Chemical and reagents
The RWF extract (generously provided by Maison Beljanski, New York, NY) was prepared with aqueous-alcoholic extraction of the root bark from the tropical shrub Rauwolfia vomitoria, followed by a spray drying to produce a free-flowing powder form. The final product is standardized using HPLC for the active component alstonine, and reserpine is undetectable. Quality control includes tests for pesticides, heavy metals, bacteria, yeast, and mold. For experiments, the RWF extract was dissolved in DMSO and further diluted with sterile phosphate buffered saline (PBS) to 50 mg/ml stock solutions. The RWF extract was stored in aliquots at -80 °C until use. Cisplatin (Cat #: HY-17394), ER inhibitor 4-phenylbutyrate (4-PBA; Cat #: HY-A0281), and autophagy inhibitor 3-methyladenine (3-MA; Cat #: HY-19312) were purchased from MedChemExpress (MCE) Co., Ltd (Shanghai, China).
Human BPH-1 cell line, derived from BPH tissues, was a gift obtained from Dr. Simon Hayward (Vanderbilt University Medical Center, Nashville, TN), while human prostate myofibroblast WPMY-1 cells were purchased from CBTCCCAS (The Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China). BPH-1 and WPMY-1 cell lines were cultured as monolayers in RPMI 1640 and DMEM respectively, supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) and penicillin (100 U/ml) plus streptomycin (100 μg/ml) (S110JV, BasalMedia, Shanghai, China) at 37 °C and 5% CO2.
BPH-1 cells (2 × 103 cells/well) and WPMY-1 cells (6 × 103 cells/well) were seeded in 96-well plates, respectively. On the 2nd day, cells were treated with 0, 125, 250, and 500 μg/ml RWF extract for 0, 24, 48, and 72 h. Cell viability was measured by staining cells with 10 μl of 5 mg/ml MTT (Cat #: 143315, Biofroxx) to each well in a 37℃ incubator for 4 h, followed by dissolution with 100 μl DMSO for each well. Optical density (OD) was measured by a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA) at 490 and 680 nm. For the rescue assay, cells were pre-treated with an ER stress inhibitor 4-PBA (250 µM) or an autophagy inhibitor 3-MA (500 µM) for 2 h, followed by the addition of RWF extract (500 µg/ml).
Flow cytometry analysis
Cell apoptotic rates were examined by FITC Annexin V Apoptosis Detection Kit I (Cat #556547, BD Biosciences, Franklin Lakes, NJ), according to the manufacturer’s instructions. Briefly, BPH-1 and WPMY-1 cells were cultured in RPMI1640 and DMEM (containing 10% FBS) in 6-well plates at densities of 50,000 cells/well and 100,000 cells/well, respectively. The cells were then treated with various concentrations of RWF extract (0, 125, 250, and 500 μg/ml) for 72 h. The 72 h treatment of cisplatin (5 μM) served as the positive control. Both the floating and adherent cells were harvested and washed with PBS. Afterwards, cells were stained with both Annexin V-FITC and propidium iodide (PI) in a dark place for 15 min. The cell death patterns were examined by a flow cytometer with 10,000 events and analyzed by FlowJo software (ver10.0.7r2, Ashland, OR).
Cells were lysed using a modified RIPA Lysis Buffer (150 mM NaCl, 1 mM Na3VO4, 50 mM Tris–HCl-pH 7.5, 25 mM NaF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1% phosphatase inhibitor cocktails) on ice for 30 min. After lysates were centrifuged at 12,000 g to remove debris, protein concentrations were measured by BCA Protein Assay Kit (Cat #: P0011, Beyotime, Shanghai, China). 10–25 μg proteins were fractionated by SDS-PAGE, followed by the transfer to nitrocellulose membrane (Millipore, Billerica, MA). The membranes were then cropped based on the prestained protein marker for the subsequent incubations of corresponding primary antibodies (Supplementary Table 1) at 4 ℃ for 12 h. The incubation of secondary antibody was performed at room temperature for 1 h after TBST solution washing. Blots were visualized by adding SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA) in the Mini Chemiluminescent Imaging and Analysis System (Beijing Sage Creation Science, Beijing, China).
Gene expression profiling
Total RNA was collected by TRIzol reagent (Thermo Fisher Scientific) from BPH-1 and WPMY-1 cells exposed to the 250 μg/ml of RWF extract for 24 h. Gene expression profiling analyses were performed by Shanghai Baygene Biotechnologies (Shanghai, China) using the Affymetrix gene chips (Human Transcriptome Array 2.0). The microarray data were deposited in the NCBI GEO Database with the accession number: GSE151643.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Total RNA from BPH cells or human BPH tissues treated with the indicated concentration of RWF extract was extracted by TRIzol. Afterwards, 1 μg of total RNA was used for cDNA synthesis using a Hifair® II 1st strand cDNA Synthesis SuperMix for qPCR (Cat #: 11123ES60, YEASEN, Shanghai, China). qRT-PCR was performed in triplicates using designated gene-specific primers (Supplementary Table 2) by CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The expression of ACTB gene was used as an internal control.
XBP1 splicing assay
The total RNA and cDNA from BPH-1 and WPMY-1cells were prepared as described above. The splicing variants of XBP1 were amplified using specific primers (Supplementary Table 3) by RT-PCR. The PCR products were then separated on 2.0% agarose gel and visualized by the staining with Ultra GelRed dye (Cat #: GR501-01, Vazyme Biotech, Nanjing, China).
GFP-LC3 lentiviral particles were produced by co-transfection of with GPF-LC3 plasmid together with psPAX2 packaging plasmid and pMD2.G envelope plasmid to 293 T cells using the transfection reagent polyethyleneimine (PEI; Cat #: 408727, Sigma-Aldrich, St. Louis, IL). 48 h after transfection, lentivirus-containing supernatant was collected and filtered using Millex-HV syringe filter unit, low adsorption 0.45 μM (Cat #: SLHU033RB, MERCK, Burlington, MA). BPH-1 cells were infected three times and WPMY-1 cells were infected twice with the virus-containing media in the presence of 8 μg/ml polybrene (Cat #: TR-1003, Sigma-Aldrich). Puromycin (Cat #: 58–58-2, Selleck, Houston, TX) was used to select infected cells (2 μg/ml for WPMY-1 and 15 μg/ml for BPH-1 cells). GFP-LC3 stably transduced BPH-1 and WPMY-1 cells were treated with/without RWF (250 μg/ml and 500 μg/ml), respectively. After 24 h growing on cover glasses, cells were washed in PBS, followed by the fixation in 4% paraformaldehyde before the visualization by LEICA SP8 confocal microscope. The number of GFP-LC3 puncta per cell was counted in 30 individual cells for each group. Brown-Forsythe and Welch ANOVA test was performed to determine the statistical significance of differences among multiple groups.
Human BPH ex vivo assay
Human BPH ex vivo explants (n = 6) were obtained from BPH patients with radical prostatectomy at Shanghai General Hospital, Shanghai Jiao Tong University (China). Briefly, fresh tissues were immersed in sterile DMEM/F-12 medium (Cat #: 10–920-CV, Corning, New York, NY) on ice and transported to the laboratory immediately. After the burnt part of the fresh tissue was removed, BPH tissue was dissected into 3–5 mm3 pieces. The BPH explants were cultured on the sterile gelatin sponge (Cat #: HSD-B, Hushida Medical Care Technology, Nanchang, China), which was pre-soaked in DMEM-F12 medium containing 10% FBS, 0.01 mg/ml hydrocortisone (Cat #: H0135, Sigma-Aldrich), antibiotic/antimycotic solution (Cat #: S120JV, BasalMedia), and 0.01 mg/ml insulin (Cat #: I1882, Sigma-Aldrich) in 6-well-plates at 37 °C, as described previously [20,21,22]. The next day, tissues were treated with various concentrations of RWF extract (0, 250, and 500 μg/ml) in fresh medium for 24 h (to induce ER stress and autophagy) or 48 h (to induce apoptosis).
The experiments were performed in three independent replicates, and the statistical analyses were performed using Student’s t test in GraphPad Prism 7.0 software (GraphPad, San Diego, CA).ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.