RP-HPLC-UV Method for Simultaneous Quantification of Second Generation Non-Steroidal Antiandrogens Along with their Active Metabolites in Mice Plasma: Application to a Pharmacokinetic Study.

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 - 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56-13.5 and 1.04-13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

Removal of novel antiandrogens identified in biological effluents of domestic wastewater by activated carbon.

Environmental antiandrogenic (AA) contaminants in effluents from wastewater treatment plants have the potential for negative impacts on wildlife and human health. The aim of our study was to identify chemical contaminants with likely AA activity in the biological effluents and evaluate the removal of these antiandrogens (AAs) during advanced treatment comprising adsorption onto granular activated carbon (GAC). In this study, profiling of AA contaminants in biological effluents and tertiary effluents was conducted using effect-directed analysis (EDA) including high performance liquid chromatography (HPLC) fractionation, a recombinant yeast screen containing androgen receptor (YAS), in combination with mass spectrometry analyses. Analysis of a wastewater secondary effluent from a membrane bioreactor revealed complex profiles of AA activity comprising 14 HPLC fractions and simpler profiles of GAC effluents with only 2 to 4 moderately polar HPLC fractions depending on GAC treatment conditions. Gas chromatography-mass spectrometry and ultra-high performance liquid chromatography-nanospray mass spectrometry analyses of AA fractions in the secondary effluent resulted in detection of over 10 chemical contaminants, which showed inhibition of YAS activity and were potential AAs. The putative AAs included biocides, food additives, flame retardants, pharmaceuticals and industrial contaminants. To our knowledge, it is the first time that the AA properties of N-ethyl-2-isopropyl-5-methylcyclohexanecarboxamide (WS3), cetirizine, and oxcarbazepine are reported. The EDA used in this study was proven to be a powerful tool to identify novel chemical structures with AA activity in the complex aquatic environment. The adsorption process to GAC of all the identified antiandrogens, except WS3 and triclosan, fit well with the pseudo-second order kinetics models. Adsorption to GAC could further remove most of the AAs identified in the biological effluents with high efficiencies.

Soy isoflavones exert beneficial effects on letrozole-induced rat polycystic ovary syndrome (PCOS) model through anti-androgenic mechanism.

CONTEXT: Soy is the main source of phytoestrogens, which has long been used as traditional food. One major subtype of phytoestrogens includes isoflavones and they are scientifically validated for their beneficial actions on many hormone-dependent conditions. OBJECTIVE: The present study examines the effect of soy isoflavones on letrozole-induced polycystic ovary syndrome (PCOS) rat model. MATERIALS AND METHODS: PCOS was induced in Sprague-Dawley rats with of 1 mg/kg letrozole, p.o. once daily for 21 consecutive days. Soy isoflavones (50 and 100 mg/kg) was administered for 14 days after PCOS induction. Physical parameters (body weight, oestrous cycle determination, ovary and uterus weight) metabolic parameters (oral glucose tolerance test, total cholesterol), steroidal hormone profile (testosterone and 17ß-oestradiol), steroidogenic enzymes (3ß-hydroxy steroid dehydrogenase (HSD) and 17ß-HSD), oxidative stress and histopathology of ovary were studied. RESULTS: Soy isoflavones (100 mg/kg) treatment significantly altered the letrozole-induced PCOS symptoms as observed by decreased body weight gain (p < 0.05), percentage diestrous phase (p < 0.001), testosterone (p < 0.001), 3ß-HSD (p < 0.01) and 17ß-HSD (p < 0.001) enzyme activity and oxidative stress. Histological results reveal that soy isoflavones treatment in PCOS rats resulted in well-developed antral follicles and normal granulosa cell layer in rat ovary. DISCUSSION: Treatment with soy isoflavones exerts beneficial effects in PCOS rats (with decreased aromatase activity) which might be due to their ability to decrease testosterone concentration in the peripheral blood. CONCLUSION: Analysis of physical, biochemical and histological evidences shows that soy isoflavones may be beneficial in PCOS.

The role of Cucurbita pepo in the management of patients affected by lower urinary tract symptoms due to benign prostatic hyperplasia: A narrative review.

OBJECTIVE: Phytotherapeutic compounds are largely used in the treatment of lower urinary tract symptoms (LUTS) related to benign prostatic hyperplasia (BPH) due to low side-effect profiles and costs, high level of acceptance by patients and a low rate of dropout. Here, we aimed to analyze all available evidence on the role of Cucurbita pepo in the treatment of LUTS-BPH. MATERIAL AND METHODS: In May 2016 a systematic search was carried out thorough National Library of Medicine Pubmed, Scopus database and the ISI Web of Knowledge official website in order to identify all published studies on Cucurbita pepo and BPH. The following search strings were used: "Cucurbita pepo" OR "pumpkin seed" AND "prostate"; "Cucurbita pepo" AND "antiandrogen" OR "antiproliferative" OR "anti-inflammatory" OR "antioxidant activities"; "cucurbita pepo" OR "pumpkin seed" AND "LUTS" AND "symptoms improvement" OR "quality of life". We consider for the present analysis only studies related to LUTS-BPH. RESULTS: Among all 670 screened, 16 were related to LUTSBPH and finally analyzed. Among all, ten of them were performed in "in vitro setting" showing anti-inflammatory and antiandrogen effect, and a reduction in prostate growth and detrusor activity, while six were clinical studies. In all studies an improvement in International Prostatic Symptoms Score (IPSS) and uroflowmetry parameters has been reported. In 4 studies, an improvement in quality of life has been reported. CONCLUSION: On the basis of our narrative review, the use of Cucurbita pepo in the management of patients affected by LUTS-BPH seems to be useful for improving symptoms and quality of life. However, future clinical trials are requested to confirm these promising results.

Relación entre el uso del antígeno prostático específico y tratamiento de bloqueo androgénico. Un estudio ecológico / Relationship between prostate-specific antigen testing and androgen deprivation therapy use. An ecological study

Objetivo: Determinar si un mayor número de determinaciones de antígeno prostático específico (nPSA) se asocia a un mayor número de tratamientos de bloqueo androgénico (nTBA). Diseño del estudio: Estudio transversal de tipo ecológico. Emplazamiento: Atención Primaria de ámbito provincial. Participantes: Facultativos titulares con al menos 1 año de permanencia en su plaza. Mediciones principales: Se determinó, para cada cupo de medicina de familia de la provincia de Ourense, el número de varones mayores de 50 años (V50) y su edad, nPSA y nTBA en 2012. Se calculó un tamaño muestral de 113 médicos. La asociación entre nTBA y nPSA se analizó mediante correlación de Spearman. El nTBA se consideró variable dependiente en un análisis de regresión lineal múltiple, incluyendo como covariables sexo del facultativo, ámbito de ejercicio, V50, edad de los pacientes y nPSA. Se consideró significativo un valor de p<0,05. Resultados: Se estudiaron 265 facultativos, 54,1 % varones. La media de V50 era 272,6 (68,6) y el nTBA era 8,5 (DE 4,0) por cupo, siendo nPSA 90,9 (52,4)/año. Existía relación entre número de V50 y nPSA (Rho de Spearman=0,4; IC95 %: 0,3-0,7; p=0,01), así como entre nTBA y edad de V50 (Rho de Spearman=0,2; IC95 %: 0,04-0,31; p<0,001). Se demostró asociación entre nTBA y nPSA (Rho de Spearman=0,2; IC95 %: 0,04-0,31; p=0,01) y entre número de V50 y nTBA (Rho de Spearman 0,5; IC95 %: 0,75-0,84; p<0,001). La regresión lineal mostró relación entre nTBA y edad de los varones (p<0,001) y número de V50 (p<0,001). Conclusiones: Una mayor frecuencia de PSA no se sigue de un mayor diagnóstico de CP medido por el número de TBA instaurados, estando asociados a la edad y número de varones mayores de 50 años (AU)

Objective: To determine if a larger number of determinations of prostate-specific antigen (PSAn) is associated with an increased number of androgen deprivation therapies (ADTn). Study Design: Transversal ecological study. Setting: Primary care at provincial level. Participants: Permanent general practitioners with at least 1-year tenure. Main measurements: The number of men over 50 (V50) and their age, PSAn and ADTn in 2012 were determined for each family medicine quota in the region of Ourense (Spain). A sample size of 113 physicians was calculated. The association between ADTn and PSAn was analyzed by Spearman correlation. The ADTn was considered as a dependent variable in a multiple linear regression analysis, including as covariates gender of the physician, rural or urban context of work, V50, patient age and PSAn. A p value <0.05 was considered significant. Results: We studied 265 physicians, 54.1% men. V50 average was 272.6 (68.6), ADTn was 8.5 (4.0) per medical quota, and PSAn was 90.9 (52.4)/year. There was a relationship between the number of V50 and PSAn (Spearman´s Rho=0,4; CI95%:0.3-0.7; p=0.01) and between ADTn and age of V50 (Spearman´s Rho=0.2; CI95%:0.04-0.31; p<0.001). Association was found between ADTn and PSAn (Spearman´s Rho=0.2; CI95%:0.04-0.31; p=0.01) and number of V50 and ADTn (Spearman´s Rho=0,5; CI95%: 0,75-0,84; p < 0,001) . Linear regression showed a relationship between ADTn and age of males (p <0.001) and number of V50 (p <0.001). Conclusions: A higher frequency of PSA testing does not follow from an increased diagnosis of PCa measured by the number of ADT, that is associated with the age and number of males over 50 (AU)

Anti-Androgenic Activity of Nardostachys jatamansi DC and Tribulus terrestris L. and Their Beneficial Effects on Polycystic Ovary Syndrome-Induced Rat Models.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a major hyperandrogenic disorder. Many drugs prescribed specifically to treat PCOS have side effects; however, previous studies suggest that natural therapeutics including botanicals may be less invasive and equally effective for the management of PCOS. METHODS: In the present study, plants were screened for antiandrogenic activity using the RIKILT yeast Androgen bioAssay (RAA). Selected positive plants were subsequently tested for their efficacy against PCOS induced by estradiol valerate (EV) in rat models. RESULTS: RAA revealed the antiandrogenic property of Nardostachys jatamansi DC (NJ), Tribulus terrestris L. (TT), and Embelia tsjeriam-cottam DC (EJ), whereas Whithania somnifera Dunal (WS), Symplocos racemosa Roxb. (SR), and Helicteres isora L. (HI) exhibited androgenic properties. EJ also exhibited mild androgenic activity and therefore was excluded from further study. EV administration reduced the weight gain and disrupted cyclicity in all rats. NJ and TT extract treatment normalized estrous cyclicity and steroidal hormonal levels and regularized ovarian follicular growth. CONCLUSION: The in vitro antiandrogenic activity of plant extracts and their positive effects on different parameters of PCOS were proved in vivo.

Ellagic acid, a component of pomegranate fruit juice, suppresses androgen-dependent prostate carcinogenesis via induction of apoptosis.

BACKGROUND: Ellagic acid (EA), a component of pomegranate fruit juice (PFJ), is a plant-derived polyphenol and has antioxidant properties. PFJ and EA have been reported to suppress various cancers, including prostate cancer. However, their chemopreventive effects on development and progression of prostate cancer using in vivo models have not been established yet. METHODS: The transgenic rat for adenocarcinoma of prostate (TRAP) model was used to investigate the modulating effects of PFJ and EA on prostate carcinogenesis. Three-week-old male transgenic rats were treated with EA or PFJ for 10 weeks. In vitro assays for cell growth, apoptosis, and Western blot were performed using the human prostate cancer cell lines, LNCaP (androgen-dependent), PC-3 and DU145 (androgen-independent). RESULTS: PFJ decreased the incidence of adenocarcinoma in lateral prostate, and both EA and PFJ suppressed the progression of prostate carcinogenesis and induced apoptosis by caspase 3 activation in the TRAP model. In addition, the level of lipid peroxidation in ventral prostate was significantly decreased by EA treatment. EA was able to inhibit cell proliferation of LNCaP, whereas this effect was not observed in PC-3 and DU145. As with the in vivo data, EA induced apoptosis in LNCaP by increasing Bax/Bcl-2 ratio and caspase 3 activation. Cell-cycle related proteins, p21(WAF) , p27(Kip) , cdk2, and cyclin E, were increased while cyclin D1 and cdk1 were decreased by EA treatment. CONCLUSIONS: The results indicate that PFJ and EA are potential chemopreventive agents for prostate cancer, and EA may be the active component of PFJ that exerts these anti-cancer effects.

Multiple readout assay for hormonal (androgenic and antiandrogenic) and cytotoxic activity of plant and fungal extracts based on differential prostate cancer cell line behavior.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostate cancer is one of the most diagnosed forms of cancer among men in western regions. Many traditional applications or phytotherapeutic concepts propose to inhibit the proliferation of prostate cancer cells. In order to detect influences of plant or fungal extracts and derived fractions on androgen receptor signaling pathways, a differentiating cell proliferation assay was established, which enables the simultaneous detection of hormonal and cytotoxic effects. MATERIAL AND METHODS: The well characterized prostate cancer cell lines LNCaP and PC-3 were used in a multiple readout assay. In all, 186 fractions of 23 traditionally used organisms were screened regarding their effects on proliferation of the two prostate cancer cell lines. The fractions were prepared by accelerated solvent extraction followed by gradient extrography. Extracts of the potential hormonally active plants Cibotium barometz, Heteropterys chrysophylla, and Sideroxylon obtusifolium (= Bumelia sartorum) were phytochemically investigated. RESULTS: Fractions from Cibotium barometz, Cortinarius rubellus, Cyrtomium falcatum, Heteropterys chrysophylla, Nephrolepis exaltata, Salvia miltiorrhiza, Sideroxylon obtusifolium, Trichilia emetica, and Trimeria grandifolia exhibited hormonal influences on prostate cancer cells. Cytotoxic activity towards human cell lines was detected for the first time for fractions from Aglaia spectabilis (A. gigantea), Nephrolepis exaltata and Cortinarius brunneus. CONCLUSIONS: The differential behavior of the two prostate cancer cell lines allows the discrimination between potential androgenic or antiandrogenic activities and effects on the estrogen or glucocorticoid receptor as well as cytotoxic activities. The combined cell lines assay can help to assess the biological activities of material used in traditional medicine.

Inhibition of the DHT-induced PSA secretion by Verbascum xanthophoeniceum and Serenoa repens extracts in human LNCaP prostate epithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Verbascum xanthophoeniceum is a mullein plant, typical of Balkan region and some parts of Turkey, traditionally used as phytotherapeutic agent due to its anti-inflammatory properties. It is rich in phenylethanoid and iridoid metabolites whose anti-inflammatory properties are under characterization. MATERIALS AND METHODS: The role of Verbascum xanthophoeniceum crude methanolic extract and its isolated phenylethanoid glycoside verbascoside have been evaluated, in comparison to a saw palmetto extract, on a human in vitro model of androgen-regulated prostate epithelium, the LNCaP cell line. Cytotoxicity and DHT-induced free and total PSA secretion have been thoroughly studied. RESULTS: We have found that similar to saw palmetto, Verbascum xanthophoeniceum extract and its isolated phenylethanoid glycoside verbascoside have no cytotoxicity in human LNCaP prostate epithelial cells, whereas an inhibitory effect on the DHT-induced free and total PSA secretion, a recognized anti-androgen like activity, has been shown in case of both Verbascum xanthophoeniceum extract and pure verbascoside. Furthermore, in the absence of the endogenous androgen DHT, an androgen-like activity in Verbascum xanthophoeniceum is detectable as it is for saw palmetto, suggesting that a mixed androgen-antiandrogen activity is present. CONCLUSIONS: For the first time, Serenoa repens and Verbascum xanthophoeniceum extracts have shown an absence of cytotoxicity and an inhibitory effect on DHT-induced PSA secretion in an in vitro model of human prostate epithelium, whereas the phenylethanoid glycoside verbascoside appeared to explain only part of the Verbascum xanthophoeniceum inhibitory activity on PSA secretion.

Anti-androgenic activity of hydroxyxanthones in prostate cancer LNCaP cells.

Anti-androgens are used to treat prostate cancer. Here, we report that hydroxyxanthones from a plant extract act as anti-androgens in androgen receptor (AR)-positive prostate cancer LNCaP cells. Anti-androgenic activity of the ethanol extract from Garcinia subelliptica was observed in a luciferase assay using LNCaP/MMTV cells with a stably integrated mouse mammary tumor virus (MMTV) promoter. HPLC-based activity profiling followed by a chemical library-based assay strategy enabled the rapid identification of several active principles bearing a xanthone core substituted with hydroxyl and isoprenyl groups. Among the active compounds, 2-(1,1-dimethyl-allyl)-1,4,5,6-tetrahydroxyxanthone (subelliptenone F) was identified as a potent inhibitor of AR transcriptional activity. The structure-activity relationship of some substituents on the xanthone core was also determined using the chemical library-based bioassay. A quantitative RT-PCR analysis revealed that treatment with the compound resulted in a significant reduction in AR-induced gene (KLK3) expression. Hydroxyxanthone may be a possible candidate for the development of a new anti-androgenic molecule.

Anti-androgenic effect of sesquiterpenes isolated from the rhizomes of Curcuma aeruginosa Roxb.

Six sesquiterpenes: germacrone (1), zederone (2), dehydrocurdione (3), curcumenol (4), zedoarondiol (5) and isocurcumenol (6) were isolated from rhizomes of Curcuma aeruginosa Roxb. (Zingiberaceae). They inhibited 5α-reductase which converts testosterone to dihydrotestosterone (DHT). Germacrone (1) was the most potent (IC(50)=0.42±0.05 mg/mL). Compound 1 was anti-androgenic in LNCaP cells when proliferation was testosterone-induced. The growth of flank gland of male Syrian hamsters is dependent on circulating androgen and when maintained with testosterone, 1 (3, 30, 100µg) inhibited growth but was ineffective against DHT. The similar activity profile was observed on the 5α-reductase inhibitor, finasteride (100 µg) treatment group. The androgen receptor binding assay showed that 1 did not bind to the androgen receptor. In conclusion, 1 showed anti-androgenic effect on in vitro and in vivo assays. One of the possible mechanisms was inhibition 5α-reductase activity. Thus, 1 is a potential lead compound for treatment of androgen-dependent disorders.

Screening for in vivo (anti)estrogenic and (anti)androgenic activities of Tropaeolum majus L. and its effect on uterine contractility.

ETHNOPHARMACOLOGICAL RELEVANCE: Tropaeolum majus L. (Tropaeolaceae) is a medicinal herb popularly used in Brazil for treatment of inflammatory and cardiovascular diseases. Despite some published data on its efficacy, there are still few toxicological data describing the safety of this plant. The aim of this study was to evaluate the (anti)estrogenic and (anti)androgenic activity of the hydroethanolic extract obtained from Tropaeolum majus L. (HETM), as well as its possible effects on uterine contractility. MATERIALS AND METHODS: Three experimental protocols were performed, (a) uterotrophic assay, (b) Hershberger assay and (c) an ex vivo test to investigate the effects of maternal administration of HETM on uterine contractility at the end of pregnancy. In all protocols three doses of the HETM were administered to Wistar rats: 3, 30 and 300mg/kg. RESULTS: In vivo tests for detection of (anti)androgenic and (anti)estrogenic activities did not show any significant alterations. Similarly, no alterations were observed on uterine contractility induced by oxytocin and arachidonic acid. CONCLUSIONS: HETM was unable to produce (anti)estrogenic or (anti)androgenic activities in the short-term in vivo screening assays performed. In addition, there was no evidence that HETM can affect uterine contractility following gestational exposure of rats.

Inhibitory activities of Puerariae Flos against testosterone 5α-reductase and its hair growth promotion activities.

Crude drugs expected to have an estrogenic effect were screened for their inhibitory activity on testosterone 5α-reductase. Testosterone 5α-reductase is an enzyme catalyzing the conversion of testosterone to dihydrotestosterone, which possesses high affinity for the androgen receptor. Among the crude drugs tested, we focused on Puerariae Flos (the flowers of Pueraria thomsonii) due to its potent inhibitory activity and suitability for commercial use. The 50% ethanolic extract of Puerariae Flos (PF-ext) showed inhibitory activity of 60.2% at 500 µg/ml against testosterone 5α-reductase. Interestingly, it was more potent than that of Puerariae Radix (roots of Pueraria lobata). PF-ext also showed in vivo anti-androgenic activity using a hair growth assay in testosterone-sensitive male C57Black/6NCrSlc strain mice. We demonstrated saponins, including soyasaponin I and kaikasaponin III, to be active components in PF-ext. In addition, hair growth promotion activity in C3H/He mice at 2 mg/mouse/day of the topical administration of PF-ext was demonstrated. Thus, Puerariae Flos is a promising crude drug for treating androgenic alopecia.

Physalins A and B inhibit androgen-independent prostate cancer cell growth through activation of cell apoptosis and downregulation of androgen receptor expression.

Androgen deprivation therapy is a common treatment strategy for advanced prostate cancer. Though effective initially, the tumor often progresses to androgen independent stage in most patients eventually after a period of remission. One of the key factors of development of resistance is reflected in expression of androgen receptor (AR). In this study, we showed that two natural compounds, physalins A and B, both secosteriods from Physalisalkekengi var. franchetii, significantly inhibited the growth of two androgen-independent cell lines CWR22Rv1 and C42B, induced apoptosis via c-Jun N-terminal kinase (JNK) and/or extracellular signal-regulated kinase (ERK) activation, and decreased AR expression. In addition, physalins A and B down-regulated the expression of prostate specific antigen (PSA) in C42B cells which is a target gene of AR. Our results suggest that physalin A and B might be useful agents in preventing the growth of androgen-independent prostate cancer (AI-PCa).

The application of reporter gene assays for the detection of endocrine disruptors in sport supplements.

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC(50) of 0.01 ng mL(-1) and 0.16 ng mL(-1) respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.

The culinary-medicinal mushroom Coprinus comatus as a natural antiandrogenic modulator.

Prostate cancer is the most common cancer diagnosed in men. Chemotherapy, androgen ablation, and androgen antagonist treatments have proven to have significant effects in the early stages of prostate cancer, whereas advanced prostate cancer is resilient to such treatments. The androgen receptor (AR), a ligand-dependent transcription factor, is the major drug target of prostate cancer therapy. Transition to the androgen-independent stage involves the activation of signaling pathways, AR gene mutations, and other mechanisms. Higher basidiomycetes mushrooms have been used since ancient times in folk medicine to treat a diversity of diseases, including cancer. The present study evaluates the antiandrogenic activity of different Coprinus comatus strains in their ability to interfere with AR function. The authors found that the most active extract was C comatus strain 734 extracted with hexane (CC734-H). This extract was able to (1) inhibit AR-mediated reporter activity, (2) inhibit the proliferation and viability of the LNCaP cell line, and (3) inhibit the colony formation of the LNCaP cell line, in comparison to the DU-145, PC-3, and MDA-Kb2 cells. In addition, CC734-H was able to reduce AR levels and prostate-specific antigen gene expression in the LNCaP-treated cell line. This study illustrates the potential of the C comatus mushroom as a natural antiandrogenic modulator that could serve in the treatment of prostatic diseases.

Isolation and structure elucidation of a novel androgen antagonist, arabilin, produced by Streptomyces sp. MK756-CF1.

In the course of screening for a new type of androgen receptor (AR) antagonist, we isolated a novel compound, arabilin, with two structural isomers, spectinabilin and SNF4435C, produced by Streptomyces sp. MK756-CF1. Structure elucidation on the basis of the spectroscopic properties showed that arabilin is a novel polypropionate-derived metabolite with a p-nitrophenyl group and a substituted γ-pyrone ring. Arabilin competitively blocked the binding of androgen to the ligand-binding domain of AR in vitro. In addition, arabilin inhibited androgen-induced prostate-specific antigen mRNA expression in prostate cancer LNCaP cells.

Androgen modulators from the roots of Paeonia lactiflora (paeoniae radix) grown and processed in nara prefecture, Japan.

The monoterpene glycoside, 3'-O-galloylpaeoniflorin (1), and four known compounds, 6'-O-galloylalbiflorin (2), pentagalloylglucose (3), 6'-O-benzoylpaeoniflorin (4) and 6'-O-galloylpaeoniflorin (5), were isolated from the roots of Paeonia lactiflora that had been grown and processed in Nara prefecture, Japan, as androgen modulators. Their structures were elucidated based on spectroscopic analysis. Compounds 2 and 3 showed strong androgen receptor (AR) binding activity (IC(50) values 33.7 and 4.1 microg/ml, respectively), 1, 4 and 5 showed weak activity (20, 31 and 12% at 120 microg/ml, respectively). However, paeoniflorin (6) and albiflorin (7), the structures of which are related to 1, 2, 4 and 5, showed no activity. These results suggested that both the structure of albiflorin and the galloyl moiety are important for 2 to show strong AR binding activity. Furthermore, compounds 1-5 inhibited growth of an androgen-dependent LNCaP-FGC (prostate cancer cell line), and were indicated to be AR antagonists. Compounds 2 and 3 might be candidates as safe, natural anti-androgens.

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators.

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newly developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.

Ganoderic acid DM: anti-androgenic osteoclastogenesis inhibitor.

Prostate cancer is the most common cancer in men in Western countries, with a high incidence of bone metastasis. Ganoderic acid DM, with 5alpha-reductase inhibitory and androgen receptor (AR) binding activity, isolated from the ethanol extracts of Ganoderma lucidum, can inhibit prostate cancer cell growth and block osteoclastogenesis.