Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases in the aged male population throughout the world. It is reported that 50% of men over the age of 50 have enlarged prostates, with the incidence increasing with age, and reaching 90% in men over 90 [1
]. BPH is characterized by a histological change in the prostate architecture and variable growth of the prostate size. Increase in prostate size tightens the urethra and induces lower urinary tract symptoms such as nocturia, dysuria, and bladder obstruction [3
Although the worldwide prevalence of BPH is high, the etiology of BPH has yet to be fully elucidated. The progression of BPH involves various factors including aging, hormonal changes, metabolic syndrome, oxidative stress, and inflammation [4
]. Among these, androgen dysregulation is particularly important, as it can induce prostate cell proliferation. In the prostate, testosterone is converted to dihydrotestosterone (DHT) by 5α-reductase. Both DHT and testosterone bind to androgen receptor (AR) and promote protein synthesis involved in the cellular growth of prostate cells [7
]. Since androgen/AR signaling plays an important role in the pathogenesis of BPH, many therapeutic agents are being developed to target this pathway.
AR is a receptor of androgen normally present in the cytoplasm that translocates into the nucleus upon binding the androgen. In the nucleus, AR regulates gene expression by binding to androgen receptor response elements (AREs) located in the promoter and enhancer region of various target genes [8
]. Androgen-mediated gene expression is affected by a number of different factors. Activating transcriptional factor 3 (ATF3), known as a common stress response mediator, is a member of the ATF/cAMP response element-binding protein (CREB) family of transcriptional factor [9
]. ATF3 represses AR transactivation by binding to the transcription domain of the AR. It was reported that the loss of ATF3 results in increased prostate cell proliferation as well as the transcription of androgen-target genes [10
]. Calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ), which is activated by Ca2+
/calmodulin, is also known to influence AR signaling. It has been reported that CaMKKβ overexpression inhibits AR-mediated gene expression, whereas the knockdown of CaMKKβ enhances AR-signaling and proliferation [11
The most frequently prescribed drugs for BPH are alpha blockers and 5α-reductase inhibitors (5ARIs). Alpha blockers are used in initial therapy for improving urine flow, but they do not reduce prostate size [12
]. 5ARIs, represented by finasteride, block prostate enlargement by blocking the conversion of testosterone to DHT. 5ARIs are more effective and have a longer lasting effect than alpha-blockers, but have been reported to produce side effects such as the loss of libido and ejaculation, and impotence [13
]. As such, there is a strong need for the development of safer and more efficacious agents for BPH.
Because BPH is a chronic disease caused by multiple factors, plants may provide an interesting source of developing therapeutic agents. Because botanical medicines contain multiple components, they can potentially interact with multiple cellular targets. Since many plants have traditionally been used as human medicines, historical evidence of their safety and efficacy is also readily available [15
]. HX109 is an ethanol extract prepared from three plants: Taraxacum officinale
, Cuscuta australis
, and Nelumbo nucifera
. These plants were selected on the basis of previous publications describing their activities and functions, and information available from traditional phytomedical practices in Asia. Taraxacum officinale
has been widely used to treat urinary and renal diseases because of its diuretic, choleretic, anti-inflammatory, and anti-carcinogenic effects [16
]. It has also been reported to inhibit prostate cancer cell proliferation and counteract inflammation [17
]. Cuscuta australis
has been used as a tonic to treat urinary complaints, such as frequent urination and involuntary ejaculation [19
]. The aqueous extract of Nelumbo nucifera
has been reported to have antioxidant and anti-steroid properties that may inhibit androgen signaling [20
]. Therefore, we hypothesized that the combination of these three plants might be effective in ameliorating BPH symptoms.
In this study, we investigated the therapeutic potential of HX109 in a TP-induced BPH rat model by measuring prostate weight and the protein level of prostate specific antigens (PSAs). After observing significant improvements in BPH condition in rat model, LNCaP cells, a human prostate epithelial cell line, were used to understand the underlying mechanism.
2. Materials and Methods
2.1. Preparation of HX109
All plants used in the preparation of HX109 were purchased from Humanherb (Gyeongsan, Korea) and authenticated through the plant identification services of Plant DNA Bank in Korea (PDBK, Seoul, Korea) using their genome sequences. HX109 was prepared by mixing Taraxacum officinale, Cuscuta australis, and Nelumbo nucifera at a ratio of 2:1:1. The combination of plants (total dry weight, 60 g) was extracted with 600 mL of 25% EtOH at 20 °C for 8 h. The extract was filtered with 10-μm cartridge paper and concentrated using a rotary evaporator (Eyela, Tokyo, Japan), followed by a freeze-drying process. This process generally produced approximately 8.5 g of brown powder with a yield of about 14%. The voucher specimens used in this study were deposited in the herbarium of ViroMed Co., Ltd. (Seoul, Korea).
2.2. High-Performance Liquid Chromatography (HPLC) Analysis
High-performance liquid chromatography analysis was employed to validate the quality of HX109. Reference standards for chicoric acid, maltol, dihydrophaseic acid, and isoschaftoside were used for qualitative and quantitative analyses of HX109. Analytical samples of HX109 were studied by HPLC-PDA (Waters, Millford, MA, USA) with Capcell PAK C18 MG column (4.6 mm × 250 mm, 5 µm, Shiseido, Japan). Water (0.05% trifluoroacetic acid) for solvent A and acetonitrile (0.01% trifluoroacetic acid) for solvent B was used for the mobile phase. The mobile phase gradient was 5–27% B (0–10 min), 27–35% B (10–25 min), 35–100% B (25–30 min); the flow rate was 1.0 mL/min, and the injection volume was 5 µL at the concentration of 20 mg/mL. The samples were analyzed at a wavelength of 280 nm and the optimum temperature for HPLC separation was 25 °C.
Ten-week old male Sprague Dawley (SD) rats weighing 330 ± 20 g were obtained from Orient Bio (Seongnam, Korea) for animal studies. Animals were housed under controlled environmental conditions: constant temperature (25 ± 2 °C), humidity (60 ± 10%), and a 12 h light/ dark cycle. All experiments were performed according to the guidelines set by the International Animal Care and Use Committee at Seoul National University (Approval Number: SNU-131111-5-1).
2.4. TP-Induced Benign Prostate Hyperplasia Rat Model
Rats were acclimatized for 1 week, followed by bilateral orchiectomies to prevent the influence of endogenous testosterone. After 1 week, rats were divided into five groups: NC, BPH, HX200, HX300, and Fina (n = 5 per group). Prostatic hyperplasia was induced in four groups (BPH, HX200, HX300, Fina) by subcutaneous injection of 3 mg/kg of testosterone propionate (TP) (Tokyo Chemical Industry, Tokyo, Japan) dissolved in cottonseed oil (Sigma-Aldrich, St. Louis, MO, USA) every three days. The NC group received only cottonseed oil in order to provide similar subcutaneous injection conditions in all groups. During the induction of prostate hyperplasia, rats orally received respective reagents on a daily basis for 4 weeks. The HX200 group and HX300 group were orally administrated 200 mg/kg of HX109 or 300 mg/kg of HX109. The Fina group was orally administrated 5 mg/kg of finasteride as a positive control. The NC group and BPH group were orally administrated distilled water as a vehicle. Body weight was measured once a week during the experiment. After 4 weeks, rats were sacrificed, and prostates were immediately removed and weighed.
2.5. H&E Staining
Prostates were fixed in 10% normalized buffered formalin (Sigma-Aldrich, St. Louis, MO, USA) and embedded in the paraffin block. Then, 6-μm paraffin sections of the prostate were stained with Hematoxylin&Eosin to analyze acinar areas. The size of each acinus was measured by ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).
2.6. Enzyme-Linked Immunosorbent Assay (ELISA)
To measure DHT and PSA levels, ELISA kits specific to DHT (ALPCO Diagnostics, Salem, NH, USA) and PSA (Cusabio, Houston, TX, USA) were used according to the manufacturer’s instructions. When in vivo samples were prepared, sera were used to detect DHT, and levels were expressed as pg/mL. Prostate samples were homogenized using T-PER tissue protein lysis buffer (Thermo Fisher Scientific, Woburn, MA, USA) containing a protease inhibitor (Roche, Basel, Switzerland) and a phosphatase inhibitor (Roche, Basel, Switzerland). After preparation, samples were centrifuged at 12,000 rpm for 10 min at 4 °C and the supernatants were used to detect DHT and PSAs. Values from prostate proteins were normalized by total proteins and expressed as pg/mg protein.
2.7. Cell Culture and Reagents
LNCaP human prostate cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium supplemented with a 10% heat-inactivated fetal bovine serum, HEPES (10 mM), penicillin, and streptomycin in a humidified 5% CO2 atmosphere at 37 °C. To examine the effects of TP, cells were cultured in phenol red-free RPMI 1640 containing 5% charcoal stripped serum (CSS) (TCB, Long Beach, CA, USA) for 24 h, and 100 nM TP was then added to the medium. STO-609 (a CaMKKβ inhibitor, Tocris Bioscience, Ellisville, MO, USA) was used at 30 μM and BAPTA-AM (a calcium chelator, Sigma-Aldrich, St. Louis, MO, USA) was used at 20 μM for the experiment.
2.8. RNA Isolation and qRT-PCR
Total RNAs were prepared from LNCaP cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. One microgram of RNA was converted to cDNA using oligo dT primers (QIAGEN, Hilden, Germany) and Reverse Transcriptase XL (avian myeloblastosis virus (AMV)) (Takara, Kusatsu, Japan). Real-time quantitative RT-PCR was performed with SYBR Premix (Takara, Kusatsu, Japan) and Thermal Cycler Dice Real Time System TP800 (Takara, Kusatsu, Japan). PCR conditions were denaturation at 95 °C for 5 s, followed by annealing and extension at 60 °C for 30 s. The sequences of synthesized PCR primer sets (Bioneer Co. Ltd., Seoul, Korea) for KLK3 (hPSA) were 5′-GTGTGTGGACCTCCATGTTATT-3′ and 5′-CCACTCACCTTTCCCCTCAAG-3′; for ATF3 were 5′-AAGAACGAGAAGCAGCATTTGA-3′ and 5′-TTCTGAGCCCGGACAATACAC-3′; for KLK2 were 5′-ATGTGTGCTAGAGCTTACTC-3′ and 5′-AAGTGGACCCCCAGAATCAC-3′; for TMPRSS2 were 5′-GGACAGTGTGCACCTCAAAGAC-3′ and 5′-TCCCACGAGGAAGGTCCC-3′; for DHCR24 were 5′-GAGGCAGCTGGAGAAGTTTG-3′ and 5′-CTTGTGGTACAAGGAGCCATC-3′; for NKX3.1 were 5′-CCTCCCTGGTCTCCGTGTA-3′ and 5′-TGTCACCTGAGCTGGCATTAC-3′.
2.9. Western Blot
After treatment with TP and HX109, LNCaP cells were washed with cold PBS lysed with radioimmunoprecitation(RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing a protease inhibitor (Roche, Basel, Switzerland) and a phosphatase inhibitor (Roche, Basel, Switzerland). Equal amounts of protein were then separated by 10% SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% bovine serum albumin (Gibco, Waltham, MA, USA) in TBST buffer (1 M Tris-HCl (pH 7.4), 0.9% NaCl, and 0.1% Tween 20) for 1 h and incubated with primary antibodies diluted in a 3% BSA blocking solution overnight at 4 °C. Membranes were then treated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:100,000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h, and protein bands were visualized with an enhanced chemiluminescence solution (Millipore, Burlington, MA, USA) and X-Omat film (Kodak, Rochester, NY, USA).
2.10. Luciferase Reporter Plasmid Assay
An inducible AREs-responsive luciferase reporter assay kit was purchased from QIAGEN (Valencia, CA, USA), and the assay was performed as described previously [14
]. LNCaP cells were briefly transfected with ARE-reporter plasmid or negative control plasmid using lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. Twenty-four hours after transfection, the cells were treated with TP (100 nM) and various concentrations of HX109 for 18 h. Cell lysates were prepared, and a luciferase activity assay was performed using the dual luciferase reporter assay system (Promega, Madison, WI, USA) and microplate luminometer (MicroLumat Plus LB96V, Berthold, Germany) according to the manufacturer’s protocol. The data are shown as the ratio of firefly luciferase activity to Renilla luciferase activity (Fluc/Rluc).
2.11. Extraction of Nuclear and Cytoplasmic Fractions
Fractionation and extraction of nuclear and cytoplasmic proteins from LNCaP cells treated with TP and HX109 for 3 h were performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Woburn, MA, USA) according to the manufacturer’s protocol.
2.12. siRNA Transfection
The siRNA specific to ATF3 and scrambled siRNA (Thermo Fisher Scientific, Woburn, MA, USA) were transfected into LNCaP cells using RNAiMAX (Thermo Fisher Scientific, Woburn, MA, USA) according to the manufacturer’s instructions. Twenty-four hours after the siRNA mediated knockdown of ATF3, cells were briefly treated with TP and HX109 and then subjected to further analysis. Knockdown efficiency was evaluated using an antibody against ATF3 (1:1000, Cell Signaling Technology, Danvers, MA, USA).
2.13. Calcium Assay
For the calcium assay, LNCaP cells were plated at 5 × 104 cells per well in a 24-well CellBIND plate containing phenol red-free RPMI with 10% fetal bovine serum (FBS). Twenty-four hours later, cells were treated with TP and 1 mg/mL HX109. After 1 and 5 min, cells were washed with PBS and lysed with PBS containing 0.5% Triton X. The calcium levels of the cell lysates were measured using a calcium assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol.
2.14. Statistical Analysis
All values are presented as mean ± S.E.M. from three independent experiments. Statistical significance was determined using unpaired Student’s t-test or one-way ANOVA with Turkey correction, provided by the GraphPad Prism software version 7 (GraphPad, San Diego, CA, USA). Data were considered statistically significant if the p-value was <0.05.
In this study, we showed that the botanical formulation HX109 could ameliorate TP-induced prostate hyperplasia by controlling androgen receptor signaling. Oral administration of HX109 reduced the TP-induced increase in weight and PSA levels of the prostate. Furthermore, in LNCaP cells, HX109 inhibited androgen-induced proliferation and repressed androgen receptor-mediated gene expression. It appeared that these effects were mediated through an increase in the levels of ATF3 expression, phosphorylation of CaMKKβ, and also an increase in calcium levels, as the effects of HX109 were attenuated by treatment with ATF3-specific siRNA, CaMKKβ inhibitor, or calcium chelator.
Androgen binds to AR, translocates to the nucleus, and binds to ARE present in the promoters of various genes, eventually leading to cell proliferation. Various molecules known to inhibit androgen signaling exert their effects by blocking AR-androgen interaction, AR degradation, or AR translocation. However, HX109 did not seem to use conventional pathways to exert its effect. Instead, HX109 appeared to repress AR transactivation by upregulating the AR-interacting factors ATF3 and CaMKKβ. This indirect regulation of AR signaling by HX109 might have advantages, as it might produce lesser side effects than finasteride, which directly modulates AR signaling by inhibiting DHT [30
It has been reported that intracellular calcium levels are regulated by the influx of external calcium, by calcium channel openings, and by the release of calcium stored in endoplasmic reticulum (ER) [31
]. In this study, pretreatment with EGTA, an extracellular calcium chelator, exerted no effect, while BAPTA-AM suppressed HX109-mediated PSA reduction. Therefore, the HX109-mediated increase in intracellular calcium appears to be the result of calcium release from ER, rather than influx from the outside. It has been reported that Taraxacum officinale
can raise calcium levels through the regulation of ER [32
]. Further studies are needed to clarify the exact mechanism underlying the HX109-mediated regulation of calcium levels in the cells.
Increases in intracellular calcium levels resulting from HX109 action may activate CaMKKβ by increasing its phosphorylation, as evidenced by our data. Since CaMKKβ is well known to repress AR-mediated gene expression [11
], the activation of CaMKKβ may play a crucial role in the HX109-mediated suppression of androgen signaling. In addition, CaMKKβ regulates the phosphorylation of AMP-activated protein kinase (AMPK), which has been shown to inhibit prostate cell growth and AR activity [33
]. Therefore, it is possible that HX109 may control AMPK through the activation of CaMKKβ, thereby producing the therapeutic effects observed in this study.
ATF3 is expected to target BPH pathogenesis by mitigating oxidative stress and inflammation, or inhibiting androgen signaling [35
]. Furthermore, the levels of ATF3 expression in the prostates of BPH patients have been shown to be lower than those of healthy control groups [29
]. We showed that HX109 upregulated ATF3 expression, suppressing AR-mediated gene expression and eventually prostate enlargement. It would be interesting to investigate how HX109 would upregulate ATF3 expression at molecular levels.
We have not yet identified the compounds responsible for the observed effects of HX109 described in this study. The effects of HX109 might result from the complex actions of several components rather than the actions of one specific compound. For instance, flavonoids like quercetin and astragalin from Cuscuta australis
have been shown to reduce oxidative stress in various cell types [36
]. Notably, 7-hydroxydehydronuciferine and dauricine from Nelumbo nucifera
have been reported to inhibit the proliferation of prostate cancer cells and urinary tract tumor cells [39
]. Therefore, it is possible that the combined actions of various compounds contained in HX109 resulted in an inhibition of prostate enlargement. Given the significant effects of HX109, further studies are warranted to identify the active compounds, or at least a fraction with concentrated bioactivity, from this botanical extract.
Taken together, our data indicate that HX109 ameliorated TP-induced prostate enlargement and histological development. In the in vitro cell culture system, HX109 controlled AR-mediated gene expression and proliferation through the upregulation of ATF3, CaMKKβ, and intracellular calcium levels. The safety of the plants used for the preparation of HX109 has been established by a long history of human use. Indeed, no toxic effects of HX109 have been observed in acute or repeated-dose toxicity studies involving rats and dogs (unpublished data). Our data suggest that HX109 may have the potential to be a safe and efficacious therapeutic agent for BPH.