The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes.

Lysosomes mediate degradation of macromolecules to their precursors for cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. Chédiak-Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein and an effector of the minor Rab5 isoform Ypt52. Strikingly, bph1Δ mutant cells have lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy-inducing conditions. In agreement with this, proteomic analysis of bph1Δ vacuoles reveals an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1Δ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When Bph1 is lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases.

Evaluation of the Antioxidant and Antiangiogenic Activity of a Pomegranate Extract in BPH-1 Prostate Epithelial Cells.

Benign prostatic hypertrophy (BPH) is a noncancerous enlargement of the prostate gland that develops from hyper-proliferation of the stromal and epithelium region. Activation of pathways involving inflammation and oxidative stress can contribute to cell proliferation in BPH and tumorigenesis. Agricultural-waste-derived extracts have drawn the attention of researchers as they represent a valid and sustainable way to exploit waste production. Indeed, such extracts are rich in bioactive compounds and can provide health-promoting effects. In particular, extracts obtained from pomegranate wastes and by-products have been shown to exert antioxidant and anti-inflammatory effects. This study focused on the evaluation of the anti-angiogenic effects and chemopreventive action of a pomegranate extract (PWE) in cellular models of BPH. In our experimental conditions, we observed that PWE was able to significantly (p < 0.001) reduce the proliferation and migration rates (up to 60%), together with the clonogenic capacity of BPH-1 cells concomitantly with the reduction in inflammatory cytokines (e.g., IL-6, PGE2) and pro-angiogenic factor (VEGF-ADMA) release. Additionally, we demonstrated the ability of PWE in reducing angiogenesis in an in vitro model of BPH consisting in transferring BPH-1-cell-conditioned media to human endothelial H5V cells. Indeed, PWE was able to reduce tube formation in H5V cells through VEGF level reduction even at low concentrations. Overall, we confirmed that inhibition of angiogenesis may be an alternative therapeutic option to prevent neovascularization in prostate tissue with BPH and its transformation into malignant prostate cancer.

Differential impact of paired patient-derived BPH and normal adjacent stromal cells on benign prostatic epithelial cell growth in 3D culture.

BACKGROUND: Benign prostatic hyperplasia (BPH) is an age-related disease characterized by nonmalignant abnormal growth of the prostate, which is also frequently associated with lower urinary tract symptoms. The prostate with BPH exhibits enhanced growth not only in the epithelium but also in the stroma, and stromal-epithelial interactions are thought to play an important role in BPH pathogenesis. However, our understanding of the mechanisms of stromal-epithelial interactions in the development and progression of BPH is very limited. METHODS: Matched pairs of glandular BPH and normal adjacent prostate specimens were obtained from BPH patients undergoing simple prostatectomy for symptomatic BPH. Tissues were divided further into fresh specimens for culture of primary prostatic stromal cells, and specimens were embedded in paraffin for immunohistochemical analyses. Proliferation assays, immunohistochemistry, and immunoblotting were used to characterize the primary prostate stromal cells and tissue sections. Coculture of the primary stromal cells with benign human prostate epithelial cell lines BHPrE1 or BPH-1 was performed in three-dimensional (3D) Matrigel to determine the impact of primary stromal cells derived from BPH on epithelial proliferation. The effect of stromal-conditioned medium (CM) on BHPrE1 and BPH-1 cell growth was tested in 3D Matrigel as well. RESULTS: BPH stromal cells expressed less smooth muscle actin and calponin and increased vimentin, exhibiting a more fibroblast and myofibroblast phenotype compared with normal adjacent stromal cells both in culture and in corresponding paraffin sections. Epithelial spheroids formed in 3D cocultures with primary BPH stromal cells were larger than those formed in coculture with primary normal stromal cells. Furthermore, CM from BPH stromal cells stimulated epithelial cell growth while CM from normal primary stromal cells did not in 3D culture. CONCLUSIONS: These findings suggest that the stromal cells in BPH tissues are different from normal adjacent stromal cells and could promote epithelial cell proliferation, potentially contributing to the development and progression of BPH.

BPH1, a novel substrate receptor of CRL3, plays a repressive role in ABA signal transduction.

KEY MESSAGE: BPH1 acts as a substrate receptor of CRL3 complex and negatively regulates ABA-mediated cellular responses. The study on its function provides information that helps further understand the relationship between ABA signaling and UPS. Abscisic acid (ABA) plays a crucial role in a variety of cellular processes, including seed dormancy, inhibition of seedling growth, and drought resistance in plants. Cullin3-RING E3 ligase (CRL3) complex is a type of multi-subunit E3 ligase, and BTB/POZ protein, a component of CRL3 complex, functions as a receptor to determine a specific substrate. To elucidate the CRL3 complex that participates in ABA-mediated cellular processes, we first investigated ABA-inducible BTB/POZ genes based on data from the AtGenExpress Visualization Tool (AVT). We then isolated an ABA-inducible gene encoding a potential CRL3 substrate receptor in Arabidopsis, BPH1 (BTB/POZ protein hypersensitive to ABA 1). The isolate gene has a BTB/POZ domain and a NPH3 domain within its N-terminal and C-terminal region, respectively. Yeast two-hybrid and co-immunoprecipitation assays showed that BPH1 physically interacted with cullin3a, a scaffold protein of CRL3, suggesting that it functions as an Arabidopsis CRL3 substrate receptor. The functional mutation of BPH1 caused delayed seed germination in response to ABA and enhanced sensitivity by NaCl and mannitol treatments as ABA-related stresses. Moreover, bph1 mutants exhibited enhanced stomatal closure under ABA application and reduced water loss when compared with wild-type, implying their enhanced tolerance to drought stress. Based on the information from microarray/AVT data and expression analysis of various ABA-inducible genes between wild-type and bph1 plants following ABA treatments, we concluded loss of BPH1 resulted in hyper-induction of a large portion of ABA-inducible genes in response to ABA. Taken together, these results show that BPH1 is negatively involved in ABA-mediated cellular events.

Inflammatory Responses in a Benign Prostatic Hyperplasia Epithelial Cell Line (BPH-1) Infected with Trichomonas vaginalis.

Trichomonas vaginalis causes the most prevalent sexually transmitted infection worldwide. Trichomonads have been detected in prostatic tissues from prostatitis, benign prostatic hyperplasia (BPH), and prostate cancer. Chronic prostatic inflammation is known as a risk factor for prostate enlargement, benign prostatic hyperplasia symptoms, and acute urinary retention. Our aim was to investigate whether T. vaginalis could induce inflammatory responses in cells of a benign prostatic hyperplasia epithelial cell line (BPH-1). When BPH-1 cells were infected with T. vaginalis, the protein and mRNA of inflammatory cytokines, such as CXCL8, CCL2, IL-1ß, and IL-6, were increased. The activities of TLR4, ROS, MAPK, JAK2/STAT3, and NF-κB were also increased, whereas inhibitors of ROS, MAPK, PI3K, NF-κB, and anti-TLR4 antibody decreased the production of the 4 cytokines although the extent of inhibition differed. However, a JAK2 inhibitor inhibited only IL-6 production. Culture supernatants of the BPH-1 cells that had been incubated with live T. vaginalis (trichomonad-conditioned medium, TCM) contained the 4 cytokines and induced the migration of human monocytes (THP-1 cells) and mast cells (HMC-1 cells). TCM conditioned by BPH-1 cells pretreated with NF-κB inhibitor showed decreased levels of cytokines and induced less migration. Therefore, it is suggested that these cytokines are involved in migration of inflammatory cells. These results suggest that T. vaginalis infection of BPH patients may cause inflammation, which may induce lower urinary tract symptoms (LUTS).

Effects of a proprietary mixture of extracts from Sabal serrulata fruits and Urtica dioica roots (WS® 1541) on prostate hyperplasia and inflammation in rats and human cells.

Introduction: Phytotherapeutics, particularly extracts from Sabal serrulata (saw palmetto) fruit or Urtica dioica (stinging nettle) root, are popular for the treatment of male lower urinary symptoms in many countries, but their mechanism of action is poorly understood. We performed in vivo and in vitro studies to obtain deeper insight into the mechanism of action of WS® 1541, a proprietary combination of a Sabal serrulata fruit and an Urtica dioica root extract (WS® 1473 and WS® 1031, respectively) and its components. Methods: We used the sulpiride model of benign prostatic hyperplasia in rats and tested three doses of WS® 1541 in comparison to finasteride, evaluating weight of prostate and its individual lobes as well as aspects of inflammation, oxidative stress, growth and hyperplasia. In human BPH-1 cells, we studied the effect of WS® 1473, WS® 1031, WS® 1541 and finasteride on apoptosis, cell cycle progression and migrative capacity of the cells. Results: WS® 1541 did not reduce prostate size in sulpiride treated rats but attenuated the sulpiride-induced changes in expression of most analyzed genes and of oxidized proteins and abrogated the epithelial thickening. In vitro, WS® 1473 and WS® 1031 showed distinct profiles of favorable effects in BPH-1 cells including anti-oxidative, anti-proliferative and pro-apoptotic effects, as well as inhibiting epithelial-mesenchymal-transition. Conclusion: This data supports a beneficial effect of the clinically used WS® 1541 for the treatment of lower urinary tract symptoms associated with mild to moderate benign prostate syndrome and provides a scientific rationale for the combination of its components WS® 1473 and WS® 1031.

Transforming growth factor beta 1 impairs benign prostatic luminal epithelial cell monolayer barrier function.

Our recent studies identifying the presence of luminal secretory protein PSA in the stroma, decreased E-cadherin expression, and reduced number of tight junction kiss points in benign prostatic hyperplasia (BPH) tissues suggest that epithelial barrier permeability is increased in BPH. However, the cause of increased epithelial permeability in BPH is unclear. Transforming growth factor beta 1 (TGF-ß1) has been reported to be up-regulated in clinical BPH specimens and TGF-ß1 overexpression induced fibrosis and inflammation in a murine model. TGF-ß1 was reported to repress the expression of E-cadherin in benign prostatic cells. However, whether and how TGF-ß1 up-regulation affects epithelial barrier permeability is unknown. Here, in vitro benign prostatic epithelial cell lines BHPrE1 and BPH-1 were utilized to determine the impact of TGF-ß1 treatment on epithelial barrier, tight junctions, and expression of E-cadherin and claudin 1 by transepithelial electrical resistance (TEER) measurement, FITC-dextran trans-well diffusion assays, qPCR, as well as transmission electron microscopy (TEM) observation. Laser capture micro-dissection (LCM) combined with reverse transcription-polymerase chain reaction (qPCR) were utilized to determine the expression of E-cadherin and claudin 1 in BPH patient specimens. TGF-ß1 treatment decreased TEER, increased FITC-dextran diffusion, and reduced the mRNA expression of junction protein claudin 1 in cultured cell monolayers. Claudin 1 mRNA but not E-cadherin mRNA was down-regulated in the luminal epithelial cells in BPH nodules compared to normal prostate tissues. Our studies suggest that TGF-ß1 could increase the permeability through decreasing the expression of claudin 1 and inhibiting the formation of tight junctions in BHPrE1 and BPH-1 monolayers. These results suggest that TGF-ß1 might play an important role in BPH pathogenesis through increasing the permeability of luminal epithelial barrier in the prostate.

High-Aspect-Ratio SU-8-Based Optofluidic Device for Ammonia Detection in Cell Culture Media.

Miniaturization of sensing technology has led to the development of multifunctional micro total analysis systems (µTAS) that benefit from microfluidic technology. Optical sensing is one of the most commonly used sensing approaches integrated into µTAS devices and features high sensitivity and low detection limits. Different materials have been used for the fabrication of µTAS devices, each having their advantages and disadvantages. Herein, a high-aspect-ratio optofluidic waveguide fabricated from SU-8 is presented for the first time. The suitable optical properties and chemical inertness of SU-8 provide a durable device made by a flexible and cost-efficient fabrication process. The optofluidic device was used for colorimetric ammonia (NH3) sensing with a dynamic range of 3-70 µM, a detection limit of 2.5 µM, a response time of 8 min, and close to 10 times better analytical performance compared to using a standard microplate reader. The µTAS device was capable of monitoring NH3 accumulating in the cell culture media of prostatic epithelial cell (BPH-1) culture.

6'-Sialyllactose Ameliorates In Vivo and In Vitro Benign Prostatic Hyperplasia by Regulating the E2F1/pRb-AR Pathway.

BACKGROUND: 6'-Sialyllactose (6SL) displays a wide range of the bioactive benefits, such as anti-proliferative and anti-angiogenic activities. However, the therapeutic effects of 6SL on benign prostatic hyperplasia (BPH) remain unknown. METHODS: Six-week-old male Wistar rats (n = 40) were used for in vivo experiments. All rats were castrated and experimental BPH was induced in castrated rats by intramuscular injection of testosterone, with the exception of those in the control group. Rats with BPH were administrated finasteride and 0.5 or 1.0 mg/kg 6SL. Furthermore, the inhibitory effects of 6SL on human epithelial BPH cell line (BPH-1) cells were determined in vitro. RESULTS: Rats with BPH exhibited outstanding BPH manifestations, including prostate enlargement, histological alterations, and increased prostate-specific antigen (PSA) levels. Compared to those in the BPH group, rats in the 6SL group showed fewer pathological changes and normal androgen events, followed by restoration of retinoblastoma protein (pRb) and cell cycle-related proteins. In BPH-1 cells, treatment with 6SL significantly suppressed the effects on the androgen receptor (AR), PSA, and E2F transcription factor 1 (E2F1)-dependent cell cycle protein expression. CONCLUSIONS: 6SL demonstrated anti-proliferative effects in a testosterone-induced BPH rat model and on BPH-1 cells by regulating the pRB/E2F1-AR pathway. According to our results, we suggest that 6SL may be considered a potential agent for the treatment of BPH.

Paljung-San, a traditional herbal medicine, attenuates benign prostatic hyperplasia in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Paljung-san is a traditional herbal medicine used widely for the treatment of urogenital diseases in East Asia. However, scientific evidence of the efficacy of Paljung-san and its mechanisms of action against benign prostatic hyperplasia (BPH) is not clearly established. AIM OF THE STUDY: We investigated the inhibitory effect of Paljung-san water extract (PSWE) and its mechanisms against BPH in vitro and in vivo. MATERIALS AND METHODS: Active compounds of PSWE were analyzed quantitatively by High-performance liquid chromatography (HPLC). For in vitro study, PSWE treated BPH-1 cells were used to perform western blot analysis, cell cycle analysis and enzyme-linked immunosorbent assay. For in vivo BPH model, male rats were subcutaneously injected with 10 mg/kg of testosterone propionate (TP) every day for four weeks. 200 and 500 mg/kg of PSWE was administrated daily by oral gavage with s.c. injection of TP, respectively. RESULTS: HPLC revealed that PSWE contains 1.21, 1.18, 2.27, 3.56, 4.23, 3.00, 6.78, and 0.004 mg/g of gallic acid, 5-caffeoylquinic acid, chlorogenic acid, geniposide, liquiritin apioside, liquiritin, glycyrrhizin, and chrysophanol components, respectively. In human BPH-1 cells, PSWE treatment reduced cell proliferation through arresting the cell cycle in the DNA synthesis phase. Moreover, PSWE suppressed prostaglandin E2 production with reduced cyclooxygenase-2 expression. In TP -induced BPH rat model, PSWE administration showed reduced prostate weights and dihydrotestosterone levels and led to a restoration of normal prostate morphology. PSWE also decreased TP-induced Ki-67 and cyclin D1 protein levels in the prostatic tissues. Decreased glutathione reductase activity and increased malondialdehyde levels in the BPH groups were reversed by PSWE administration. CONCLUSION: PSWE attenuates the progression of BPH through anti-proliferative, anti-inflammatory and anti-oxidant activities in vitro and in vivo. Therefore, these data provide the scientific evidence of pharmacological efficacy of PSWE against BPH.

Yongdamsagan-tang, a traditional herbal formula, inhibits cell growth through the suppression of proliferation and inflammation in benign prostatic hyperplasia epithelial-1 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Benign prostatic hyperplasia (BPH), also called benign enlargement of the prostate, is a progressive disease that is observed in most elderly men. Yongdamsagan-tang, a traditional herbal formula, is used commonly for the treatment of inflammation-related diseases. Although the therapeutic efficacy of Yongdamsagan-tang against BPH in vivo was reported previously, its underlying mechanisms are not clearly understood. AIM OF THE STUDY: In this study, we investigated the effect of Yongdamsagan-tang water extract (YSTE) and its mechanism on the growth of human BPH epithelial BPH-1 cells. MATERIALS AND METHODS: YSTE was extracted from 11 herbaceous plants and its chemical composition was analyzed by High-performance liquid chromatography (HPLC). YSTE was treated in the epithelial BPH-1 cell line and then cell lysates or supernant were used to evaluate cell viability, cell cycle, proliferation and cytokine production. RESULTS: HPLC revealed that Baicalin and gentiopicroside were involved as the major compounds of YSTE. YSTE treatment in BPH-1 cells repressed cell viability in a dose-dependent manner. Regarding the inhibitory mechanisms of YSTE on cell growth, YSTE inhibited cell proliferation via a decrease in endogenous cyclin D1 protein levels and arrest at the S phase during cell-cycle progression. Furthermore, YSTE treatment in BPH-1 cells suppressed prostaglandin E2 production and cyclooxygenase-2 (COX-2) protein levels. The secretion of the proinflammatory cytokines, interleukin-8 and interleukin-6, was also reduced by YSTE treatment. CONCLUSIONS: YSTE in BPH-1 cells showed antiproliferative and anti-inflammatory activities via cell-cycle arrest and downregulation of COX-2 expression, respectively. Taken together, the results of the present study will enhance our understanding of the mechanisms underlying the effect of YSTE in BPH.

Antiproliferative activity of aqueous leaf extract of Annona muricata L. on the prostate, BPH-1 cells, and some target genes.

BACKGROUND: Annona muricata L. has been reported to possess antitumor and antiproliferative properties. Not much work has been done on its effect on BPH-1 cell lines, and no in vivo studies targeting the prostate organ exist. The study determined the effect of A muricata on human BPH-1 cells and prostate organ. METHODS: The MTT assay was performed on BPH-1 cells using the aqueous leaf extract of A muricata. Cells (1 × 10(5) per well) were challenged with 0.5, 1.0, and 1.5 mg/mL extract for 24, 48, and 72 hours. Cell proliferation and morphology were examined microscopically. BPH-1 cells (1 × 10(4) per well) were seeded into 6-well plates and incubated for 48 hours with 0.5, 1.0, and 1.5 mg/mL A muricata extract. Reverse transcriptase polymerase chain reaction was performed using mRNA extracted from the cells. Possible target genes, Bax and Bcl-2, were examined. Twenty F344 male rats (≈200 g) were gavaged 30 mg/mL (10 rats) and 300 mg/mL (10 rats) and fed ad libitum alongside 10 control rats. Rats were sacrificed after 60 days. The prostate, seminal vesicles, and testes were harvested for histological examination. RESULTS: Annona muricata demonstrated antiproliferative effects with an IC50 of 1.36 mg/mL. Best results were obtained after 48 hours, with near cell extinction at 72 hours. Bax gene was upregulated, while Bcl-2 was downregulated. Normal histological architecture was observed for all testes. Seminal vesicle was significantly reduced in test groups (P < .05) and demonstrated marked atrophy with increased cellularity and the acinii, empty of secretion. Prostate of test groups were reduced with epithelial lining showing pyknotic nucleus, condensation, and marginalization of the nuclear material, characteristic of apoptosis of the glandular epithelium. Furthermore, scanty prostatic secretion with flattening of acinar epithelial lining occurred. CONCLUSION: Annona muricata has antiproliferative effects on BPH-1 cells and reduces prostate size, possibly through apoptosis.

Quantification of the Bioactive Components of the Rhizomes of Curcuma wenyujin and Assessment of Its Anti-inflammatory Effect in Benign Prostatic Hyperplasia-1 Cells

In this study, the marker compounds of Curcumae Rhizoma (CR) were simultaneously quantified by high-performance liquid chromatography equipped with a photodiode array detector and the anti-inflammatory effects of CR extract and marker compounds in human benign prostatic hyperplasia epithelial-1 (BPH-1) cell lines were investigated. The marker components (4S,5S)-(+)-germacrone-4,5-epoxide, furanodienone, and germacrone, were separated on Gemini C₁₈ columns (250 mm × 4.6 mm, 5 µm) at 40 ℃ by using a gradient of two mobile phases eluting at 1.0 mL/min. Prostaglandin E₂ (PGE₂) levels in Human BPH-1 cells were determined with an ELISA kit. The coefficients of determination in a calibration curve of each analyte were all 0.9997. The limits of detection and quantification of the three compounds were 0.10 – 0.32 µg/mL and 0.30 – 0.98 µg/mL, respectively. The content of three compounds, (4S,5S)-(+)-germacrone-4,5-epoxide, furanodienone, and germacrone, in the CR sample were found to be 5.79 – 5.92 mg/g, 4.72 – 4.86 mg/g, and 1.06 – 1.09 mg/g, respectively. Regarding pharmacological activity against benign prostatic hyperplasia, CR and its components significantly suppressed PGE₂ levels of BPH-1 cells. The established analysis method will help to improve quality assessment of CR samples and related products. In addition, CR and its components exhibit anti-inflammatory activity in BPH-1 cells, suggesting the inhibitory efficacy of these compounds against the pathogenesis of BPH.

Inflammatory Responses in a Benign Prostatic Hyperplasia Epithelial Cell Line (BPH-1) Infected with Trichomonas vaginalis

Trichomonas vaginalis causes the most prevalent sexually transmitted infection worldwide. Trichomonads have been detected in prostatic tissues from prostatitis, benign prostatic hyperplasia (BPH), and prostate cancer. Chronic prostatic inflammation is known as a risk factor for prostate enlargement, benign prostatic hyperplasia symptoms, and acute urinary retention. Our aim was to investigate whether T. vaginalis could induce inflammatory responses in cells of a benign prostatic hyperplasia epithelial cell line (BPH-1). When BPH-1 cells were infected with T. vaginalis, the protein and mRNA of inflammatory cytokines, such as CXCL8, CCL2, IL-1β, and IL-6, were increased. The activities of TLR4, ROS, MAPK, JAK2/STAT3, and NF-κB were also increased, whereas inhibitors of ROS, MAPK, PI3K, NF-κB, and anti-TLR4 antibody decreased the production of the 4 cytokines although the extent of inhibition differed. However, a JAK2 inhibitor inhibited only IL-6 production. Culture supernatants of the BPH-1 cells that had been incubated with live T. vaginalis (trichomonad-conditioned medium, TCM) contained the 4 cytokines and induced the migration of human monocytes (THP-1 cells) and mast cells (HMC-1 cells). TCM conditioned by BPH-1 cells pretreated with NF-κB inhibitor showed decreased levels of cytokines and induced less migration. Therefore, it is suggested that these cytokines are involved in migration of inflammatory cells. These results suggest that T. vaginalis infection of BPH patients may cause inflammation, which may induce lower urinary tract symptoms (LUTS).