Pao Pereira extract suppresses benign prostatic hyperplasia by inhibiting inflammation-associated NFκB signaling

Background Our previous study revealed the extract from the bark of an Amazonian tree Pao Pereira can suppress benign prostatic hyperplasia (BPH) in a rat model. Herein, we examined its inhibitory effects on human BPH cells and dissect its molecular mechanism. Methods We applied Pao extract to human BPH epithelial BPH-1 and prostate myofibroblast WPMY-1 cells. Cell viability, apoptosis and immunoblotting were performed, followed by gene expression profiling and gene set enrichment analysis (GSEA) to detect the differentially expressed genes and signaling pathway induced by Pao extract. Human ex vivo BPH explant organ culture was also used to examine the effects of Pao extract on human BPH tissues. Results Pao extract treatment inhibited viability and induced apoptosis in human BPH-1 and WPMY-1 cells. Gene expression profiling and the following validation indicated that the expression levels of pro-apoptotic genes (eg. PCDC4, CHOP and FBXO32) were induced by Pao extract in both two cell lines. GSEA further revealed that Pao extract treatment was negatively associated with the activation of NFκB signaling. Pao extract suppressed the transcriptional activity of NFκB and down-regulated its target genes involved in inflammation (CXCL5, CXCL6 and CXCL12) and extracellular matrix (ECM) remodeling (HAS2, TNC and MMP13) in both cultured cells and human ex vivo BPH explants. Conclusion In both BPH epithelial and stromal cells, Pao extract induces apoptosis by upregulating the pro-apoptotic genes and inhibiting the inflammation-associated NFκB signaling via reducing phosphorylation of NFκB subunit RelA. Our data suggest that Pao extract may be a promising phytotherapeutic agent for BPH.


Background
Benign prostatic hyperplasia (BPH) is a non-malignant enlargement of the prostate gland that is common in older males. About 70% of men over 70 will develop BPH [1]. Due to the excessive proliferation of the epithelial and stromal cells in the transition zone and periurethral glands, the enlarged prostate ultimately induces lower urinary tract symptoms (LUTS) including urgency and difficulty of urination [2,3]. Moderate-to-severe LUTS have significant influences on the patients' quality of life. The etiology of BPH is multi-factorial, including sex hormones, smooth muscle and inflammation [4]. Currently, medications for BPH/LUTS primarily target sex hormone synthesis and relief of tension in smooth muscle. However, alpha-blockers and 5α-reductase inhibitors (5-ARIs) induce non-trivial adverse side effects including asthenia and ejaculatory dysfunction [5]. Hence, more effective agents with fewer side effects are needed.
Chronic inflammation has been implicated in BPH development, as manifested by the infiltration of immune cells, including activated T cells and macrophages into human BPH tissues [6]. It was reported that chronic prostatic inflammation was associated with a larger prostate volume and a higher International Prostate Symptom Score (IPSS) [7].
Pao pereira (Geissospermum vellosii) is an Amazon rainforest tree, and its bark extract is used to treat malaria, digestive disorders and cancers. Pao extract is rich in β-carboline alkaloids [8] and exhibited antiproliferative activities against melanoma and glioblastoma, ovarian cancer and pancreatic cancer [9][10][11][12][13]. Our previous study showed that Pao extract decreased human prostate cancer cell growth via inducing apoptosis [14]. Recently we found that Pao extract can attenuate BPH development in a rat model [15]. However, the molecular mechanism remains largely unclear.
In this study, we investigated the effects of Pao extract on human BPH epithelial cells (BPH-1) and stromal cells (WPMY-1). We found that Pao extract suppressed the growth of BPH-1 and WPMY-1 cells, and induced apoptosis via inhibition of NFκB signaling pathway. An ex vivo human BPH explant was also exploited to test the effects of Pao extract. These findings suggest that Pao extract may be a very promising therapeutic agent for BPH.

Cell lines, human BPH tissues and reagents
Human BPH-derived prostate epithelial BPH-1 cell line was kindly provided by Dr. Simon Hayward (Vanderbilt University Medical Center, Nashville, TN, USA). Human prostate myofibroblast WPMY-1 cell line was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). BPH-1 and WPMY-1 cells were cultured in RPMI 1640 (Corning, New York, NY, USA) and DMEM medium (Corning), respectively, containing 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA) and penicillin-streptomycin (S110JV, BasalMedia, Shanghai, China). Passage number of BPH-1 and WPMY-1 cell lines was less than 20 for all the cell-based experiments. The study protocol using human BPH tissues was approved by the Ethics Committee of Shanghai General Hospital, Shanghai Jiaotong University. Human BPH tissues were collected also with patients' consent. Pao extract was from Natural Source International (New York, NY, USA). Briefly, the extract was prepared with aqueous alcoholic extraction of the bark of the plant Pao Pereira, which was then transformed into a free-flowing powder by spray drying. It contained 54% β-carboline alkaloids, including flavopereirine, by high-performance liquid chromatography [13,15]. The Pao extract was dissolved in DMSO and diluted with sterile phosphate buffered saline (PBS) to 50 mg/ml as a stock solution and stored at − 80°C until use. The final concentration of DMSO was less than 6% (v/v).

Antibodies
Primary antibodies targeting the following proteins were Cell cytotoxicity assay BPH-1 cells (1.5 × 10 3 cells/well) and WPMY-1 cells (2.5 × 10 3 cells/well) were seeded into 96-well plates in triplicate. The next day Pao extract with different concentrations was added into culture medium. 24, 48 and 72 h later, the cells were fixed with 10% trichloroacetic acid (TCA; Sigma-Aldrich) over 4 h and stained with 4 mg/ml sulforhodamine B (SRB; Sigma-Aldrich) in 1% acetic acid. 10 mM Tris base solution was used to dissolve the protein-bound dye. Optical density (OD) at 560 nm was determined by a microplate reader (Spectra-Max M5, Molecular Devices, Sunnyvale, CA, USA).

Apoptosis assay
Apoptosis assay was performed using FITC Annexin V Apoptosis Detection Kit I (#556547, BD Biosciences, Franklin Lakes, NJ, USA). BPH-1 and WPMY-1 cells were seeded into 6-well plates at densities of 5 × 10 4 cells/well and 1 × 10 5 cells/well, respectively. The next day, the medium was replaced with fresh medium and the cells were treated with Pao extract or cisplatin (5 μM, a positive control) for 72 h. Then the floating and adherent cells were collected, washed with PBS twice and re-suspended in 1 × Binding Buffer as 1 × 10 6 cells/ ml. 100 μl cell suspension was stained with 5 μl of FITC-Annexin V and 5 μl propidium iodide (PI) for 15 min at room temperature in the dark. Flow cytometry analysis was performed with a FACS Calibur flow cytometer (BD Biosciences) and cell death patterns were quantified ( Figure S1) with FlowJo software (version 10.0.7r2, Ashland, OR, USA).

Gene expression profiling analysis
BPH-1 and WPMY-1 cells were treated with Pao extract for 24 h, and total RNA was extracted with TRIzol reagent (Life Technologies). Affymetrix GeneChips (Human Transcriptome Array 2.0) were used for the gene expression profiling analysis (Shanghai Baygene Biotechnologies, Shanghai, China). The raw and normalized microarray data from this study can be accessed at GSE128856 in NCBI GEO Datasets. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA software (v3.0, http://software.broadinstitute.org/gsea/ index.jsp) [16] to determine the association between the priori defined gene sets in the Molecular Signatures Database (MSigDB) and the different changes of genes induced by Pao extract treatment. The number of permutations was 1000. Enrichment statistic was weighted and the ranking metric was the difference of class means (Diff_of_Classes). Normalized enrichment score (NES) ≥ 1 and the false discovery rate (FDR) < 0.25 were used as the cutoff.

Western blotting
Cells were lysed with 2% SDS lysis buffer and total protein was quantified with the Enhanced BCA Protein Assay Kit (P0009, Beyotime, Shanghai, China). 102 0 μg total protein was separated on an SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA; FA016, Gen-view Scientific, Houston, TX, USA) and incubated with the primary antibodies overnight at 4°C. Then the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature.

Statistical analysis
Statistical analyses were performed using GraphPad Prism Software (version 8.0.1, GraphPad, San Diego, CA, USA). Two-tailed Student's t test was used to compare the difference between two groups, and P < 0.05 was considered statistically significant.

Pao extract inhibited proliferation of BPH-1 and WPMY-1 cells
To evaluate the effect of Pao extract on the growth of cells derived from the prostate, BPH-1 and WPMY-1 cells were exposed to Pao extract with dosages ranging from 125 to 500 μg/ml, and the cell proliferation were assessed by SRB assay. Pao extract treatment significantly reduced the adherent cell number at 48 h (Fig. 1a) and viability within 72 h (p < 0.05) in a concentration-dependent manner in both cell lines (Fig. 1b).

Pao extract induced apoptosis in BPH-1 and WPMY-1 cells
Flow cytometry analyses were performed to determine whether Pao extract could induce apoptosis in BPH-1 and WPMY-1 cells. After 72 h treated with Pao extract, both cells showed that the percentages of apoptotic cells were significantly increased in a concentration-dependent manner by Annexin V-FITC/PI double staining (Fig. 2a). Notably, apoptosis was detected in~43% BPH cells and2 4% WPMY-1 cells under the Pao extract treatment at 500 μg/ml (Fig. 2b). Moreover, the levels of cleaved Caspase-3 and cleaved PARP were consistently increased in the Pao extract-treated cells (Fig. 2c).

Pao extract regulated the pro-apoptotic and inflammation-associated genes in BPH-1 and WPMY-1 cells
To investigate which genes were regulated by Pao extract in both BPH-1 and WPMY-1 cells, gene expression profiling was performed using the Affymetrix microarray chips. Using 1.4-fold as the cutoff, 106 up-regulated genes and 68 down-regulated genes were identified in Pao extract-treated BPH-1 cells, comparing to the vehicle-treated cells (Fig. 3a, left panel); 212 up-regulated genes and 511 down-regulated genes were identified in Pao extract-treated WPMY-1 cells (Fig. 3a, right panel). Among them, several pro-apoptotic and inflammation-associated genes were induced by 250 μg/ml Pao extract treatment (Fig. 3b). We further confirmed the induction of the pro-apoptotic genes (PDCD4, FBXO32 and DDIT3) in both BPH-1 and WPMY-1 cells by Pao extract at mRNA level (Fig. 3c). Western blotting data also indicated that Pao extract increases PDCD4 and DDIT3/CHOP at protein level (Fig. 3d).

Pao extract inhibited NFκB signaling pathway in human BPH tissues
To validate the effects of Pao extract on human BPH, an ex vivo explant culture system was established for the tissues from BPH patients (Fig. 5a). BPH explants were treated with 0, 250 and 500 μg/ml Pao extract for 48 h, following the assessment of NFκB signaling. Consistent with the data from cultured cell lines, the phosphorylation levels of RelA were markedly decreased and the protein levels of PDCD4 and DDIT3/CHOP were increased in Pao extract-treated BPH explants (Fig. 5b). In addition, the mRNA expression levels of the downstream target genes of NFκB signaling, including CXCL6, HAS2, CXCL5, CXCL12, MMP13 and TNC, were also significantly reduced (p < 0.01), as well as the up-regulation of the apoptosis-associated genes PDCD4, DDIT3 and FBXO32 (p < 0.05), in BPH explants treated with Pao extract (Fig. 5c). Taken together, it was demonstrated that Pao extract inhibited the NFκB signaling pathway in human BPH tissues.

Discussion
Our previous study demonstrated that Pao extract suppresses testosterone-induced BPH in a rat model. To examine its effects on human BPH sample and delineate its molecular mechanism, herein we found that Pao extract inhibited the viabilities of BPH epithelial cells and stromal cells in a dose-dependent manner, due to the increase of apoptosis and suppression of NFκB signaling. Downregulation of inflammation-and ECM remodelingrelated genes via NFκB signaling was further demonstrated in human BPH cell lines in vitro and in human BPH explants ex vivo (Fig. 6). The canonical NFκB pathway is frequently upregulated during the progression of BPH, and the severity of BPH is correlated with activation of NFκB [19], which makes it an interesting target for BPH. For example, Elocalcitol was reported to inhibit BPH stromal cells proliferation by targeting NFκB/p65 nuclear translocation [20]. Here, we showed that Pao extract could inhibit the phosphorylation level of RelA (Ser536), leading to the decrease of transcriptional activity of NFκB complex. In addition, Pao extract increased the mRNA and protein expression levels of two pro-apoptotic proteins, PDCD4 and DDIT3/CHOP. PDCD4 was reported to directly bind with NFκB/p65 and suppressed NFκB-dependent transcription in human glioblastoma [21]. Hence, it is suggested that Pao extract may target NFκB activity in BPH at different levels.
As an age-related disease, BPH progression is accompanied by chronic inflammation and ECM remodeling around BPH nodules. The inflammatory BPH microenvironment contains various secreted cytokines and chemokines, including CXCL5, CXCL6 and CXCL12, which are the direct NFκB target genes [22][23][24] and can promote the proliferation of both prostatic epithelial cells and stromal fibroblasts [25][26][27]. For the ECMassociated genes, such as HAS2, MMP13 and TNC, they are also target genes of NFκB signaling. HAS2 gene Fig. 5 Pao extract inhibited NFκB signaling pathway in human BPH tissues. a Schematic diagram of the ex vivo BPH explant culture process. BPH explants were treated with 0, 250, 500 μg/ml Pao extract for 48 h. b The phosphorylation of RelA and protein expression levels of pro-apoptotic and inflammation-associated genes survival-associated proteins (PDCD4 and DDIT3/CHOP) were analyzed by Western blotting in BPH explants with Pao extract treatment. c The mRNA expression levels of target genes from NFκB signaling pathway, pro-apoptotic and inflammationassociated genes in BPH explants with Pao extract treatment by qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns p ≥ 0.05

Dong et al. BMC Complementary Medicine and Therapies
(2020) 20:150 Page 8 of 10 encodes hyaluronan synthase 2, an enzyme that synthesizes hyaluronan (HA) in BPH tissues [28,29]. When BPH-1 cells were cultured in 3D gel containing collagen, they proliferated faster in the collagen from aged mice (high level of HA) than that from young mice (low level of HA). Previous studies also showed that MMP13 promoted ECM degradation, and the elevated ECM glycoprotein Tenascin-C was associated with myofibroblast in BPH tissues [30,31]. Here, we observed that Pao extract downregulates the expression levels of CXCL5, CXCL6, CXCL12, HAS2, MMP13 and TNC in BPH-1 and WPMY-1 cells, thus indicating that Pao extract attenuates the inflammation and ECM-remodeling via inhibition of NFκB signaling in BPH.

Conclusions
Our data have proved the inhibitory effect of Pao extract on NFκB signaling pathway in two cell lines derived from human BPH and ex vivo explants from human BPH patients. Using Pao extract as a negative regulator of NFκB signaling may be a promising phytotherapeutic agent for BPH.