Viscosalactone B, a natural LSD1 inhibitor, inhibits proliferation in vitro and in vivo against prostate cancer cells

Lysine-specific demethylase 1 (LSD1) has been a promising target to treat prostate cancer, and discovery of novel LSD1 inhibitors would have great clinical significance. In this work, viscosalactone B was first identified as a novel LSD1 inhibitor. Viscosalactone B isolated from Withania Somnifera displayed antiproliferative activity against PC3, DU145, C42B, PC3/MDVR, DU145/MDVR, and C42B/MDVR cells with IC50 values of 1.17, 0.72, 3.86, 2.06, 0.96 and 1.15 μM, respectively. In comparison, it was a selective LSD1 inhibitor with an IC50 value of 970.27 nM and could induce a significant accumulation of LSD1 substrates H3K9me1, H3K9me2, and H3K4me1 in a concentration-dependent manner in DU145 cells. According to docking studies, it formed hydrogen bonds with the Thr11, Lys14, and Arg8 residues of LSD1. Importantly, while it displayed potent antitumor efficacy in vivo, it did not show obvious cytotoxicity on the major organs of nude mice. Therefore, viscosalactone B, as a novel LSD1 inhibitor, is a potential candidate that can be used for the treatment of prostate cancer in clinics.


Introduction
Lysine-specific demethylase 1 (LSD1), as an epigenetic enzyme, could remove the methyl group from lysine residues on the histone H3 protein via flavin adenine dinucleotidedependent oxidative reaction [1][2][3]. LSD1 is overexpressed in various cancer and has been identified as a druggable target for cancer therapy [4]. Nowadays, many LSD1 inhibitors are undergoing clinical assessment for the treatment of prostate cancer, gastric cancer, acute myeloid leukemia, lung cancer, breast cancer, and liver cancer [5][6][7]. Importantly, some natural products with different chemical scaffolds exhibit potent inhibitory effects against LSD1 [8]. Therefore, developing novel LSD1 inhibitors from natural products has been an effective strategy in prostate cancer drug discovery.
Withania Somnifera is an important medicinal plant used in traditional medicine in many countries for beneficial health activities [9]. It exhibits potential pharmacologic properties, including anti-inflammatory, anticancer, antimicrobial, neuroprotective and anti-stress activities [10].
Natural products used as traditional medicines from ancient times are readily applicable and inexpensive; moreover, they possess minimum cytotoxicity [11]. In the last two decades, some natural products isolated from Withania Somnifera have shown excellent antiproliferative ability in the treatment of several cancers, including colon, lung, prostate, liver, ovarian, breast, and skin cancers [12]. Viscosalactone B, withanolideA, withalongolide A, withanolide D, and withanone isolated from Withania Somnifera, as potential anti-carcinogenic agents, can provide useful scaffolds for the discovery of small molecule inhibitors for cancer treatment [13,14].
In order to develop novel chemical scaffolds as LSD1 inhibitors and anti-prostate agents, high throughput screening was effectively utilized in our group to screen LSD1 inhibitors from ZINC data library. As a result, viscosalactone B (Fig. 1) from Withania Somnifera was screened as a potential LSD1 inhibitor. In this work, the antiproliferative effects of viscosalactone B were evaluated against prostate cancer cells and MDV3100-resistant prostate cancer cells. Its enzymatic activity and selectivity were also analyzed against LSD1. Finally, the anticancer effects in vivo and molecular docking studies of viscosalactone B were investigated.

Viscosalactone B inhibits the proliferation of prostate cancer cells and MDV3100-resistant prostate cancer cells
The antiproliferative effects of viscosalactone B against prostate cancer cells and MDV3100-resistant prostate cancer cells were examined by thiazolyl blue tetrazolium bromide assay. All these cancer cell lines were treated with different concentrations of viscosalactone B (0.5, 1, 2 and 4 μM) for 72 h. Viscosalactone B could markedly inhibit the proliferation of prostate cancer cells and MDV3100-resistant prostate cancer cells in a concentration-dependent manner (Fig. 2). As a natural product isolated from Withania Somnifera, viscosalactone B exhibited excellent antiproliferative activity against PC3, DU145, C42B, PC3/MDVR, DU145/ MDVR and C42B/MDVR cells with IC 50 values ranging from 0.72 μM to 3.86 μM. Specifically, viscosalactone B showed the best antiproliferative effect against prostate cancer DU145 cells with an IC 50 value of 0.72 μM.

In vivo antitumor effects of viscosalactone B
To determine the in vivo antitumor effects of viscosalactone B, we first established prostate cancer xenograft mouse models bearing DU145 cells. Intragastric administration of viscosalactone B was used to perform these animal experiments. After treatment with viscosalactone B, tumor volume, tumor weight, and weight of mice were recorded. Compared to the control group, viscosalactone B at a concentration of 30 mg/kg could significantly suppress DU145 tumor growth (Fig. 3a). As shown in Fig

Viscosalactone B exhibits no obvious cytotoxicity on the major organs of nude mice
Based on the above results of mice weight, viscosalactone B might display weak cytotoxicity. In order to further investigate the cytotoxicity of viscosalactone B, hematoxylin-eosin staining was performed. Major organs were dissected from the BALB/c nude mice, and sectioned into 5-μm slices, which were then stained with hematoxylin and eosin according to the manufactor's protocol. Finally, the stained slices were observed under a microscope and analyzed to determine the cytotoxicity of viscosalactone B. As shown in Fig. 4, hematoxylin-eosin staining analysis suggested that viscosalactone B displayed no obvious in vivo cytotoxicity on the major organs, including the spleen, heart, lung, liver, and kidney.

Selective inhibition of viscosalactone B against LSD1
We next evaluated the inhibitory activity of viscosalactone B against LSD1. In order to investigate the enzymatic selectivity of viscosalactone B, MOA-A, MOA-B, CDK1, CDK2, and CDK3 were also tested. All these enzymes were treated with viscosalactone B at 2 μM. The inhibitory rates of viscosalactone B against MOA-A, MOA-B, CDK1, and CDK2 were < 20%. However, the inhibitory rate of viscosalactone B at the same concentration against LSD1 was 68.3%. As shown in Fig. 5a, the IC 50 value of viscosalactone B against LSD1 was 970.27 nM. All these results showed that viscosalactone B was an inhibitor selective for LSD1. As illustrated in Fig. 5b, viscosalactone B could inhibit LSD1 in a time-and concentration-dependent manner.

Antiproliferative effects of viscosalactone B against DU145 LSD1 knockdown cells
In view of the potent inhibitory activity against LSD1 and the strong antiproliferative activity against DU145 cells, viscosalactone B was chosen for further studies. In this work, LSD1 knockdown DU145 cell line (DU145&shLSD1) and control cell line (DU145&shControl) were established to investigate the antiproliferative effects of viscosalactone B in vitro. The expression levels of LSD1 in DU145&shLSD1 and DU145&shControl cells were detected. As shown in DU145&shLSD1 and DU145&shControl cells were examined by thiazolyl blue tetrazolium bromide assay. As shown in Fig. 6b,

Viscosalactone B induced the accumulation of LSD1 substrates
To evaluate the cellular activity of viscosalactone B against LSD1, diverse substrates of methylated histone 3 (H3K4me1, H3K9me1 and H3K9me2) in prostate cancer cells were screened. DU145 cell line was treated with different concentrations of viscosalactone B (0.3, 0.6 and 0.9 μM) for 48 h. As shown in Fig. 7, viscosalactone B induced the significant accumulation of LSD1 substrates H3K9me1/2 and H3K4me1 in DU145 cells in a concentration-dependent manner.

Molecular docking studies of viscosalactone B
In order to investigate the binding models and interactions of viscosalactone B with LSD1, molecular docking studies were performed using the Autodock software. The LSD1 structure file was downloaded from the RCSB protein database and the PDB code was 2V1D. As shown in Fig. 8a, viscosalactone B could bind to the active pocket of LSD1. As shown in Fig. 8b, the carbonyl group of viscosalactone B formed a hydrogen bond with the Thr11 residue in LSD1. In addition, two hydroxyl groups and the lactone unit formed three hydrogen bonds with the Lys14 and Arg8 residues in LSD1. Viscosalactone B could

Concentration (μM)
exert hydrophobic effects by binding with the Leu677, Pro171, Trp531, and Ala541 residues in LSD1, respectively. Moreover, viscosalactone B could also exert hydrophilic effects by binding with the Gln358 and Asn535 residues.

Discussion
Prostate cancer as a frequently diagnosed cancer affects men's health in the world [15][16][17]. Treatments for prostate cancer are becoming significantly aggressive and intensive, with a corresponding increase in toxicity and drug resistance [18]. Chemopreventive agents containing natural products and synthetic compounds could be used for prostate cancer therapy [19]. Natural products, including chalcone, coumarin, polyphenols, alkaloids and sesquiterpenes exhibit the potent antiproliferative activity against prostate cancer [20]. Therefore, developing natural products has been a new strategy for the treatment of prostate cancer. LSD1, as one of the flavin-dependent oxidases, could control the methylation of histone H3 and affect many cellular processes in cancer [21]. In the last two decades, LSD1 has been an attractive target for cancer treatment [22]. Synthetic LSD1 inhibitors (ORY-1001, tranylcypromine, IMG-7289 and GSK2879552) entered in clinical trials to treat breast cancer, prostate cancer, lung cancer and acute myeloid leukemia [23]. Natural LSD1 inhibitors, such as protoberberine alkaloids, flavones, xanthones, and diarylheptanoids, also exhibit potent activity to inhibit LSD1 [24]. Compared with synthetic LSD1 inhibitors, natural LSD1 inhibitors showed better safety. Therefore, discovering novel LSD1 inhibitors from natural products to treat prostate cancer would be of significance.
In the last decade, high throughput screening has been developed as a promising strategy to screen antitumor agents such as small molecule inhibitors [25]. In our previous work, viscosalactone B, a natural product isolated from Withania Somnifera, was screened as a potential LSD1 inhibitor via the high throughput screening method. In this work, viscosalactone B displayed potent antiproliferative activity against PC3, DU145, C42B, PC3/MDVR, DU145/MDVR and C42B/MDVR cells with IC 50 values ranging from 0.72 μM to 3.86 μM. It also effectively inhibited tumor growth in the xenograft mouse models. As a selective LSD1 inhibitor, the IC 50 value of viscosalactone B against LSD1 was 970.27 nM. Importantly, it induced the significant accumulation of LSD1 substrates H3K9me1, H3K9me2 and H3K4me1 in DU145 cells in a concentration-dependent manner. Based on molecular docking results, the carbonyl group, lactone unit and hydroxyl groups of viscosalactone B formed hydrogen bonds with the Thr11, Lys14 and Arg8 residues, respectively.
In summary, viscosalactone B displayed excellent antitumor activity against prostate cancer in vivo and in vitro without cytotoxicity. Compared with the related enzymes (MOA-A, MOA-B, CDK1, and CDK2), viscosalactone B selectively and potently inhibited LSD1 at the enzymatic level. Antiproliferative activity of viscosalactone B against DU145 LSD1 knockdown cells suggested that viscosalactone B exerts antitumor effects partly through the inhibition of LSD1. Therefore, viscosalactone B might be a starting scaffold for the structure-based design of LSD1 inhibitors to treat prostate cancer.

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 °C and fixed by 95% ethanol. Nextly, they were stained by hematoxylin solution and eosin solution using the above kit. The stained slides were observed under 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][29][30][31]. Viscosalactone B was dissolved in dimethyl sulfoxide and added into 48-well plates. Then, the solution of the targeted enzyme was treated and incubated for 60 min at room temperature.

Western blot
After treatment of viscosalactone B at different concentrations for 48 h, proteins of DU145 cells were harvested by adding the RIPA lysis buffer (Thermo Fisher, Shanghai, China). 20 μg of proteins were electrophoresed on SDS-PAGE gel and transferred to PVDF membrane (Thermo Fisher, Shanghai, China). Anti-H3K4me1, anti-H3K9me1, anti-H3K9me2, and anti-H3 antibodies 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 using the AutoDock 4.2 software (The Scripps Research Institute, California, USA). Crystal structure files of LSD1 was downloaded from the Protein Data Bank (https:// www. rcsb. org/, PDB code: 2V1D). PDB file of viscosalactone B was obtained from ChemBio3D Ultra 14.0. The autogrid and autodock functions were used to establish a binding model between LSD1 and viscosalactone B. Hydrogen bonds, hydrophobic effects, and hydrophilic interactions of viscosalactone B with LSD1 were analyzed by the Pymol software.