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Journal of Advanced Research logoLink to Journal of Advanced Research
. 2023 Apr 14;57:135–147. doi: 10.1016/j.jare.2023.04.006

Targeting benign prostate hyperplasia treatments: AR/TGF-β/NOX4 inhibition by apocynin suppresses inflammation and proliferation

Bo-Ram Jin a, Hyo-Jung Kim a, Jung-Hyun Na b, Won-Kyu Lee c, Hyo-Jin An a,d,⁎
PMCID: PMC10918329  PMID: 37061215

Graphical abstract

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Keywords: Apocynin, Androgen receptor; Benign prostatic hyperplasia; Dihydrotestosterone; NOX4; Prostate cancer prevention

Highlights

  • •

    Androgen-dependent benign prostatic hyperplasia (BPH) model exerts excessive ROS, which was revealed by the involvement of the AR and NOX4 networks.

  • •

    In BPH in vitro and in vivo model, the NADPH oxidase inhibitor Apocynin (Apo) suppresses inflammation and proliferation to suppress BPH through the AR/NOX4/TGF-β signaling.

  • •

    Apo exerts anti-proliferative, anti-inflammatory, and anti-oxidant effects, highlighting the potential of Apo as a therapeutic agent for treatment of BPH.

Abstract

Introduction

Apocynin (Apo), an NADPH oxidase (NOX) inhibitor, has been widely used to treat various inflammatory diseases. However, the therapeutic effects of Apo on benign prostatic hyperplasia (BPH), a multifactorial disease associated with chronic inflammation and hormone imbalance, remain unknown.

Objectives

The link between androgen signaling, reactive oxygen species (ROS), and prostate cell proliferation may contribute to the pathogenesis of BPH; therefore, the aim of this study was to identify the specific signaling pathway involved and to demonstrate whether the anti-oxidant Apo plays a role in the prevention and treatment of BPH.

Methods

Ingenuity pathway analysis and si-RNA transfection were conducted to demonstrate the androgen receptor (AR) and NOX4 linkage in BPH. Pathological markers of BPH were measured by H&E staining, immunoblotting, ELISA, qRT-PCR, and immunofluorescence to examine the effect of Apo. Rats stimulated with testosterone and BPH-1 cells were used as BPH models.

Results

AR and NOX4 network-mediated oxidative stress was upregulated in the BPH model. Next, we examined the effects of Apo on oxidative stress and chronic prostatic inflammation in BPH mouse models. In a testosterone-induced BPH rat model, Apo alleviated pathological prostate enlargement and suppressed androgen/AR signaling. Apo suppressed the upregulation of proinflammatory markers and promoted the expression of anti-oxidant factors. Furthermore, Apo regulated the TGF-β/Glut9/activin pathway and macrophage programming. In BPH-1 cells, Apo suppressed AR-mediated proliferation and upregulation of TGFB and NOX4 expression by alleviating oxidative stress. Apo activated anti-oxidant and anti-inflammatory systems and regulated macrophage polarization in BPH-1 cells. AR knockdown partially abolished the beneficial effects of Apo in prostate cells, indicating AR-dependent effects of Apo.

Conclusion

In contrast with existing BPH therapies, Apo may provide a new application for prostatic disease treatment, especially for BPH, by targeting the AR/TGF-β/NOX4 signaling pathway.

Introduction

Benign prostatic hyperplasia (BPH), a nonmalignant enlargement of the prostate gland, is the most common disease in men aged > 50 years. The incidence of BPH is reported to be 30–40%, while the prevalence of BPH among men aged > 70 years is approximately 80–90% [1], [2]. The etiology of BPH remains unclear. Various studies have reported that factors such as age, hormone imbalance, chronic inflammation, and excess oxidative stress mediate the pathogenesis of BPH [3]. Dysregulation of androgen metabolism is considered the main contributor to the development and progression of prostate diseases. Additionally, previous studies have proposed partially overlapping multilateral and complementary theories regarding the etiology of BPH. Prostatic inflammation, which is reported to be regulated by androgens and metabolic disturbances, directly regulates prostate cell proliferation by inducing oxidative stress [4]. Aging-associated chronic inflammation promotes the production of reactive oxygen species (ROS). Aged cells exhibit increased levels of oxidative DNA damage [5]. A previous study reported that androgens induce oxidative stress in prostate cancer cells via NADPH oxidase (NOX) [6]. NOX4 has been reported to be critical for the proliferation of androgen receptor (AR)+ prostate cancer cells, suggesting a correlation between androgen signaling, ROS, and prostate cell proliferation [7]. ROS regulate multiple physiological processes, including proliferation, through the modulation of redox‐sensitive proteins, which subsequently results in the induction of conformational changes that alter the activity of enzymes or transcription factors [8].

Progression of BPH toward increased lower urinary tract symptoms is associated with an increased number of inflammatory cell infiltrates [9]. In BPH, inflammatory infiltrates mainly include activated T cells, lymphocytes, and macrophages, which may contribute to BPH development [10]. As the M1/M2 ratio in BPH changes over time, the ratio of the number of CD68 cells to that of CD163 cells increases more than two-fold as BPH progresses [11]. The M1 phenotype is mainly involved in the production of proinflammatory cytokines and ROS. Stimulated macrophages exhibit upregulated levels of oxidative biomarkers such as malondialdehyde (MDA) and nitric oxide (NO) [12]. Inducible NO synthase (iNOS), which is upregulated in macrophages and other cell types under diverse inflammatory conditions, promotes excess production of NO and ROS [13]. Previous studies have examined alternative activation of macrophages in prostate cancer (PCa). However, the role of macrophages in BPH has not been completely elucidated.

Over the past decade, many studies have suggested a link between BPH and PCa [14], [15]. Conversely, other studies have reported a reverse correlation between prostate size and the incidence of PCa [16]. Despite many controversies on the link between the two diseases, a major pathophysiological driving factor involves chronic inflammation, implying that the modulation of inflammation in prostatic disease may provide a potential treatment for PCa [17]. According to GLOBOCAN 2020, PCa is the most frequently diagnosed cancer in men in 112 countries and the leading cause of cancer-related deaths in 48 countries [18]. Additionally, the incidence of PCa in young populations is rapidly increasing and is considered a long-term contributor to the development of metastatic PCa later in life [19]. In addition, the rate of metastatic PCa, which requires extensive and expensive therapy, is constantly increasing in young adults. As a result, the corresponding PCa-related economic burden is immense. In fact, the cost of treating PCa has increased more rapidly than that of any other cancer in the last 30 years [20].

Currently, to reverse the trends of a dramatic increase in PCa incidence and medical care cost, the paradigm change from reactive treatments of clinically manifested PCa to predictive, preventive, and personalized medicine (3PM/PPPM) is preferable [21]. For predictive diagnostics, disease-relevant phenotyping and risk factor-relevant molecular signaling were analyzed. Here, a strong predisposition, such as low-grade inflammation, may be indicative for specific phenotyping and genotyping of PCa. Generalized anticancer prevention or personalized prevention are applied as mitigating measures. In personalized prevention, applying multiparametric analysis is an important optimal and cost-effective approach. The corresponding approach can be illustrated by excessive production of ROS, upregulation of AR signaling, and inactivation of anti-oxidant genes in the initiation of PCa, which can be recovered by natural anti-oxidant supplements (phytochemicals) with a potent anti-tumor effect in the prostate tissue [22].

The pharmacological activities of apocynin (4-hydroxy-3-methoxyacetophenone; Apo), an acetovanillone isolated from several plants, have been previously reported [23]. Apo is one of the most promising NOX inhibitors for the alleviation of ROS-related diseases. The NOX-inhibitory properties of Apo can be used for the treatment of oxidative stress-induced inflammatory diseases [24], [25]. Apo exerts potent antiproliferative and antitumor effects by regulating oxidative stress [26]. A previous study demonstrated that Apo inhibits cell proliferation and suppresses the progression of PCa via the inhibition of ROS production [27]. Another study revealed that Apo represses androgen-independent PCa progression with no direct relationship with oxidative stress [28]. Thus, although the mechanism of Apo in PCa is unclear, the inhibitory effects of Apo on the proliferation of prostate cells have been clearly demonstrated. However, the therapeutic effect and underlying molecular signaling have not been completely elucidated for BPH, which is linked to PCa.

As the linkage between androgen signaling, ROS, and prostate cell proliferation might contribute to BPH pathogenesis, the aim of this study was to identify specific signaling pathways and investigate whether the anti-oxidant Apo plays a role in the prevention and treatment of BPH. We showed that upregulation of AR and NOX signaling mediated oxidative stress in the BPH model. Additionally, using IPA tool and si-RNA transfection system, we verified the correlation between AR, TGF-β, and NOX4. We demonstrated the therapeutic efficacy of Apo against BPH using a rat BPH model and BPH-1 cells and examined the underlying molecular mechanisms.

Materials and methods

Chemicals and reagents

Recombinant human TGF-β1 and TGF-β2 proteins were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against AR, CD68, NOX4, nuclear receptor coactivator 1 (NCOA1), proliferating cell nuclear antigen (PCNA), tumor protein P53 (TP53, also known as P53), transforming growth factor beta (TGFB), actin beta (ACTB), and 8-hydroxy-2-deoxyguanosine (8-OHdG) were purchased from Santa Cruz Biotechnology, Inc. (TX, USA). Boster Biological Technology (CA, USA) provided antibodies against solute carrier family 2 member 9 (SLC2A9, also known as Glut9) and kallikrein-related peptidase 3 (KLK3, also known as PSA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (PA, USA). SYBR Green Master Mix was purchased from Applied Biosystems (CA, USA). Oligonucleotide primers were purchased from Bioneer (Daejeon, Republic of Korea). Testosterone propionate was purchased from Wako Pure Chemical Industries (Osaka, Japan). Finasteride (Fina) was obtained from Merck & Co., Inc. (NJ, USA).

Cell culture

BPH-1 cells (human BPH cell line), which were purchased from the Leibniz Institute DSMZ (Braunschweig, Germany), were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Waltham, MA, USA) supplemented with 20% fetal bovine serum (Life Technologies Inc.) and 100 mg/mL penicillin–streptomycin (GE Healthcare Life Sciences Inc., Chicago, IL, USA) in a humidified atmosphere of 5% CO2 at 37℃.

Cell counting kit (CCK)-8 assay

Cell proliferation was examined using CCK-8 (Dojindo Molecular Technologies, Inc., Doc, USA). BPH-1 cells were seeded into 96-well plates (1 × 105 cells/well) and cultured for 24 h. Next, the cells were treated without or with various concentrations of Apo (7.81–500 µM) for 24 h, followed by incubation with CCK-8 solution for 4 h. The number of viable cells was monitored by measuring the absorbance at 450 nm using an Epoch microplate reader (Biotek, Winooski, VT, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis

Total RNAs was isolated using the Easy-Blue® reagent (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) and reverse-transcribed into complementary DNA, following the manufacturer’s instructions. qRT-PCR analysis was performed as previously described [29].

Western blot analysis

Total proteins extracted from rat prostate tissues and BPH-1 prostate cells were subjected to western blot analysis, as described previously [30].

Immunofluorescence

BPH-1 cells treated with H2O2 and/or Apo were fixed in 100% methanol, blocked with 10% normal goat serum (Gibco®, Big Cabin, OK, USA), and incubated overnight with primary antibodies against 8-OHdG. The cells were then washed and incubated with Alexa Fluor 488-conjugated anti-mouse IgG secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Life Technologies). Images of the cells were captured using an optical microscope (ECLIPSE Ni-U, Nikon, Tokyo, Japan).

Short-interfering RNA (si-RNA) transfection

BPH-1 cells were transfected with siRNA against AR (si-AR) (Bioneer Corporation) or the pmaxGFPtm vector using a 4D-Nucleofectortm system (Lonza, Basel, Switzerland) for 4 h. The si-AR sequences used were as follows: CUCUCUUCACAGCCGAAGA = tt(6-AS) (forward) and UCUUCGGCUGUGAAGAGAG = tt(6-AA) (reverse). The cells were then treated with or without Apo 500 μM. The efficiency of AR knockdown was examined using qRT-PCR.

Upstream regulator analysis

Ingenuity Pathway Analysis IPA (version 62089861, QIAGEN), a bioinformatics tool used for molecular network analysis [31], [32], was used to analyze the molecular interactions between proteins that were upregulated in the in vitro and in vivo BPH models. The datasets of upregulated proteins were imported into IPA and subjected to Regulator Effect algorithms to predict upstream regulators and their downstream effects.

Ethics statement

All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Sangji University before the initiation of the study (IACUC Animal Approval Protocol #2018–27).

Animals

Male Sprague-Dawley rats (n = 40; 200 ± 20 g) aged 6 weeks were purchased from Daehan Biolink Co. (Daejeon, Korea). The animals were housed in cages (four rats/cage) under conditions recommended in the guidelines for the care and use of laboratory animals, which were adopted and promulgated by Sangji University, according to the requirements of the National Institutes of Health. Rats were allowed to acclimatize to the laboratory conditions for one week before the initiation of the experiments. To induce BPH, rats were subcutaneously injected with testosterone propionate (TP) after castration. Briefly, the rats were randomly divided into the following four groups (n = 10 rats per group): control (Con, non-pathological prostate), subcutaneously administered 200 mL of vehicle (corn oil); BPH, subcutaneously administered TP (10 mg/kg bodyweight/day) after castration; Fina, BPH animals orally administered Fina (5 mg/kg bodyweight/day); and Apo, BPH animals orally administered Apo (5 mg/kg bodyweight/day). For castration, the rats were anesthetized by intraperitoneal injection of zoletil 50 (10 mg/kg bodyweight), and their testicles were removed. Rats in the control group were anesthetized and dissected without testicles removal. All rats were allowed to recover for 1 week. The treatment duration was 4 weeks (treatment was not performed on weekends). At 24 h after the administration of the final treatment dose, all rats were anesthetized by intramuscular injection of zoletil 50 (20 mg/kg bodyweight). Blood samples were collected via cardiac puncture and centrifuged to obtain the serum, which was stored at –80℃.

MDA assay

The serum levels of MDA were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Abcam, Cambridge, United Kingdom), following the manufacturer’s instructions.

Histological analysis

Prostate tissues from different groups were fixed in 4% formalin, embedded in paraffin, and cut into 4-mm thick sections. Next, the sections were stained with hematoxylin and eosin for histological examination. Images of the stained sections were acquired using a Leica microscope (Leica DFC 295; Wetzlar, Germany). The thickness of the epithelium in prostate tissue (TETP) was measured using the Leica Application Suite software (ver. 3.3.0; Leica Microsystems, Inc., Buffalo Grove, IL, USA).

Analysis of serum dihydrotestosterone (DHT) levels

Serum dihydrotestosterone (DHT) levels were quantified using commercial ELISA kits (Abcam, Cambridge, United Kingdom), following the manufacturer’s instructions.

Uric acid assay

The intracellular and extracellular levels of uric acid were determined using commercial ELISA kits (CUSABIO Life Science, Houston, TX, USA), following the manufacturer’s instructions.

Statistical analysis

The results are expressed as the mean ± standard deviation of triplicate experiments. The means of different groups were compared using analysis of variance (ANOVA), followed by Dunnett’s post-hoc test. Differences were considered statistically significant at P < 0.05.

Results

Apo exerts anti-proliferative, anti-oxidant and anti-inflammatory effects in BPH-1 cells

To investigate the pharmacological effects of Apo on prostate cancer cells, an in vitro BPH model was established using BPH-1 cells. Using the CCK-8 assay, we confirmed that the effective dose of Apo to suppress the proliferation of BPH-1 cells was > 7.81 μM (Fig. 1A). To investigate whether Apo exerts anti-oxidant activity in BPH-1 cells, an immunofluorescence assay was performed to detect 8-OHdG, a biomarker of oxidative stress. BPH-1 cells exhibited green fluorescence in the nucleus and cytosol, indicating oxidative stress. Treatment with 500 μM Apo mitigated the enhanced green fluorescence observed in the BPH-1 cells. In contrast to the untreated group, the group stimulated with exogenous H2O2 exhibited markedly increased green fluorescence intensity. Treatment with Apo suppressed H2O2-induced upregulation of 8-OHdG fluorescence intensity (Fig. 1B). In addition, Apo significantly upregulated the levels of the anti-oxidant genes HMOX1, SOD1, and GPX1 (Fig. 1C). Treatment with Apo also significantly downregulated NOS2, PTGS2, IL6, and TNFA levels in BPH-1 cells (Fig. 1D). Furthermore, Apo significantly upregulated the levels of the M2 macrophage markers MRC1 and IL10 (Fig. 1E) and suppressed the upregulated levels of the M1 macrophage markers CD68, CD80, and CD86 in BPH-1 cells (Fig. 1F). NOX4 is a major source of oxidative stress and has pro-inflammatory actions [33]. As shown in Fig. 1G and 1H, treatment with Apo significantly suppressed the expression of NOX4. Thus, Apo exerts anti-proliferative, anti-oxidant, and anti-inflammatory effects on BPH-1 cells via inhibition of NOX4 expression.

Fig. 1.

Fig. 1

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Anti-proliferative, anti-oxidant and anti-inflammatory effects of the NOX inhibitor Apo on BPH-1 cells. Effect of Apo on the viability of BPH-1 cells. BPH-1 cells were treated with different concentrations (7.81–500 μM) of Apo for 24 h. (B) Effect of Apo on oxidative stress in BPH-1 cells. BPH-1 cells were treated without or with 500 µM Apo for 24 h, followed by treatment with or without H2O2 for 30 min. (C-F) BPH-1 cells were treated without or with Apo (125, 250, or 500 μM). mRNA levels of HMOX1, SOD1, and GPX1 (C) and NOS2, PTGS2, IL6, and TNFA (D) and MRC1, IL10 (E) and CD68, CD80, CD86 (F) were quantified using quantitative real-time polymerase chain reaction. The expression levels of target genes were normalized to those of GAPDH (housekeeping gene). (G) Protein level of NOX4 was determined. (H) The levels of target protein were normalized to that of ACTB (internal control) and are presented as relative protein levels. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the vehicle-treated group; analysis of variance (ANOVA), followed by Dunnett’s post-hoc test.

Apo inhibits proliferation in BPH-1 cells through the inhibition of AR/TGF-β pathways

IPA was used to confirm the interaction between proteins, and the correlation between AR and NOX4 was verified. Interestingly, upstream regulator analysis using IPA revealed that AR is an upstream regulator of TGF-β, which indirectly regulates the expression of NOX4 (Fig. 2A). We used immunoblotting to investigate the effects of Apo on the AR signaling pathway. The KLK3 gene, also known as prostate-specific antigen (PSA), is a specific cluster of AR-regulated genes. NCOA1, also known as steroid receptor coactivator-1 (SRC-1), is a coactivator of AR. As shown in Fig. 2B and 2C, treatment with Apo significantly suppressed the protein expression of AR, KLK3, NCOA1, and PCNA. Next, the effects of Apo on TGFB levels in BPH-1 cells were examined. As shown in Fig. 2D and 2E, TGFB levels in Apo-treated cells were significantly lower than those in vehicle-treated cells.

Fig. 2.

Fig. 2

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Inhibitory effect of the Apo on AR-dependent proliferation of BPH-1 cells. (A) Link between AR, TGF-β, and NOX4. Solid lines represent a direct interaction between the two gene products, while the dotted lines indicate an indirect interaction. (B) BPH-1 cells were treated without or with Apo (125, 250, or 500 μM). The cell lysates were subjected to immunoblotting with primary antibodies against AR, KLK3, NCOA1, and PCNA. (C) The levels of target proteins were normalized to those of ACTB (internal control) and expressed as relative protein levels. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the vehicle-treated group. (D and E) BPH-1 cells were treated without or with Apo (125, 250, or 500 μM). (D) TGFB level was determined. (E) The level of target protein was normalized to those of ACTB (internal control) and is presented as relative protein level. ***P < 0.001 compared with the vehicle-treated group; analysis of variance (ANOVA), followed by Dunnett’s post-hoc test.

Apo regulates AR/TGF-β/NOX4 signaling pathway in BPH-1 cells

To explore the exact role of Apo, BPH-1 cells were transfected with AR siRNA. Transfection with si-AR downregulated the expression of AR, TGFB, and NOX4 (Fig. 3A). To demonstrate the correlation between AR and Apo, cells transfected with si-AR were treated with 500 μM Apo for 12 h. The AR, NCOA1, and TGFB mRNA levels in si-AR-transfected cells were significantly lower than those in vector-transfected cells. Meanwhile, the knockdown of AR reversed the inhibitory effects of Apo on AR, NCOA1, and TGFB in BPH-1 cells (Fig. 3B). Indeed, transfection with si-AR mitigated the inhibitory effects of Apo on TGFB and NOX4 protein expression, confirming the linkage between AR, TGFB, and NOX4 (Fig. 3C and 3D). Based on these results, we performed a subsequent experiment where si-AR-transfected BPH-1 cells were stimulated with exogenous TGF-β1 or TGF-β2 and then treated with Apo. The mRNA level of NOX4 in si-AR-transfected cells was significantly lower than that in GFP-transfected cells, whereas treatment with TGF-β1 or TGF-β2 significantly upregulated NOX4 mRNA. Meanwhile, treatment with Apo significantly downregulated the levels of NOX4, which suggested that the inhibitory effect of Apo on NOX4 is AR/TGF-β-dependent (Fig. 3E).

Fig. 3.

Fig. 3

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Effect of Apo on TGFB/NOX4 signaling in BPH-1 cells. BPH-1 cells were transfected with green fluorescent protein (GFP)-encoding vector or short-interfering RNAs against AR (AR siRNA), and (A) the mRNA levels of AR, TGFB, and NOX4 in BPH-1 cells were determined using qRT-PCR. (B-D) Transfected BPH-1 cells were treated without or with 500 µΜ Apo for 4 h. The cells were harvested, and (B) the mRNA levels of AR, NCOA1, and TGFB and (C) the protein levels of TGFB and NOX were determined. (D) The levels of target proteins were normalized to those of ACTB (internal control) and are presented as relative protein levels. **P < 0.01 and ***P < 0.001 compared with the GFP group. (E) Transfected BPH-1 cells treated with exogenous TGF-β1 or TGF-β2, and then treated without or with 500 µΜ Apo for 4 h. The cells were harvested, and the mRNA levels of NOX4 were determined. ###P < 0.001 compared with the GFP group; * P < 0.05 compared with the AR siRNA group; $$ P < 0.01 compared with the AR siRNA + TGF-β1 group; ££ P < 0.01 compared with the AR siRNA + TGF-β2 group.

NOX4 is upregulated in the TP-induced BPH rat model, suggesting the effects of Apo on BPH

In this study, a TP-induced BPH rat model was established. As shown in Fig. 4A, the MDA level in the BPH group was significantly higher than that in the Con group. Compared with those in the Con group, prostate levels of NOX2 and NOX4 were upregulated in the BPH group (Fig. 4B and 4C). The expression of NOX1, NOX3, DUOX1, and DUOX2 was not detected (data not shown). Next, the NADPH oxidase inhibitor Apo was used to investigate the effects of Apo in a rat model of TP-induced BPH. Compared to the Con group, the TP-treated groups exhibited marked prostate swelling and significantly increased prostate weight. However, treatment with Fina or Apo 5 significantly mitigated TP-induced prostate enlargement (Fig. 4D and 4E). Rats in the BPH group exhibited histological changes, including a thickened and varifold layer of epithelium and a decreased glandular luminal area, which were mitigated upon treatment with Fina and Apo 5. Furthermore, the TETP value in the Fina and Apo groups decreased by 21.86% and 34.57%, respectively, compared to that in the BPH group (Fig. 4F and 4G).

Fig. 4.

Fig. 4

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Inhibitory effects of Apo on prostate gland enlargement in the testosterone-induced BPH rat model. (A-C) Rats were divided into the following two groups: control (Con) and BPH (testosterone-induced BPH). (A) Serum malondialdehyde (MDA) levels were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit. ###P < 0.001 compared with the Con group. (B) Prostate tissue lysates were subjected to immunoblotting with antibodies against NOX2 and NOX4. ACTB served as an internal control. (C) The density of target protein bands was normalized to that of Actb protein band using ImageJ and is presented as mean ± standard deviation. ##P < 0.01 and ###P < 0.001 compared with the Con group. (D-G) The rats were randomly divided into the following four groups: control (Con), BPH (testosterone-induced BPH), Fina (BPH animals treated with finasteride (5 mg/kg bodyweight)), and Apo (BPH animals treated with Apo (5 mg/kg bodyweight)). (D) Representative photographs of prostate tissues from the testosterone-induced BPH rat model. (E) The ratio of prostate weight/bodyweight in the experimental groups. (F) Prostate tissue sections were subjected to hematoxylin and eosin staining. Original magnification: 100×. (G) Based on histological analysis, the thickness of the epithelium from the prostate tissue (TETP) was measured. ### P < 0.001 compared with the Con group; * P < 0.05, and *** P < 0.001 compared with the BPH group; analysis of variance, followed by Dunnett’s post-hoc test.

Apo exerts therapeutic effects on BPH by inhibiting the androgen/Ar signaling pathway

To investigate the role of Ar signaling in Apo-mediated inhibition of prostate enlargement, Srd5a2 mRNA levels, DHT production, and AR and NCOA1 levels were examined. The SRD5A2 gene, also known as 5α-reductase type 2, is involved in pathways responsible for the pathogenesis of BPH. In this study, the 5α-reductase inhibitor Fina, which is the first-line therapy for BPH, was used as a positive control. As shown in Fig. 5A, Srd5a2 mRNA levels in the BPH group were upregulated when compared with those in the Con group. However, treatment with Fina and Apo 5 significantly mitigated the TP-induced upregulation of Srd5a2 mRNA levels. DHT production in the BPH group was upregulated compared to that in the Con group. However, treatment with Apo significantly mitigated TP-induced upregulation of DHT production (Fig. 5B). As shown in Fig. 5C-5E, the mRNA and protein levels of AR and NCOA1 in the BPH group were higher than those in the Con group. However, the mRNA and protein levels of AR and NCOA1 in the Fina and Apo groups were significantly downregulated compared to those in the BPH group.

Fig. 5.

Fig. 5

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Inhibitory effects of Apo on Androgen/AR signaling in the testosterone-induced BPH rat model. (A) The mRNA level of Srd5a2 was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of Srd5a2 was normalized to that of Gapdh (housekeeping gene). (B) Serum levels of dihydrotestosterone (DHT) were analyzed using an enzyme-linked immunosorbent assay kit. (C) Prostate tissue lysates were subjected to immunoblotting with antibodies against AR and NCOA1. (D) The density of target protein bands was normalized to that of ACTB band using ImageJ and presented as mean ± standard deviation. (E) The Ar and Ncoa1 mRNA levels in the prostate were quantified using qRT-PCR. ## P < 0.01 and ### P < 0.001 compared with the Con group; * P < 0.05, * P < 0.01, and *** P < 0.001 compared with the BPH group; analysis of variance, followed by Dunnett’s post-hoc test.

Apo exerts anti-oxidant and anti-inflammatory effects in the TP-induced BPH rat model

Next, we determined the anti-oxidant and anti-inflammatory effects of Apo in the TP-induced BPH rat model. As shown in Fig. 6A, treatment with Fina and Apo significantly suppressed the TP-induced upregulation of MDA. In addition, treatment with Apo significantly mitigated the TP-induced downregulation of the mRNA expression levels of the anti-oxidant genes Hmox1, Sod1, and Gpx1 (Fig. 6B). As shown in Fig. 6C, NOX4 and CD68 levels in the BPH group were upregulated when compared with those in the Con group. However, Apo significantly downregulated the TP-induced upregulation of CD68 and NOX4, whereas Fina only suppressed the TP-induced upregulation of Nox4 (Fig. 6C and 6D). As shown in Fig. 6E and 6F, the regulatory effects of Apo on the polarization of M1 and M2 macrophage subsets were examined. Compared to those in the Con group, the mRNA levels of the M1 macrophage markers Cd68, Cd80, and Cd86 were significantly upregulated, whereas Mrc1, Retnla, Il10, and Chi3l3 mRNA levels were downregulated in the BPH group. Treatment with Apo increased the proportion of M2 macrophages twofold relative to M1 macrophages, indicating that Apo alleviated BPH through its anti-oxidant effect. Compared to those in the Con group, the mRNA levels of the pro-inflammatory factors Il6, Il1b, Tnfa, Nos2, and Ptgs2 were significantly upregulated in the BPH group. Treatment with Fina or Apo mitigated the TP-induced changes in the mRNA levels of pro-inflammatory factors (Fig. 6G and 6H).

Fig. 6.

Fig. 6

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Anti-oxidant and anti-inflammatory effects of Apo in the BPH rat model. (A) Serum malonaldehyde (MDA) concentrations were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit. (B) mRNA levels of Hmox1, Sod1, and Gpx1 in the prostate tissues were determined using quantitative real-time polymerase chain reaction. (C) NOX4 and CD68 levels in the prostate tissue lysates were determined using western blotting with specific antibodies. ACTB served as an internal control. (D) Densitometric analysis of each protein was performed, and the relative protein levels are presented as mean ± standard deviation. mRNA levels of Cd68, Cd80, Il6 (E), Mrc1, Retnla, Il10, and Chi3l3 (F) were quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Il6, Il1b, Tnfa (G), Nos2, and Ptgs2 (H) mRNA levels in the prostate tissues were determined using qRT-PCR. # P < 0.05, ## P < 0.01, and ### P < 0.001 compared with the Con group; * P < 0.05, * P < 0.01, and *** P < 0.001 compared with the BPH group; analysis of variance, followed by Dunnett’s post-hoc test.

Apo regulates TGF-β/Glut9 signaling pathway in the TP-induced BPH rat model

One recent study suggested that SLC2A9, which encodes GLUT9, plays an important role in the extracellular effects of uric acid, which are associated with BPH [34]. Compared to those in the Con group, TGFB and SLC2A9 levels were upregulated, whereas TP53 levels were downregulated in the BPH group. Treatment with Fina and Apo significantly mitigated the TP-induced upregulation of TGFB and SLC2A9 and downregulation of TP53 (Fig. 7A and 7B). Apo treatment also regulated SLC2A9 levels in BPH-1 cells (Supplementary Fig. 1). The level of activin A, which belongs to the TGF-β family and plays a pivotal role in cellular proliferation, was upregulated in the BPH group. Treatment with Fina and Apo significantly mitigated TP-induced production of activin A (Fig. 7C). Next, the effects of Apo on uric acid levels in the TP-induced BPH rat model were determined. Compared with those in the Con group, plasma uric acid levels were downregulated and intracellular uric acid levels were upregulated in the BPH group. However, treatment with Fina and Apo upregulated plasma uric acid levels and mitigated the intracellular accumulation of uric acid in the TP-induced BPH rat model (Fig. 7D and 7E).

Fig. 7.

Fig. 7

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Effects of Apo on Tgfb/activin/Glut9 pathway in the BPH rat model. (A) TGFB, TP53, and SLC2A9 levels in the prostate tissue lysates were examined using western blotting with specific antibodies. ACTB served as an internal control. (B) Densitometric analysis of each protein was performed, and the relative protein levels are presented as mean ± standard deviation. (C) Serum Activin A concentrations were determined using an ELISA kit. (D) The intracellular and (E) plasma levels of uric acid were determined using an ELISA kit. ### P < 0.001 compared with the Con group, *** P < 0.001 compared with the BPH group; analysis of variance, followed by Dunnett’s post-hoc test.

Discussion

The link between BPH and PCa

The age-related chronic disease BPH and PCa present major health burdens for patients. The pathophysiological risk factors for both BPH and PCa include chronic inflammation-induced oxidative stress and hormonal imbalance [35]. Epidemiological studies have revealed that the pathological progression of BPH and PCa is dependent on hormones, based on the increased incidence and prevalence of the two diseases with age and chronic prostatic inflammation [36]. Aberrant activation of the AR by androgens leads to enhanced proliferation and angiogenic events, which subsequently mediate the pathogenesis of BPH and PCa. Androgen deprivation therapy is the primary treatment for BPH and PCa;, it inhibits androgen production and/or blocks AR activity [37]. Inhibitors of 5α-reductase, including Fina, achieve desired clinical outcomes in BPH. However, these therapies are not completely beneficial and are associated with serious adverse effects, including depression, sexual dysfunction, and progression to high-grade PCa [38].

Involvement of Apo in AR/TGF-β /NOX4 signaling in BPH

Androgen signaling can regulate multiple overlapping cellular and molecular pathways involved in inflammation. Inflammation can affect androgen signaling during the initiation and progression of BPH [39]. Khurana et al. demonstrated crosstalk between oxidative stress, inflammation, and AR. This suggests that anti-inflammatory and anti-oxidant phytochemicals, which also inhibit AR, are potential therapeutic agents for PCa [40]. Despite growing evidence of an association between oxidative stress and PCa progression, the molecular mechanisms underlying BPH have not been completely elucidated. Further studies are required to understand the role of oxidative stress in BPH.

In this study, network analysis using the IPA tool revealed that AR, TGF-β, and NOX4, which are upregulated in BPH, are significantly related to protein expression (Fig. 2). The effect of the NOX inhibitor Apo on BPH and the underlying mechanisms were examined in vitro. Apo downregulated proliferation and inflammation-related factors, upregulated anti-oxidant markers, and promoted the polarization of macrophages from the M1 to M2 phenotype in BPH-1 cells. AR knockdown partially mitigated the effects of Apo on the expression of NCOA1 and TGFB in BPH-1 cells. This indicates that the therapeutic effects of Apo on BPH are partially dependent on AR. Notably, AR siRNA transfection also counteracted the effects of Apo on the protein levels of TGFB and NOX4, which suggests the involvement of Apo on AR/TGF-β /NOX4 signaling in BPH (Fig. 3).

In the animal studies, treatment with Apo also repressed inflammation and proliferation, thus suppressing BPH via inhibition of the AR/TGF-β/NOX4 pathway. The administration of TP promoted the development of BHP in rats, as evidenced by the upregulation of the AR signaling pathway. In contrast, Apo markedly alleviated AR-dependent BPH. These findings suggest that Apo-mediated AR inhibition represses the proliferation of prostate cells and consequently suppresses BPH in vivo (Fig. 4, Fig. 5). Treatment with Apo promoted macrophage polarization from the M1 to M2 phenotype and consequently exerted anti-oxidant effects in the BPH rat model (Fig. 6). Previous studies have targeted ROS signaling and macrophage reprogramming for drug development [41]. The induction of HO-1 has been reported to determine the switch from the M1 to M2 phenotype and to regulate NO activity [42]. In contrast to M1 macrophage activation, M2 macrophage activation stimulates ARG1 expression and downregulates ROS and NO [43]. Treatment with Apo upregulated Hmox1 expression and promoted the polarization of macrophages from the M1 to M2 phenotype, exerting anti-oxidant effect of Apo in BPH. NOX4 is a novel source of inducible ROS in macrophages. NOX4-dependent ROS production is activated in response to extracellular stimuli (including TGF-β) and mediates various biological functions [44]. TGFB, NOX4, and CD68 (macrophage-specific markers) were upregulated in rats with BPH. However, Apo treatment downregulated the levels of TGFB, NOX4, and CD68. This suggests that Apo exerts anti-oxidant and anti-inflammatory effects through TGF-β-dependent NOX4 signaling.

Involvement of Apo on TGF-β/Activin/Glut9 signaling in BPH

Activin plays a critical role in the pathogenesis of androgen-dependent diseases. A previous study reported that activin A promoted PCa metastasis through the activation and overexpression of AR [45]. An emerging body of evidence indicates that activin A upregulates KLK3 production via an AR-independent pathway. This suggests that activins may cooperate with androgen to upregulate KLK3 [46]. Activins, which are members of the TGF-β superfamily, regulate the production of dominant inflammatory mediators, including TNF-α, IL-6, and iNOS, and promote the onset of the inflammatory response [47]. The mRNA levels of Il6, Il1b, and Tnfa in the Apo group were markedly lower than those in the BPH group. Although further studies will be needed to clarify the molecular mechanism underlying the effects of Apo on BPH, it is possible that the anti-inflammatory effect of Apo could be involved in TGF-β signaling.

The extracellular transport of uric acid is mediated by Glut9, the expression of which is observed in BPH, prostatitis, and normal prostatic tissues at varying degrees [48]. Sangkop et al. also demonstrated that uric acid is a modulator of prostate cells and activin sensitivity, suggesting that Glut9 can counter intracellular ROS by transporting urate in all prostate pathologies [49]. A retrospective cohort study revealed that patients with gout had a markedly increased risk of incident BPH, indicating a correlation between inflammation and BPH [50]. Another cohort study demonstrated that the highest level of serum uric acid was related to the lowest risk of LUTS, emphasizing the role of uric acid in BPH [34]. We found that Apo upregulated serum uric acid levels and regulated the level of Glut9. Considering the role of Glut9 in prostatic diseases, it is likely that the effect of Apo on BPH involves the regulation of uric acid and Glut9.

The correlation between AR, TGF-β, and NOX4 in BPH

Pathway analysis revealed molecular interactions between the upregulated proteins, including TNF, P53, COX2, and IL-1β, in the experimental model (Supplementary Fig. 2). AR modulated the downstream factors KLK3, PCNA, and IL-6, directly regulating TGF-β expression, and consequently regulating the expression of COX2 and IL-1β, which are downstream factors of TGF-β and NOX4. P53 expression was significantly correlated with AR, TGF-β, and NOX4. Additionally, AR, TGF-β, and NOX4 directly and indirectly regulated TNF, a pro-inflammatory cytokine. These results support the in vivo and in vitro findings, suggesting a reciprocal role of AR and NOX4 in BPH and the therapeutic effects of Apo on BPH.

Clinical relevance of Apo

Overall, our study highlights that Apo, which exerts anti-inflammatory and anti-oxidant activities and inhibits AR/TGF-β/NOX4 signaling, is a potential therapeutic target for BPH. Numerous clinical and pre-clinical studies have shown the effectiveness of Apo in many inflammatory disorders without any adverse effects, even with long-term administration [25]. Apo has a good safety profile (oral LD50 of 9 g/kg in mice), and no adverse effects have been reported [51]. The use of phytochemicals has been proposed for their potent anti-cancer effects targeting each step of carcinogenesis that begins with suboptimal health status. As mentioned above, in terms of 3PM/PPPM, the use of Apo as a preventive agent for PCa and treatment for BPH can be considered. Apo can not only overcome the limitations of existing BPH treatments but may also exert its potential as a preventive agent for PCa.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Acknowledgements

Not applicable.

Funding:

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF2019R1A2C4070234 and NRF-2021R1A6A3A01086659), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (HF20C0080).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2023.04.006.

Contributor Information

Bo-Ram Jin, Email: wlsqh92@khu.ac.kr.

Hyo-Jung Kim, Email: hyojung_95@khu.ac.kr.

Jung-Hyun Na, Email: jhna@sungshin.ac.kr.

Won-Kyu Lee, Email: gre7@kbiohealth.kr.

Hyo-Jin An, Email: hjan@khu.ac.kr.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (185.5KB, docx)

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