that multivalent antigen-expressing aAVCs present a promising therapeutic option and could be a more comprehensive therapy for treating cold tumors like prostate cancer.

Aberrant Mannosylated and Highly Fucosylated Glycoepitopes of Prostatic Acid Phosphatase as Potential Ligands for Dendritic-Cell Specific ICAM-Grabbing Nonintegrin (DC-SIGN) in Human Seminal Plasma-A Step towards Explaining Idiopathic Infertility.

Semen prostatic acid phosphatase (PAP) has been proposed as an endogenous ligand for dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), which plays a critical immuno-modulating role in maintaining homeostasis in the female reproductive tracts. In the current study, we assumed that semen PAP bears a set of fucosylated and mannosylated glycans, which may mediate the efficient binding of PAP to DC-SIGN. To investigate this hypothesis, we developed ELISA assays using Galanthus nivalis and Lotus tetragonolobus lectins capable of binding mannose-containing glycans or LewisX and LewisY motifs, respectively. In our assay with Galanthus nivalis, we detected that the relative reactivity of PAP mannose-presenting glycans in the normozoospermic idiopathic group was significantly higher than in the asthenozoospermic, oligozoospermic and oligoasthenozoospermic groups. Simultaneously, we observed slight differences in the relative reactivities of PAP glycans with Lotus tetragonolobus lectin among groups of patients with abnormal semen parameters. Subsequently, we examined whether DC-SIGN interacts with seminal plasma PAP glycans, and we detected a significantly higher relative reactivity in the normozoospermic group compared to the oligozoospermic group. Finally, we concluded that the significantly aberrant abundance of mannosylated functional groups of PAP among patients with semen disorders can suggest that PAP may thereby be engaged in modulating the immune response and promoting a tolerogenic response to male antigens in the female reproductive system.

Elusive physiological role of prostatic acid phosphatase (PAP): generation of choline for sperm motility via auto-and paracrine cholinergic signaling.

Prostatic acid phosphatase (PAP) exists as two splice variants, secreted PAP and transmembrane PAP, the latter of which is implicated in antinociceptive signaling in dorsal root ganglia. However, PAP is predominantly expressed in the prostate gland and the physiological role of seminal PAP, first identified in 1938, is largely unknown. Here, the author proposes that PAP, following ejaculation, functions to hydrolyze phosphocholine (PC) in seminal fluid and generate choline, which is imported by sperm via a choline transporter and converted to acetylcholine (ACh) by choline acetyltransferase. Auto- and paracrine cholinergic signaling, or choline directly, may subsequently stimulate sperm motility via α7 nicotinic ACh receptors (nAChRs) and contractility of the female reproductive tract through muscarinic ACh receptors (mAChRs). Consistent with a role of PAP in cholinergic signaling, 1) seminal vesicles secrete PC, 2) the prostate gland secretes PAP, 3) PAP specifically catalyzes the hydrolysis of PC into inorganic phosphate and choline, 4) seminal choline levels increase post-ejaculation, 5) pharmacological inhibition of choline acetyltransferase inhibits sperm motility, 6) inhibition or genetic deletion of α7 nAChRs impairs sperm motility, and 7) mAChRs are expressed in the uterus and oviduct (fallopian tube). Notably, PAP does not degrade glycerophosphocholine (GPC), the predominant choline source in the semen of rats and other mammals. Instead, uterine GPC phosphodiesterases may liberate choline from seminal GPC. In summary, the author deduces that PAP in humans, and uterine GPC phosphodiesterases in other mammals, function to generate choline for sperm cholinergic signaling, which promotes sperm motility and possibly contractility of the female reproductive tract.

Sequence-based identification of amyloidogenic β-hairpins reveals a prostatic acid phosphatase fragment promoting semen amyloid formation

β-Structure-rich amyloid fibrils are hallmarks of several diseases, including Alzheimer’s (AD), Parkinson’s (PD), and type 2 diabetes (T2D). While amyloid fibrils typically consist of parallel β-sheets, the anti-parallel β-hairpin is a structural motif accessible to amyloidogenic proteins in their monomeric and oligomeric states. Here, to investigate implications of β-hairpins in amyloid formation, potential β-hairpin-forming amyloidogenic segments in the human proteome were predicted based on sequence similarity with β-hairpins previously observed in Aβ, α-synuclein, and islet amyloid polypeptide, amyloidogenic proteins associated with AD, PD, and T2D, respectively. These three β-hairpins, established upon binding to the engineered binding protein β-wrapin AS10, are characterized by proximity of two sequence segments rich in hydrophobic and aromatic amino acids, with high β-aggregation scores according to the TANGO algorithm. Using these criteria, 2505 potential β-hairpin-forming amyloidogenic segments in 2098 human proteins were identified. Characterization of a test set of eight protein segments showed that seven assembled into Thioflavin T-positive aggregates and four formed β-hairpins in complex with AS10 according to NMR. One of those is a segment of prostatic acid phosphatase (PAP) comprising amino acids 185–208. PAP is naturally cleaved into fragments, including PAP(248−286) which forms functional amyloid in semen. We find that PAP(185−208) strongly decreases the protein concentrations required for fibril formation of PAP(248−286) and of another semen amyloid peptide, SEM1(86−107), indicating that it promotes nucleation of semen amyloids. In conclusion, β-hairpin-forming amyloidogenic protein segments could be identified in the human proteome with potential roles in functional or disease-related amyloid formation.

In-Depth Glycoproteomic Assay of Urinary Prostatic Acid Phosphatase.

Prostate-specific antigen (PSA) is a well-known clinical biomarker in prostate cancer (PCa) diagnosis, but a better test is still needed, as the serum-level-based PSA quantification exhibits limited specificity and comes with poor predictive value. Prior to PSA, prostatic acid phosphatase (PAP) was used, but it was replaced by PSA because PSA improved the early detection of PCa. Upon revisiting PAP and its glycosylation specifically, it appears to be a promising new biomarker candidate. Namely, previous studies have indicated that PAP glycoforms differ between PCa and non-PCa individuals. However, an in-depth characterization of PAP glycosylation is still lacking. In this study, we established an in-depth glycoproteomic assay for urinary PAP by characterizing both the micro- and macroheterogeneity of the PAP glycoprofile. For this purpose, PAP samples were analyzed by capillary electrophoresis coupled to mass spectrometry after affinity purification from urine and proteolytic digestion. The developed urinary PAP assay was applied on a pooled DRE (digital rectal examination) urine sample from nine individuals. Three glycosylation sites were characterized, namely N94, N220, and N333, via N-glycopeptide analysis. Taking sialic acid linkage isomers into account, a total of 63, 27, and 4 N-glycan structures were identified, respectively. The presented PAP glycoproteomic assay will enable the determination of potential glycomic biomarkers for the early detection and prognosis of PCa in cohort studies.

Activation of Dendritic Cells Isolated from the Blood of Patients with Prostate Cancer by Ex Vivo Fluid Shear Stress Stimulation.

Prostate cancer is one of the most common cancers among men in the United States and a leading cause of cancer-related death in men. Treatment options for patients with advanced prostate cancer include hormone therapies, chemotherapies, radioligand therapies, and immunotherapies. Provenge (sipuleucel-T) is an autologous cancer-vaccine-based immunotherapy approved for men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Administration of sipuleucel-T involves leukapheresis of patient blood to isolate antigen-presenting cells (APCs), including dendritic cells (DCs), and subsequent incubation of isolated APCs with both an antigen, prostatic acid phosphatase (PAP), and granulocyte macrophage-colony stimulating factor (GM-CSF) before their infusion back into the patient. Although sipuleucel-T has been shown to improve overall survival, other meaningful outcomes, such as prostate-specific antigen (PSA) levels and radiographic response, are inconsistent. This lack of robust response may be due to limited ex vivo activation of DCs using current protocols. Earlier studies have shown that many cell types can be activated ex vivo by external forces such as fluid shear stress (FSS). We hypothesize that novel fluid shear stress technologies and methods can be used to improve ex vivo efficacy of prostate cancer DC activation in prostate cancer. Herein, we report a new protocol for activating DCs from patients with prostate cancer using ex vivo fluid shear stress. Ultimately, the goal of these studies is to improve DC activation to expand the efficacy of therapies such as sipuleucel-T. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample collection and DC isolation Basic Protocol 2: Determination and application of fluid shear stress Basic Protocol 3: Flow cytometry analysis of DCs after FSS stimulation.