Partialsynthesen von Cardenoliden und Cardenolid-Analogen. VII. Synthese A/B-cis- und C/D-cis-verknüpfter Steroidmono- und -bis(α-methylen-γ-butyrolactone)

Partial Syntheses of Cardenolides and Cardenolide Analogues. VII. Synthesis of A/B-cis- and C/D-cis-linked Steroidal Mono- and Bis (α-methylene-γ-butyrolactones) The synthesis of A/B-cis- and C/D-cis-linked steroidal mono- and bis(α-methylene-γ-butyrolactones) 10–13 , 18 and 24 by Reformatsky-type reaction of 3β-acetoxy-14-hydroxy-5β, 14β-androstan-17-one( 6 ), 14-hydroxy-5β, 14β-androstane-3,17-dione ( 8 ), 3β-acetoxy-14-hydroxy-5β,14β-pregnan-20-one ( 17 ) and 14-hydroxy-5β,14β-pregnane-3,20-dione ( 23 ), respectively, with ethyl α-(bromomethyl)acrylate is described. All steroidal lactones exhibit strong inhibitory activity on the proliferation of Ehrlich ascites carcinoma cells in suspension culture. For inhibitory activity the lactone moiety at C-17 of the steroid molecule is more important than the lactone moiety at C–3 and C–20, respectively.     

Synthesis of deuterium labeled cholesterol and steroids and their use for metabolic studies

A simple method is described for the preparation of [6,7,7⁻²H₃] sterols and steroids. The synthesis starts with a Δ⁵-sterol or steroid and involves preparation of the 6-oxo-3α,5α-cyclosteroid, base exchange in the presence of deuterium oxide to introduce two deuteriums at the C-7 position and sodium borodeuteride reduction of the 6-oxo group to introduce the third deuterium atom at C-6. Rearrangement of the [6,7,7⁻²H₃]6α-hydroxy-3α,5α-cyclosteroid then gives the desired [6,7,7^-2H₃]-Δ⁵ sterol or steroid. [6,7,7⁻²H₃]Cholesterol, [6,7,7⁻²H₃]pregnenolone and [6,7,7⁻²H₃]3β-hydroxyandrost-5-en-17-one were synthesized in this fashion and [6,7,7⁻²H₃]progesterone was prepared from the [6,7,7⁻²H₃]pregnenolone. Three examples of the use of these deuchromatography-mass spectrometry. The chrysophyte alga,Ochromonas malhamensis, was shown to be capable of introducing an extra methyl or ethyl group at C-24 of the side chain of [6,7,7⁻²H₃]cholesterol to yield brassicasterol and poriferasterol, respectively. The ovary of the echinoderm,Asterias rubens, was demonstrated to metabolize [6,7,7⁻²H₃]progesterone to yield mainly the 5α-isomers of pregnane-3,20-dione and 3β-hydroxypregnan-20-one. However, the 5β-isomers of these compounds were also detected as minor products for the first time as progesterone metabolites in this animal. Isolated oocytes of the frog,Xenopus laevis, produced a number of metabolites of [6,7,7⁻²H₃]progesterone. In this report, two of them were shown to be 17α-hydroxy-pregn-4-en-3,20-dione and 20α-hydroxypregn-4-en-3-one. Presented at the “Sterol Symposium” of the American Oil Chemists' Annual International Conference, New Orleans, LA, May 1981.     

Gonadotropin-Releasing Hormone Receptors in Prostate Cancer: Molecular Aspects and Biological Functions

Pituitary Gonadotropin-Releasing Hormone receptors (GnRH-R) mediate the activity of the hypothalamic decapeptide GnRH, thus playing a key role in the regulation of the reproductive axis. Early-stage prostate cancer (PCa) is dependent on serum androgen levels, and androgen-deprivation therapy (ADT), based on GnRH agonists and antagonists, represents the standard therapeutic approach for PCa patients. Unfortunately, the tumor often progresses towards the more aggressive castration-resistant prostate cancer (CRPC) stage. GnRH receptors are also expressed in CRPC tissues, where their binding to both GnRH agonists and antagonists is associated with significant antiproliferative/proapoptotic, antimetastatic and antiangiogenic effects, mediated by the Gαi/cAMP signaling cascade. GnRH agonists and antagonists are now considered as an effective therapeutic strategy for CRPC patients with many clinical trials demonstrating that the combined use of these drugs with standard therapies (i.e., docetaxel, enzalutamide, abiraterone) significantly improves disease-free survival. In this context, GnRH-based bioconjugates (cytotoxic drugs covalently linked to a GnRH-based decapeptide) have been recently developed. The rationale of this treatment is that the GnRH peptide selectively binds to its receptors, delivering the cytotoxic drug to CRPC cells while sparing nontumor cells. Some of these compounds have already entered clinical trials.     

Androgen Receptor Phosphorylation at Serine 308 and Serine 791 Predicts Enhanced Survival in Castrate Resistant Prostate Cancer Patients

We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR⁹⁴), 308 (pAR³⁰⁸), 650(pAR⁶⁵⁰) and 791 (pAR⁷⁹¹). No correlations with clinical parameters were observed for pAR⁹⁴ or pAR⁶⁵⁰ in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR³⁰⁸ is significantly associated with a longer time to disease specific death (p = 0.011) and high pAR⁷⁹¹ expression significantly associated with a longer time to disease recurrence (p = 0.018) in HNPC tumours and longer time to death from disease recurrence (p = 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p = 0.022) and low proliferating tumours (p = 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer.     

High eEF1A1 Protein Levels Mark Aggressive Prostate Cancers and the In Vitro Targeting of eEF1A1 Reveals the eEF1A1–actin Complex as a New Potential Target for Therapy

Although the eukaryotic elongation factor eEF1A1 plays a role in various tumours, there is little information on its prognosis/therapeutic value in prostate carcinoma. In high-grade and castration-resistant prostate carcinoma (CRPC), the identification of novel therapeutic markers/targets remains a priority. The expression of eEF1A1 protein was determined in formalin-fixed, paraffin-embedded prostate cancer and hyperplasia tissue by IHC. The role of eEF1A1 was investigated in a cellular model using a DNA aptamer (GT75) we previously developed. We used the aggressive CRPC cancer PC-3 and non-tumourigenic PZHPV-7 lines. Cytotoxicity was measured by the MTS assay and eEF1A1 protein levels by in-cell Western assays. The mRNA levels of eEF1A1 were measured by qPCR and ddPCR. Higher expression of eEF1A1 was found in Gleason 7–8 compared with 4–6 tissues (Gleason ≥ 7, 87% versus Gleason ≤ 6, 54%; p = 0.033). Patients with a high expression of eEF1A1 had a worse clinical outcome. In PC-3, but not in PZHPV-7, GT75 decreased cell viability and increased autophagy and cell detachment. In PC-3 cells, but not in PZHPV-7, GT75 mainly co-localised with the fraction of eEF1A1 bound to actin. Overexpression of the eEF1A1 protein can identify aggressive forms of prostate cancer. The targeting of eEF1A1 by GT75 impaired cell viability in PC-3 cancer cells but not in PZHPV-7 non-tumourigenic cells, indicating a specific role for the protein in cancer survival. The eEF1A1–actin complexes appear to be critical for the viability of PC-3 cancer cells, suggesting that eEF1A1 may be an attractive target for therapeutic strategies in advanced forms of prostate cancer.     

Breast Cancer: Targeting of Steroid Hormones in Cancerogenesis and Diagnostics

Breast cancer is the most common malignancy in women with high mortality. Sensitive and specific methods for the detection, characterization and quantification of endogenous steroids in body fluids or tissues are needed for the diagnosis, treatment and prognosis of breast cancer and many other diseases. At present, non-invasive diagnostic methods are gaining more and more prominence, which enable a relatively fast and painless way of detecting many diseases. Metabolomics is a promising analytical method, the principle of which is the study and analysis of metabolites in biological material. It represents a comprehensive non-invasive diagnosis, which has a high potential for use in the diagnosis and prognosis of cancers, including breast cancer. This short review focuses on the targeted metabolomics of steroid hormones, which play an important role in the development and classification of breast cancer. The most commonly used diagnostic tool is the chromatographic method with mass spectrometry detection, which can simultaneously determine several steroid hormones and metabolites in one sample. This analytical procedure has a high potential in effective diagnosis of steroidogenesis disorders. Due to the association between steroidogenesis and breast cancer progression, steroid profiling is an important tool, as well as in monitoring disease progression, improving prognosis, and minimizing recurrence.     

Energetic analysis of binding of progesterone and 5{beta}-androstane-3,17-dione to anti-progesterone antibody DB3 using molecular dynamics and free energy calculations

Molecular dynamics simulations and molecular mechanics–Poisson–Boltzmannsurface area (MM-PBSA) free energy calculations were used tostudy the energetics of the binding of progesterone (PRG) and5ß-androstane-3,17-dione (5AD) to anti-PRG antibodyDB3. Although the two steroids bind to DB3 in different orientations,their binding affinities are of the same magnitude, 1 nM forPRG and 8 nM for 5AD. The calculated relative binding free energyof the steroids, 8.8 kJ/mol, is in fair agreement with the experimentalenergy, 5.4 kJ/mol. In addition, computational alanine scanningwas applied to study the role of selected amino acid residuesof the ligand-binding site on the steroid cross-reactivity.The electrostatic and van der Waals components of the totalbinding free energies were found to favour more the bindingof PRG, whereas solvation energies were more favourable forthe binding of 5AD. The differences in the free energy componentsare due to the binding of the A rings of the steroids to differentbinding pockets: PRG is bound to a pocket in which electrostaticantibody–steroid interactions are dominating, whereas5AD is bound to a pocket in which van der Waals and hydrophobicinteractions dominate.     

Effect of AQP9 Expression in Androgen-Independent Prostate Cancer Cell PC3

It is known that aquaporin 9 (AQP9) in the prostate was strictly upregulated by androgen and may represent a novel therapeutic target for several cancers, but whether AQP9 plays a role in the regulation of androgen-independent prostate cancer still remains unclear. In the present study, AQP9 was determined in prostate cancer and adjacent cancer tissues; AQP9-siRNA was applied to silencing AQP9 in androgen-independent prostate cancer cell PC3 cell line. Western blot and flow cytometry analysis were employed to detect changes in related-function of control and AQP9-siRNA groups. The results showed that AQP9 is significantly induced in cancer tissues than that in adjacent cancer tissues. Moreover, knockdown of AQP9 in PC3 androgen-independent prostate cancer cell prostate cancer cells increased inhibition rates of proliferation. In addition, knockdown of AQP9 resulted in a significant decrease in the expression of the Bcl-2 and with a notable increase in the expression of Bax and cleaved caspase 3, indicated that AQP9 knockdown promoted apoptosis in prostate cancer cells. From wound healing assay and matrigel invasion, we suggested that AQP9 expression affects the motility and invasiveness of prostate cancer cells. Moreover, In order to explore the pathway may be involved in AQP9-mediated motility and invasion of prostate cancer cells, the phosphorylation of ERK1/2 was significant suppressed in AQP9 siRNA-transfected cells compared with that in control cells, suggesting that AQP9 is involved in the activation of the ERK pathway in androgen-independent prostate cancer cells.     

The Crosstalk of Long Non-Coding RNA and MicroRNA in Castration-Resistant and Neuroendocrine Prostate Cancer: Their Interaction and Clinical Importance

Prostate cancer is featured by its heterogeneous nature, which indicates a different prognosis. Castration-resistant prostate cancer (CRPC) is a hallmark of the treatment-refractory stage, and the median survival of patients is only within two years. Neuroendocrine prostate cancer (NEPC) is an aggressive variant that arises from de novo presentation of small cell carcinoma or treatment-related transformation with a median survival of 1–2 years from the time of diagnosis. The epigenetic regulators, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), have been proven involved in multiple pathologic mechanisms of CRPC and NEPC. LncRNAs can act as competing endogenous RNAs to sponge miRNAs that would inhibit the expression of their targets. After that, miRNAs interact with the 3’ untranslated region (UTR) of target mRNAs to repress the step of translation. These interactions may modulate gene expression and influence cancer development and progression. Otherwise, epigenetic regulators and genetic mutation also promote neuroendocrine differentiation and cancer stem-like cell formation. This step may induce neuroendocrine prostate cancer development. This review aims to provide an integrated, synthesized overview under current evidence to elucidate the crosstalk of lncRNAs with miRNAs and their influence on castration resistance or neuroendocrine differentiation of prostate cancer. Notably, we also discuss the mechanisms of lncRNA–miRNA interaction in androgen receptor-independent prostate cancer, such as growth factors, oncogenic signaling pathways, cell cycle dysregulation, and cytokines or other transmembrane proteins. Conclusively, we underscore the potential of these communications as potential therapeutic targets in the future.     

5-雄甾烯-3β-醇-17-酮-3-醋酸酯的工艺研究

5-雄甾烯-3β-醇-17-酮-3-醋酸酯是多个甾体激素合成的中间体,如去氢表雄酮、癸酸诺龙、雌二醇、炔诺酮等雄激素、雌激素、孕激素产品的合成。通过对工艺的实验研究,得出最优化的工艺为用盐酸替代三氯氧磷,作为反应的催化剂,且加入的盐酸的量与I的质量比为0．5：1。用甲苯和丙酮的混合溶剂替代受限制使用溶剂苯,与I的质量比为2．5：2．5：1。实现了工艺绿色化,并且收率有明显提高。     

Determination of Intraprostatic and Intratesticular Androgens

Androgens represent the main hormones responsible for maintaining hormonal balance and function in the prostate and testis. As they are involved in prostate and testicular carcinogenesis, more detailed information of their active concentration at the site of action is required. Since the introduction of the term intracrinology as the local formation of active steroid hormones from inactive precursors of the adrenal gland, mainly dehydroepiandrosterone (DHEA) and DHEA-S, it is evident that blood circulating levels of sex steroid hormones need not reflect their actual concentrations in the tissue. Here, we review and critically evaluate available methods for the analysis of human intraprostatic and intratesticular steroid concentrations. Since analytical approaches have much in common in both tissues, we discuss them together. Preanalytical steps, including various techniques for separation of the analytes, are compared, followed by the end-point measurement. Advantages and disadvantages of chromatography-mass spectrometry (LC-MS, GC-MS), immunoanalytical methods (IA), and hybrid (LC-IA) are discussed. Finally, the clinical information value of the determined steroid hormones is evaluated concerning differentiating between patients with cancer or benign hyperplasia and between patients with different degrees of infertility. Adrenal-derived 11-oxygenated androgens are mentioned as perspective prognostic markers for these purposes.     

An Overview of Next-Generation Androgen Receptor-Targeted Therapeutics in Development for the Treatment of Prostate Cancer

Traditional endocrine therapy for prostate cancer (PCa) has been directed at suppression of the androgen receptor (AR) signaling axis since Huggins et al. discovered that diethylstilbestrol (DES; an estrogen) produced chemical castration and PCa tumor regression. Androgen deprivation therapy (ADT) still remains the first-line PCa therapy. Insufficiency of ADT over time leads to castration-resistant PCa (CRPC) in which the AR axis is still active, despite castrate levels of circulating androgens. Despite the approval and use of multiple generations of competitive AR antagonists (antiandrogens), antiandrogen resistance emerges rapidly in CRPC due to several mechanisms, mostly converging in the AR axis. Recent evidence from multiple groups have defined noncompetitive or noncanonical direct binding sites on AR that can be targeted to inhibit the AR axis. This review discusses new developments in the PCa treatment paradigm that includes the next-generation molecules to noncanonical sites, proteolysis targeting chimera (PROTAC), or noncanonical N-terminal domain (NTD)-binding of selective AR degraders (SARDs). A few lead compounds targeting each of these novel noncanonical sites or with SARD activity are discussed. Many of these ligands are still in preclinical development, and a few early clinical leads have emerged, but successful late-stage clinical data are still lacking. The breadth and diversity of targets provide hope that optimized noncanonical inhibitors and/or SARDs will be able to overcome antiandrogen-resistant CRPC.     

Deficiency of NEIL3 Enhances the Chemotherapy Resistance of Prostate Cancer

Acquired treatment resistance is an important cause of death in prostate cancer, and this study aimed to explore the mechanisms of chemotherapy resistance in prostate cancer. We employed castration-resistant prostate cancer (CRPC), neuroendocrine prostate cancer (NEPC), and chemotherapy-resistant prostate cancer datasets to screen for potential target genes. The Cancer Genome Atlas (TCGA) was used to detect the correlation between the target genes and prognosis and clinical characteristics. Nei endonuclease VIII-like 3 (NEIL3) knockdown cell lines were constructed with RNA interference. Prostate cancer cells were treated with enzalutamide for the androgen deprivation therapy (ADT) model, and with docetaxel and cisplatin for the chemotherapy model. Apoptosis and the cell cycle were examined using flow cytometry. RNA sequencing and western blotting were performed in the knockdown Duke University 145 (DU145) cell line to explore the possible mechanisms. The TCGA dataset demonstrated that high NEIL3 was associated with a high T stage and Gleason score, and indicated a possibility of lymph node metastasis, but a good prognosis. The cell therapy models showed that the loss of NEIL3 could promote the chemotherapy resistance (but not ADT resistance) of prostate cancer (PCa). Flow cytometry revealed that the loss of NEIL3 in PCa could inhibit cell apoptosis and cell cycle arrest under cisplatin treatment. RNA sequencing showed that the knockdown of NEIL3 changes the expression of neuroendocrine-related genes. Further western blotting revealed that the loss of NEIL3 could significantly promote the phosphorylation of ATR serine/threonine kinase (ATR) and ATM serine/threonine kinase (ATM) under chemotherapy, thus initiating downstream pathways related to DNA repair. In summary, the loss of NEIL3 promotes chemotherapy resistance in prostate cancer, and NEIL3 may serve as a diagnostic marker for chemotherapy-resistant patients.     

Isolation and Characterization of Urinary Extracellular Vesicles from Healthy Donors and Patients with Castration-Resistant Prostate Cancer

Prostate cancer (PCa) is the most commonly diagnosed malignancy among men in developed countries. The five-year survival rate for men diagnosed with early-stage PCa is approximately 100%, while it is less than 30% for castration-resistant PCa (CRPC). Currently, the detection of prostate-specific antigens as biomarkers for the prognosis of CRPC is criticized because of its low accuracy, high invasiveness, and high false-positive rate. Therefore, it is important to identify new biomarkers for prediction of CRPC progression. Extracellular vesicles (EVs) derived from tumors have been highlighted as potential markers for cancer diagnosis and prognosis. Specifically, urinary EVs directly reflect changes in the pathophysiological conditions of the urogenital system because it is exposed to prostatic secretions. Thus, detecting biomarkers in urinary EVs provides a promising approach for performing an accurate and non-invasive liquid biopsy for CPRC. In this study, we effectively isolated urinary EVs with low protein impurities using size-exclusion chromatography combined with ultrafiltration. After EV isolation and characterization, we evaluated the miRNAs in urinary EVs from healthy donors and patients with CRPC. The results indicated that miRNAs (miR-21-5p, miR-574-3p, and miR-6880-5p) could be used as potential biomarkers for the prognosis of CRPC. This analysis of urinary EVs contributes to the fast and convenient prognosis of diseases, including CRPC, in the clinical setting.     

Hydrogen Peroxide Stimulates Dihydrotestosterone Release in C2C12 Myotubes: A New Perspective for Exercise-Related Muscle Steroidogenesis?

Skeletal muscle is a tissue that has recently been recognized for its ability to produce androgens under physiological conditions. The steroidogenesis process is known to be negatively influenced by reactive oxygen species (ROS) in reproductive Leydig and ovary cells, while their effect on muscle steroidogenesis is still an unexplored field. Muscle cells are continuously exposed to ROS, resulting from both their metabolic activity and the surrounding environment. Interestingly, the regulation of signaling pathways, induced by mild ROS levels, plays an important role in muscle fiber adaptation to exercise, in a process that also elicits a significant modulation in the hormonal response. The aim of the present study was to investigate whether ROS could influence steroidogenesis in skeletal muscle cells by evaluating the release of testosterone (T) and dihydrotestosterone (DHT), as well as the evaluation of the relative expression of the key steroidogenic enzymes 5α-reductase, 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, and aromatase. C2C12 mouse myotubes were exposed to a non-cytotoxic concentration of hydrogen peroxide (H₂O₂), a condition intended to reproduce, in vitro, one of the main stimuli linked to the process of homeostasis and adaptation induced by exercise in skeletal muscle. Moreover, the influence of tadalafil (TAD), a phosphodiesterase 5 inhibitor (PDE5i) originally used to treat erectile dysfunction but often misused among athletes as a “performance-enhancing” drug, was evaluated in a single treatment or in combination with H₂O₂. Our data showed that a mild hydrogen peroxide exposure induced the release of DHT, but not T, and modulated the expression of the enzymes involved in steroidogenesis, while TAD treatment significantly reduced the H₂O₂-induced DHT release. This study adds a new piece of information about the adaptive skeletal muscle cell response to an oxidative environment, revealing that hydrogen peroxide plays an important role in activating muscle steroidogenesis.     

Nonspecific and metabolic interactions between steroid hormones and human plasma lipoproteins

Previous observations demonstrated that steroid hormones associate with plasma lipoproteins. The objective of this study was to estimate the relative importance of lipoproteins as steroid hormone binding agents in comparison to sex hormone binding globulin, corticosteroid binding globulin, and albumin in both normal and hyperlipidemic human plasma. The 16 steroid hormones and related metabolites included in the study were: androstanediol, androstenediol, androstenedione, androsterone, corticosterone, cortisol, dehydroepiandrosterone, deoxycorticosterone, dihydrotestosterone, estradiol, estriol, estrone, 17α-hydroxyprogesterone, pregnenolone, progesterone, and testosterone. The binding activity of these 16 steroid hormones with purified high density lipoprotein (HDL), low density lipoprotein and very low density lipoprotein were separately evaluated by equilibrium dialysis incubations to yield 48 steroid hormone-lipoprotein combinations for further study. In incubations with HDL, six steroid hormones (androstenediol, dehydroepiandrosterone, dihydrotestosterone, estradiol, pregnenolone, and progesterone) were identified as non-equilibrium, apparently due to metabolic conversion of the steroid hormones. The metabolic activity for the three Δ⁵-3β hydroxy steroids and estradiol appears to be fatty acid esterification by lecithin:cholesterol acyltransferase. The computer program TRANSPORT, which was used to evaluate only the nonspecific steroid hormone-lipoprotein association levels in a 16×6 matrix at simultaneous equilibrium, indicated that lipoprotein-bound steroid hormones ranged from 1% for cortisol to 56% for pregnenolone in normal human blood. Simulated projections of the increase in nonspecific steroid hormone association with lipoproteins during hyperlipidemia are also presented. These results demonstrate how lipoproteins are likely to be important in the transport and metabolism of steroid hormones in human plasma.     

PSMA,EpCAM, VEGF and GRPR as Imaging Targets in Locally Recurrent Prostate Cancer after Radiotherapy

In this retrospective pilot study, the expression of the prostate-specific membrane antigen (PSMA), the epithelial cell adhesion molecule (EpCAM), the vascular endothelial growth factor (VEGF) and the gastrin-releasing peptide receptor (GRPR) in locally recurrent prostate cancer after brachytherapy or external beam radiotherapy (EBRT) was investigated, and their adequacy for targeted imaging was analyzed. Prostate cancer specimens were collected of 17 patients who underwent salvage prostatectomy because of locally recurrent prostate cancer after brachytherapy or EBRT. Immunohistochemistry was performed. A pathologist scored the immunoreactivity in prostate cancer and stroma. Staining for PSMA was seen in 100% (17/17), EpCAM in 82.3% (14/17), VEGF in 82.3% (14/17) and GRPR in 100% (17/17) of prostate cancer specimens. Staining for PSMA, EpCAM and VEGF was seen in 0% (0/17) and for GRPR in 100% (17/17) of the specimens’ stromal compartments. In 11.8% (2/17) of cases, the GRPR staining intensity of prostate cancer was higher than stroma, while in 88.2% (15/17), the staining was equal. Based on the absence of stromal staining, PSMA, EpCAM and VEGF show high tumor distinctiveness. Therefore, PSMA, EpCAM and VEGF can be used as targets for the bioimaging of recurrent prostate cancer after EBRT to exclude metastatic disease and/or to plan local salvage therapy.