Reagents
Viscosalactone B was provided by Wuhan Pubiao Technology Co., LTD. The chemical purity was > 99% analyzed by high performance liquid chromatography (Leaps iUHPLC, Chromai, Beijing, China).
Cell Culture
Prostate cancer cell lines (PC3, DU145 and C42B) were obtained from Shanghai Yuanye Bio-Technology Co., LTD (Shanghai, China). These prostate cancer cells were cultured in RPMI1640 medium (Shanghai Yuanye Bio-Technology Co., LTD, Shanghai, China) with 10% fetal bovine serum (Shanghai Yuanye Bio-Technology Co., LTD, Shanghai, China). MDV3100-resistant prostate cancer (PC3/MDVR, DU145/MDVR and C42B/MDVR), DU145&shLSD1 and DU145&shControl cell lines were established according to reported references [8, 26, 27]. All cancer cell lines were maintained at 37 oC under 5% CO2.
Thiazolyl Blue Tetrazolium Bromide Assay
Prostate cancer cell lines and MDV3100-resistant prostate cancer cell lines were seeded in 48-well plates with RPMI1640 medium for 24 hours. Viscosalactone B was dissolved in dimethyl sulfoxide (Shanghai Yuanye Bio-Technology Co., LTD, Shanghai, China). Nextly, the solution containing viscosalactone B was added at different concentrations. With the incubation for 72 hours, thiazolyl blue tetrazolium bromide solution was added to each well for 2 hours. The supernatant was removed and dimethyl sulfoxide was added to each well. The absorbance was read at 490 nm by a microplate reader (Varioskan LUX, Beijing Prang New Technology Co., LTD, Beijing, China).
Xenograft Studies
BALB/c nude mice weighing 18–25 g were purchased from Shanghai Yuanye Bio-Technology Co., LTD (Shanghai, China). DU145 cell lines were cultured and transplanted to establish prostate cancer xenograft models. These animal experiments were treated according to the protocols established by the ethics committee of Dandong Center hospital laboratory (Number: YGB-2020-02). BALB/c nude mice bearing tumors were separated into control group and treatment group (30 mg/kg). The treatment group received viscosalactone B for 21 days by intragastric administration. Finally, tumors from BALB/c nude mice were isolated and weighted.
Hematoxylin-eosin Staining
Hematoxylin and Eosin Staining Kit was purchased from Shanghai Yuanye Bio-Technology Co., LTD (Shanghai, China). Spleen, heart, lung, liver and kidney isolated from BALB/c nude mice were treated with xylene (Aladdin, Shanghai, China) and water. These organs were reserved at -20 oC and fixed by 95% ethanol. Nextly, they were stained by hematoxylin solution and eosin solution from the above kit. The staining results were observed via a microscope (OLYMPUS, Nanjing Iruda Instrument and Equipment Co., LTD, Nanjing, China).
Enzymatic Activity Assay
Different enzymes and kits (MAO-A, MAO-B, LSD1, CDK1, CDK2 and CDK3) were purchased from Shanghai Yuanye Bio-Technology Co., LTD (Shanghai, China). Enzymatic activity assay were performed according to the manufacturer’s protocols and reported references [28–31]. Viscosalactone B was dissolved in dimethyl sulfoxide and added into 48- well plates. Then, the solution of targeted enzyme was treated and incubated for 60 minutes at room temperature.
Quantitative Real-time Pcr
Total RNA was isolated from DU145&shLSD1 and DU145&shControl cell lines and quantified with Nanodrop (Nanodrop2000, Shanghai Yuanye Bio-Technology Co., LTD, Shanghai, China). cDNA was synthesized from total RNA using PrimeScript RT Reagent Kit (Shanghai Yuanye Bio-Technology Co., LTD, Shanghai, China). The primer sequences were as follows: LSD1 forward, 5’-GTGGACGAGTTGCCACATTTC-3’; LSD1 reverse, 5’-TGACCACAGCCATAGGATTCC-3’; GAPDH forward, 5’-GCACCGTCAAGGCTGAGAAC-3’; GAPDH reverse, 5’-TGGTGAAGACGCCAGTGGA-3’. The quantitative real-time PCR procedure was performed according to the reported reference [32].
Western Blot
Western blot
With the treatment of viscosalactone B at different concentrations for 48 hours, RIPA lysis buffer (Thermo Fisher, Shanghai, China) was added to harvest DU145 cells. 20 µg of proteins were electrophoresed on SDS-PAGE gel and transferred to PVDF membrane (Thermo Fisher, Shanghai, China). H3K4me1, H3K9me1, H3K9me2 and H3 were obtained from Shanghai Yuanye Bio-Technology Co., LTD (Shanghai, China). ECL kit (Shanghai Yuanye Bio-Technology Co., LTD, Shanghai, China) was used to visualize protein blots.
Molecular Docking
Molecular docking studies of viscosalactone B targeting LSD1 were performed via the AutoDock 4.2 software (The Scripps Research Institute, California, USA). Crystal structure of LSD1 was downloaded from Protein Data Bank (https://www.rcsb.org/, PDB code: 2V1D). PDB file of viscosalactone B was obtained by ChemBio3D Ultra 14.0. Autogrid and autodock were performed to compute the binding model between LSD1 and viscosalactone B. Hydrogen bonds, hydrophobic effects and hydrophilic interactions of viscosalactone B targeting LSD1 were analyzed by the Pymol software.