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

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).

Primary antibodies targeting the following proteins were used: Caspase-3 (#9662, Cell Signaling Technology (CST), Danvers, MA, USA, 1:1000), Cleaved Caspase-3 (#9661, CST, 1:1000), PARP (#9532, CST, 1:1000), Cleaved PARP (#5625, CST, 1:1000), NFκB/p65 (#8242, CST, 1:1000), Phospho-NFκB/p65(Ser536) (#3033, CST, 1:1000), PDCD4 (12587–1-AP, Proteintech, Rosemont, IL, USA, 1:1000), β-Actin (A2228, Sigma-Aldrich, St Louis, MO, USA, 1:5000), and CHOP (#2895, CST, 1:1000). Peroxidase AffiniPure goat anti-mouse IgG (H + L) (115–035-003, 1:2500) and peroxidase AffiniPure goat anti-rabbit IgG (H + L) (111–035-003, 1:2500) were purchased from Jackson ImmunoResearch (West Grove, PA, USA) and used as the secondary antibodies.

BPH-1 cells (1.5 × 10³ cells/well) and WPMY-1 cells (2.5 × 10³ 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 (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA).

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 × 10⁴ cells/well and 1 × 10⁵ 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 × 10⁶ 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).

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.

Cells were lysed with 2% SDS lysis buffer and total protein was quantified with the Enhanced BCA Protein Assay Kit (P0009, Beyotime, Shanghai, China). 10 ~ 20 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. The signals were developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and detected by the Mini Chemiluminescent Imaging and Analysis System (Beijing Sage Creation Science, Beijing, China).

The total RNA from cells was extracted using TRIzol, according to the manufacturer’s instructions. RNA was dissolved in DEPC-treated water (Sangon Biotech, Shanghai, China). The concentration and purity of RNA were measured with a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific), and RNA was kept at − 80 °C. Residual DNAs in total RNAs were removed and cDNAs were synthesized by Hifair II 1st Strand cDNA Synthesis SuperMix (11123ES60, YEASEN, Shanghai, China) following the manufacturer’s protocol. Quantitative real-time PCR was performed by ChamQ Universal SYBR qPCR Master Mix (Q711–02, Vazyme, Nanjing, China) using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the reaction conditions: Stage 1, 95 °C for 30 s; Stage 2, 40 cycles of 95 °C for 10 s and 60 °C for 30 s; Stage 3, 95 °C for 15 s, 60 °C for 60 s and 95 °C for 15 s. Data analysis was performed using the ΔΔCt method. Fold change was determined in relative quantification units using ACTB gene for normalization. Primers were listed in Table S1.

BPH-1 and WPMY-1 cells were seeded into 24-well plates (5 × 10⁴ cells/well). 200 ng 6 × NFκB-Luc plasmid and 50 ng pRL-CMV plasmid for each well were transfected into the cells at ~ 70% confluency by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) in the presence of FBS. Eight hours after transfection, Pao extract was added for 36 h (BPH-1 cells) and 40 h (WPMY-1 cells), respectively. Cell lysates were then measured by Dual-Luciferase Reporter Assay System (E1910, Promega, Madison, WI, USA). The ratio of firefly luciferase activity versus Renilla luciferase activity was determined for NFκB transcriptional activity.

Human BPH tissues (n = 4) were obtained from patients with a transurethral resection of the prostate at Shanghai General Hospital, Shanghai Jiaotong University. The BPH ex vivo explant culture was described previously [17, 18]. In brief, the BPH tissues were subdivided into 3–5 mm³ pieces and cultured on absorbable gelatin sponges (HSD-B, HUSHIDA, Nanchang, China) in 6-well plates containing 4 ml DMEM/F-12 (Corning) with 10% FBS, antibiotic /antimycotic solution (S120, BasalMedia), 0.01 mg/ml insulin (I1882, Sigma-Aldrich) and 0.01 mg/ml hydrocortisone (H0135, Sigma-Aldrich). Tissues were treated with Pao extract for 48 h at 37 °C. The tissues were washed with PBS twice and then grinded with the High Throughput Tissue Grinder (Scientz-48, Ningbo Scientz Biotechnology, Ningbo, China) in protein lysis buffer or TRIzol.

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.

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).

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 and ~ 24% 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).

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).

We also used the aforementioned microarray data to investigate which signaling pathways were regulated by Pao extract in both BPH-1 and WPMY-1 cells by GSEA. The association between the down-regulation in gene set of NFκB signaling pathway and Pao extract treatment was identified in both BPH-1 cells (NES = 2.17, FDR < 0.001) and WPMY-1 cells (NES = 1.24, FDR = 0.12), respectively (Fig. 4a). The phosphorylation levels of RelA (a subunit NFκB transcription complex) were also shown to be reduced in BPH-1 and WPMY-1 cells by Pao extract using Western blotting (Fig. 4b). Moreover, NFκB-reporter was transiently transfected into BPH-1 and WPMY-1 cells, followed by Pao extract treatment. The luciferase activity from NFκB-reporter was suppressed by 60% in BPH-1 cells (p < 0.01) and by 62% in WPMY-1 cells (p < 0.01) using 500 μg/ml Pao extract (Fig. 4c). The expression levels of several well-known NFκB target genes involved in inflammation (CXCL5, CXCL6, and CXCL12) and extracellular matrix (ECM) remodeling (HAS2, TNC, and MMP13) were further tested. Consistently, HAS2, CXCL6, CXCL5, and MMP13 were down-regulated in BPH-1 cells and HAS2, CXCL6, CXCL12, TNC were down-regulated in WPMY-1 cells by Pao extract (Fig. 4d). Altogether, it was suggested that Pao extract suppresses the activation of NFκB signaling in both BPH epithelial and stromal cells.

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.