Botanical Formulation HX109 Ameliorates TP-Induced Benign Prostate Hyperplasia in Rat Model and Inhibits Androgen Receptor Signaling by Upregulating Ca2+/CaMKKβ and ATF3 in LNCaP Cells

Benign prostatic hyperplasia (BPH) is a common disease in the elderly male population throughout the world. Among other factors, androgen dysregulation has been known to play major roles in its pathogenesis. HX109 is a botanical formulation prepared from a mixture of Taraxacum officinale, Cuscuta australis, and Nelumbo nucifera, which have traditionally been used—usually along with other plants—to treat urinary diseases. An ethanol extract was prepared from a mixture of these three plants, and its quality was controlled through cell-based bioassays and by quantification of several marker compounds by high-performance liquid chromatography (HPLC). In the testosterone propionate (TP)-induced prostate hyperplasia rat model, oral administration of HX109 ameliorated prostate enlargement and histological changes induced by TP. In LNCaP cells, a human prostate epithelial cell line, HX109 repressed AR-mediated cell proliferation and the induction of androgen receptor (AR) target genes at the transcriptional level without affecting the translocation or expression of AR. Such effects of HX109 on AR signaling were mediated through the control of activating transcriptional factor 3 (ATF3) expression, phosphorylation of calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ), and increases in intracellular calcium, as evidenced by data from experiments involving ATF3-specific siRNA, CaMKKβ inhibitor, and calcium chelator, respectively. Taken together, our data suggest that HX109 might be used as a starting point for developing therapeutic agents for the treatment of BPH.


Introduction
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 [1,2]. 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].
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).

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.

Animals
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).

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.

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).

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.
Nutrients 2018, 10, 1946 4 of 15 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.

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% CO 2 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.

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).

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.

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).

Calcium Assay
For the calcium assay, LNCaP cells were plated at 5 × 10 4 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.

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.

Quality of HX109 Is Monitored by HPLC Analysis and Cell-Based Bioassay
To establish batch-to-batch consistency of research grade HX109, one representative marker compound from each plant (chicoric acid for Taraxacum officinale, maltol for Cuscuta australis, and dihydrophaseic acid for Nelumbo nucifera) was used ( Figure 1C), based on previously published information [23][24][25]. In addition, isoschaftoside was used as a qualitative marker of Nelumbo nucifera [26]. The marker compounds were analyzed using HPLC ( Figure 1A), and only the extracts containing these compounds within the set range (4.8 ± 0.2 mg/g for chicoric acid, 0.3 ± 0.1 mg/g for maltol, and 0.1 ± 0.05 mg/g for dihydrophaseic acid) were used for this study ( Figure 1B). The identifications of these marker compounds were further confirmed by mass spectrometer (see Figures S1 and S2 in the Supplementary Materials).
In addition, the quality of HX109 was biologically controlled using cell-based bioassays. LNCaP cells were treated with TP in the presence of various concentrations of HX109 and the effect on PSA secretions was determined to calculate the half maximal inhibitory concentration(IC 50 ) value (see Figure S3 in the Supplementary Materials). Only HX109 preparations showing IC 50 values between 2.2 to 2.4 mg/mL were used for the experiments.

HX109 Ameliorates TP-Induced Benign Prostatic Hyperplasia in Castrated Sprague Dawley Rats
To test the effects of HX109 on BPH, a testosterone propionate (TP)-induced BPH rat model was used. Injection of TP every three days into castrated Sprague Dawley rats for 4 weeks (designated as the BPH group) caused a significant (p < 0.001) increase in prostate weight and index compared to the castration-only group (NC) ( Table 1). In rats orally administrated with HX109 for 4 weeks, however, both prostate weight and index decreased in a dose-dependent manner. Prostate weight was reduced by 33% in rats receiving 300 mg/kg HX109 compared to the BPH control group, while finasteride, used as a positive control, decreased it by 43%. Body weight was not affected in all groups (Table 1).

HX109 Ameliorates TP-Induced Benign Prostatic Hyperplasia in Castrated Sprague Dawley Rats
To test the effects of HX109 on BPH, a testosterone propionate (TP)-induced BPH rat model was used. Injection of TP every three days into castrated Sprague Dawley rats for 4 weeks (designated as the BPH group) caused a significant (p < 0.001) increase in prostate weight and index compared to the castration-only group (NC) ( Table 1). In rats orally administrated with HX109 for 4 weeks, however, both prostate weight and index decreased in a dose-dependent manner. Prostate weight was reduced by 33% in rats receiving 300 mg/kg HX109 compared to the BPH control group, while finasteride, used as a positive control, decreased it by 43%. Body weight was not affected in all groups (Table 1). H&E staining was performed to investigate the effect of HX109 on histological changes in the prostate. As shown in Figure 2A, administration of TP increased the area of prostatic acinar 6-fold compared to the NC group, but the area size decreased by 35% following HX109 treatment ( Figure 2B). The effect of HX109 on the protein levels of prostate specific antigens (PSAs), a frequently used marker of prostate enlargement, was also investigated. The PSA levels in the prostate were markedly higher in the BPH group than in the NC group. However, they were reduced in the HX109-treated group compared to the BPH group ( Figure 2C).
In the prostate, the major prostatic androgen is DHT, which is converted by 5α-reductase from testosterone [27]. When DHT levels in the serum and prostate were measured, they were increased in the BPH group compared to the NC group, and HX109 treatment had little effect on DHT levels (see Figure S4A,B in the Supplementary Materials). These results indicated that 5α-reductase might not be involved in the HX109-mediated amelioration of prostate enlargement. H&E staining was performed to investigate the effect of HX109 on histological changes in the prostate. As shown in Figure 2A, administration of TP increased the area of prostatic acinar 6-fold compared to the NC group, but the area size decreased by 35% following HX109 treatment ( Figure  2B). The effect of HX109 on the protein levels of prostate specific antigens (PSAs), a frequently used marker of prostate enlargement, was also investigated. The PSA levels in the prostate were markedly higher in the BPH group than in the NC group. However, they were reduced in the HX109-treated group compared to the BPH group ( Figure 2C).
In the prostate, the major prostatic androgen is DHT, which is converted by 5α-reductase from testosterone [27]. When DHT levels in the serum and prostate were measured, they were increased in the BPH group compared to the NC group, and HX109 treatment had little effect on DHT levels (see Figure S4A,B in the Supplementary Materials). These results indicated that 5α-reductase might not be involved in the HX109-mediated amelioration of prostate enlargement. Castrated Sprague Dawley rats were injected with 3 mg/kg every three days and orally administrated with TDW (BPH) or 200 mg/kg of HX109 or 300 mg/kg of HX109 or 5 mg/kg of finasteride (Fina). Rats injected with vehicle were used as a negative control (NC) group. (A) Effects of HX109 on histological changes in prostate. After measuring the final prostate weight, rats were sacrificed and prostate tissues were fixed, sectioned, and stained with hematoxylin and eosin (H&E). (B) Relative size of acinal area. Values were calculated from three randomly captured pictures. #### p < 0.0001 (one-way ANOVA) compared with the NC group, * p < 0.05 (one-way ANOVA) compared with the BPH group. All data are shown as mean ± S.E.M. (C) Effects of HX109 on prostate PSA levels. PSA levels of rat prostate were measured by ELISA. Values were normalized to total proteins. n = 5 per group. #### p < 0.0001 (one-way ANOVA) compared with the NC group, * p < 0.05, *** p < 0.001 (one-way ANOVA) compared with BPH group. All data are shown as mean ± S.E.M.

HX109 Suppresses Androgen-Dependent Proliferation of LNCaP Cells
In the BPH rat model, the increase in prostate weight by TP is well known to be induced by prostate cell proliferation. Therefore, it was tested whether HX109 could control androgen-induced prostate cell proliferation. LNCaP cells were treated with various concentrations of HX109, with or without the addition of TP, and effects on cell proliferation were measured by WST-1 assay. As shown

HX109 Suppresses Androgen-Dependent Proliferation of LNCaP Cells
In the BPH rat model, the increase in prostate weight by TP is well known to be induced by prostate cell proliferation. Therefore, it was tested whether HX109 could control androgen-induced prostate cell proliferation. LNCaP cells were treated with various concentrations of HX109, with or without the addition of TP, and effects on cell proliferation were measured by WST-1 assay. As shown in Figure 3A, treatment with TP increased proliferation of LNCaP cells by about 56% compared to the vehicle group, but HX109 treatment inhibited TP-induced cell proliferation in a dose-dependent manner. At 2 mg/mL of HX109, TP-induced cell proliferation was inhibited by almost 50%. These effects were not due to cytotoxicity, as cell viability was not affected by HX109 ( Figure 3B). These data suggested that HX109 might inhibit the androgen-dependent proliferation of LNCaP cells. in Figure 3A, treatment with TP increased proliferation of LNCaP cells by about 56% compared to the vehicle group, but HX109 treatment inhibited TP-induced cell proliferation in a dose-dependent manner. At 2 mg/mL of HX109, TP-induced cell proliferation was inhibited by almost 50%. These effects were not due to cytotoxicity, as cell viability was not affected by HX109 ( Figure 3B). These data suggested that HX109 might inhibit the androgen-dependent proliferation of LNCaP cells.

HX109 Inhibits Androgen-Induced PSA Expression
Androgen-dependent proliferation is regulated by androgen/androgen receptor (AR) signaling. Since PSA is the main target of AR signaling, the effect of HX109 on this protein was investigated. In the absence of TP, LNCaP produced a small amount of PSA, but treatment with 100 nM TP elevated its level by 10-fold. When treated with HX109, PSA levels in the cell culture supernatant were decreased in a dose-dependent manner, maximally by 50% at 2 mg/mL ( Figure 4A). Suppression of PSA expression by HX109 was also observed by western blot ( Figure 4B).

HX109 Inhibits Androgen-Induced PSA Expression
Androgen-dependent proliferation is regulated by androgen/androgen receptor (AR) signaling. Since PSA is the main target of AR signaling, the effect of HX109 on this protein was investigated. In the absence of TP, LNCaP produced a small amount of PSA, but treatment with 100 nM TP elevated its level by 10-fold. When treated with HX109, PSA levels in the cell culture supernatant were decreased in a dose-dependent manner, maximally by 50% at 2 mg/mL ( Figure 4A). Suppression of PSA expression by HX109 was also observed by western blot ( Figure 4B).
Since PSA is the main target of AR signaling, the effect of HX109 on this protein was investigated. In the absence of TP, LNCaP produced a small amount of PSA, but treatment with 100 nM TP elevated its level by 10-fold. When treated with HX109, PSA levels in the cell culture supernatant were decreased in a dose-dependent manner, maximally by 50% at 2 mg/mL ( Figure 4A). Suppression of PSA expression by HX109 was also observed by western blot ( Figure 4B). To determine whether HX109 affects the production of PSAs at a transcriptional level, the RNA level of PSAs was measured by quantitative RT-PCR. TP-induced RNA expression of PSA was reduced by HX109 in a dose-dependent manner ( Figure 4C). At 2 mg/mL HX109, the RNA level of PSAs was lowered by 80% compared to TP only. To further verify the effects of HX109 on AR signaling, the RNA levels of other downstream target genes of AR-such as KLK2, TMPRSS2, DHCR24, and NKX3.1-were also measured. The expression of all target genes was highly downregulated when treated with HX109 (see Figure S5 in the Supplementary Materials). These results indicated that HX109 could effectively inhibit AR-mediated gene expression.

HX109 Inhibits AR Transcriptional Activity Without Affecting AR Expression and Translocation
The induction of PSAs by androgen is mediated by the binding of AR to ARE present in the PSA promoter [28]. Therefore, AR-dependent transactivation was assessed by a reporter plasmid assay in which luciferase expression is dependent on the binding of AR to ARE element. LNCaP cells were transfected with luciferase reporter construct containing three copies of ARE, and then treated with TP in the presence of HX109. As shown in Figure 5A, the level of luciferase activity was increased by TP, but was reduced by HX109 treatment in a dose-dependent manner.
AR expression levels was observed in cells treated with HX109 for 24 h ( Figure 5B). Lastly, the effects of HX109 on AR nuclear translocation were studied by isolating cytoplasmic and nuclear proteins. In the absence of TP, AR was only presented in the cytoplasmic compartment, but translocated to the nucleus upon TP treatment. There was little difference in the nuclear levels of AR between the TPonly and HX109 groups ( Figure 5C). Taken together, these results suggest that HX109 might inhibit the transcriptional activity of AR without affecting AR expression or translocation. It was also tested whether HX109 inhibited the expression of AR itself. No significant change in AR expression levels was observed in cells treated with HX109 for 24 h ( Figure 5B). Lastly, the effects of HX109 on AR nuclear translocation were studied by isolating cytoplasmic and nuclear proteins. In the absence of TP, AR was only presented in the cytoplasmic compartment, but translocated to the nucleus upon TP treatment. There was little difference in the nuclear levels of AR between the TP-only and HX109 groups ( Figure 5C). Taken together, these results suggest that HX109 might inhibit the transcriptional activity of AR without affecting AR expression or translocation.

HX109 Regulates AR Transactivation Through Upregulation of CaMKKβ and ATF3
It was previously reported that CaMKKβ and ATF3 repress the transactivation of AR [10,11], and that the expression of the ATF3 gene is downregulated in human BPH tissue [29]. We first assessed the effect of HX109 on ATF3 expression. In LNCaP cells, TP had no effect on ATF3 expression, but treatment with HX109 increased the RNA ( Figure 6A) and protein ( Figure 6B) levels of ATF3 in a dose-dependent manner.
To study the role of ATF3 in the HX109-mediated suppression of PSAs, ATF3 expression was inhibited using siRNA specific to ATF3. The knockdown efficiency of ATF3 was confirmed by western blot analysis ( Figure 6C). Consistent with previous reports showing the repression of AR transactivation by ATF3, the inhibition of ATF3 expression increased TP-induced PSA mRNA expression. However, the inhibition of ATF3 expression interrupted PSA suppression by HX109 ( Figure 6D). known to inhibit AR-mediated gene expression and AR transcriptional activity [11]. TP had no effects on CaMKKβ phosphorylation in LNCaP cells, but treatment with HX109 increased levels of phosphorylated CaMKKβ in a dose-dependent manner ( Figure 6E). When cells were pre-treated with the CaMKKβ inhibitor STO-609, HX109 did not affect PSA expression, showing the important role of CaMKKβ in the HX109-mediated inhibition of PSAs ( Figure 6F). Taken together, our data suggested that the inhibition of androgen-mediated gene expression by HX109 might be dependent on ATF3 and CaMKKβ. Next, the effects of HX109 on CaMKKβ activity were evaluated by western blot, as CaMKKβ is known to inhibit AR-mediated gene expression and AR transcriptional activity [11]. TP had no effects on CaMKKβ phosphorylation in LNCaP cells, but treatment with HX109 increased levels of phosphorylated CaMKKβ in a dose-dependent manner ( Figure 6E). When cells were pre-treated with the CaMKKβ inhibitor STO-609, HX109 did not affect PSA expression, showing the important role of CaMKKβ in the HX109-mediated inhibition of PSAs ( Figure 6F). Taken together, our data suggested that the inhibition of androgen-mediated gene expression by HX109 might be dependent on ATF3 and CaMKKβ.

Calcium Increase by HX109 Plays A Crucial Role in the Regulation of AR Transactivation
The binding of Ca 2+ /Calmodulin to CaMKKβ increases its enzymatic activity. To test whether HX109 regulates intracellular calcium levels, calcium assays were performed using cell lysates. When cells were treated with HX109, intracellular calcium levels increased at 1 min and decreased at 5 min ( Figure 7A). The role of calcium in HX109's effect on PSAs was confirmed in the Ca 2+ -free conditions. When cells were pretreated with the extracellular/intracellular calcium chelator BAPTA-AM, the HX109-mediated downregulation of PSA expression was inhibited ( Figure 7B). However, pretreatment with EGTA, an extracellular specific calcium chelator, did not affect the effect of HX109 on PSA expression (see Figure S6 in the Supplementary Materials). These results indicated that HX109 might increase intracellular calcium to inhibit AR-mediated gene expression.

Calcium Increase by HX109 Plays A Crucial Role in the Regulation of AR Transactivation
The binding of Ca 2+ /Calmodulin to CaMKKβ increases its enzymatic activity. To test whether HX109 regulates intracellular calcium levels, calcium assays were performed using cell lysates. When cells were treated with HX109, intracellular calcium levels increased at 1 min and decreased at 5 min ( Figure 7A). The role of calcium in HX109's effect on PSAs was confirmed in the Ca 2+ -free conditions. When cells were pretreated with the extracellular/intracellular calcium chelator BAPTA-AM, the HX109-mediated downregulation of PSA expression was inhibited ( Figure 7B). However, pretreatment with EGTA, an extracellular specific calcium chelator, did not affect the effect of HX109 on PSA expression (see Figure S6 in the Supplementary Materials). These results indicated that HX109 might increase intracellular calcium to inhibit AR-mediated gene expression.

Discussion
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.

Discussion
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 [33,34]. 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][37][38]. Notably, 7-hydroxydehydronuciferine and dauricine from Nelumbo nucifera have been reported to inhibit the proliferation of prostate cancer cells and urinary tract tumor cells [39,40]. 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.