INTRODUCTION
Prostate cancer (PCa) is one of the most incident and prevalent cancers in men worldwide and a leading cause of cancer-related morbidity and mortality (Siegel, et al., 2021). Current therapeutic strategies for localised PCa include androgen deprivation therapy (ADT), surgery, radio-, chemotherapy and others. ADT is the first line standard treatment of patients with newly diagnosed metastatic PCa, patients initially well respond to ADT, but drug resistance shortly develops and the disease continues to progress to castration refractory PCa (CPRC) (Litwin&Tan, 2017, Watson, et al., 2015, Gao, et al., 2010). Therefore, it is urgently needed to identify new diagnostic and therapeutic targets when the diseases develop to CRPC.
Epigenetic modifications, including aberrant DNA methylation and histone modifications contribute to the initiation and progression of PCa (Ruggero, et al., 2018, Arita, et al., 2012, Bert, et al., 2013). Ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an important epigenetic regulator that contains multiple functional domains, including the N-terminal ubiquitin-like domain (UBL), tandem Tudor domain (TTD), plant homeodomain (PHD), SET- and Ring finger-associated (SRA) and really interesting new gene (RING) domains, which are responsible for maintaining the fidelity of DNA methylation patterns during DNA replication (Arita, et al., 2012, Patnaik, et al., 2018). UHRF1 is a typical oncogene aberrantly overexpressed in a number of cancer types. In addition to gene amplification, the aberration of post translational modifications (PTMs) of UHRF1 such as phosphorylation and ubiquitination results in the dysregulation of protein degradation (Yang, et al., 2017, Chen, et al., 2013). UHRF1 overexpression suppresses the transcription of a panel of tumor suppressor genes (TSGs) by regulating DNA methylation. Reversely, inhibition of UHRF1 re-activates TSGs and induces cell cycle arrest and cellular senescence (Beck, et al., 2018, Jung, et al., 2017, Pérez-Mancera, et al., 2014). Therefore, UHRF1 is a potential therapeutic target for PCa. A lot of efforts have been made to develop novel UHRF1-targeted drugs by academics and industries, but no UHRF1-specific small compound inhibitor has been registered in the current clinical trials.
Traditional Chinese medicine (TCM) is an ancient medicine, which is based on more than 3500 years of Chinese medical practice (Su, et al., 2020). TCM has received more and more attention in recent years, and Chinese herbal extracts have immense potential for cancer treatment, and are important resources for new drug discovery (Hsieh, et al., 2014, Zhang, L., et al., 2020). Clinical studies have shown that TCM not only alleviates the symptoms of cancer patients and improves their quality of life but also diminishes adverse reactions and complications caused by chemotherapy, radiotherapy, or targeted-therapy(Duan&Wang, 2002). TCM formulas has been widely used as the complementary and alternative medicine for PCa, and many herbal extracts have demonstrated the anti-cancer efficacy in the in vitro models(Zhang, et al., 2019). Monomers extracted from Chinese herbal medicine, such as artemisinin, ginsenosides, gambogic acid and others have demonstrated significant cytotoxicity to various malignant tumors(Cheong, et al., 2020, Kim, et al., 2004, Wang, et al., 2021, Zhang, D., et al., 2020). It was reported that 65% of anticancer drugs currently on the market come from natural products (Newman&Cragg, 2016).
In view of the key roles of UHRF1 in cancer initiation and progression, several natural compounds extracted from the Chinese herbals have been reported to target UHRF1, such as Luteolin (30, 40, 5, 7-tetrahydroxyflavone)(Krifa, et al., 2014), Epigallocatechin-3-gallate (EGCG)(Achour, et al., 2013), Thymoquinone(Alhosin, et al., 2010), Limoniastrum guyonianum aqueous gall extract(Krifa, et al., 2013), red wine polyphenolic extract (RWP)(Sharif, et al., 2010), Bilberry extract (Antho 50)(Alhosin, et al., 2015), Naphthazarin (DHNQ)(Chow, et al., 2018) and Hinokitiol (4-isopropyltropolone)(Seo, et al., 2017).
In this present study, we screened a natural molecule bank for PCa treatment by using network pharmacology together with molecular docking, and Diosgenin (DSG) was identified as a novel UHRF1-targeted specific inhibitor. Furthermore, we explored its involved anticancer mechanism by using the in vitro and in vivo assays. DSG induced UHRF1 protein degradation, and then reduced the level of genomic DNA methylation, and re-activated the expression of TSGs, thereby resulted in cell cycle arrest and cell senescence. This present study provided a promising strategy to discover new molecule-targeting drug from natural compounds.
2 | MATERIALS AND METHODS
2.1 | Data Mining from the Cancer Genome Atlas (TCGA) Database
mRNA-seq and clinical data of 481 primary prostate adenocarcinoma tissues and 51 non-malignant controls were acquired from the TCGA-PRAD dataset(https://portal.gdc.cancer.gov/). After excluding those cases with incomplete data of TNM stage and survival, 397 patients were finally enrolled in this study. After normalizing raw data, we identified the differential expression genes of read counts between the normal controls and PRAD tissues using the edgeR package in R. Adjusted p value < 0.05 and |log2 (fold change) | > 1 were defined as the threshold. UHRF1 expression among different clinicopathological groups was analyzed using Student’s t-test. Receiver operating characteristic (ROC) curve was used to judge the diagnostic value of UHRF1 for PRAD, and the area under the curve (AUC) was calculated. Recurrence free survival (RFS) analysis was calculated by GEPIA2 (http://gepia2.cancer-pku.cn)(Tang, et al., 2017).
2.2 |Screening of small molecules
We searched the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and Analysis Platform database (https://old.tcmsp-e.com/tcmsp.php) for the active ingredients in the most commonly 36 botanicals used for PCa treatment in the clinic (Ru, et al., 2014, wanli&hong, 2018) . We then screened the target molecules using the following criteria. 1) The molecules exist in four or more botanicals, and 2) the molecules cannot be synthesized by human body. Finally, 75 small molecules were identified by using the online website UPSET (https://cloud.oebiotech.cn/task/detail/upset/).
2.3 | Network pharmacology together with molecular docking
The structures of 75 small molecules were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The details could be found in the Supplementary Table 1. The protein crystal structures of five domains of UHRF1 were downloaded from the protein database (http://www.rcsb.org/pdb/home/home.do) (PDB: 3ASL, PDB: 3FL2, PDB: 6W92, PDB: 2FAZ and PDB: 3BI7). Before molecule docking, the structure of target proteins was pre-processed using AutoTools and PYMOL, including removal of water molecules and ligands, addition of hydrogen, etc. Then, the size of grid matrix for blind docking was adjusted such that the protein molecules had completely been covered. Finally, the candidate ingredients were chosen for molecular docking using Autodock Vina software (Trott&Olson, 2010), and the results were visualized using PYMOL. Based on the lowest binding energy score, results were represented as heatmaps by R package pheatmap.
2.4 |Cells culture and siRNA transfection
LNCaP, C4-2, DU145 and PC3 cells were cultured in RPMI-1640 media (Gibco) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C in a 5% CO2 humidified atmosphere. siRNA transfection was performed using a Mirus Transfection Kit according to the manufacturer’s protocol (Mirus, Madison, WA). The siRNA sequences used for UHRF1 knockdown were (5’-3’): GCGCUGGCUCUCAACUGCU.
2.5 | Antibodies and chemicals
The antibodies and chemicals used in the study include anti-UHRF1 (Proteintech, 21402-1-AP), anti-His (Genscript, A00186), anti-HA (Cell Signal Technology, CST, 3724), anti-5-Methylcytosine (CST, 28692), anti-p21 (CST, 2947), anti-HDAC1 (CST, 34589) and anti-β-actin (Abclonal, AC004). Cycloheximide (CHX) was purchased from CST, MG132 was purchased from Selleck Chemicals (Houston, Texas, USA), L7G and DSG were purchased from MedChemExpress (MCE, Shanghai, China).
2.6 |Cell viability assays
Cells were cultured in 96-well flat-bottomed microtiter plates (5000 cells per well), and treated with DSG for 48 h. CCK8 stock solution was added to each well according to the manufacturer’s instructions, and the plate was incubated at 37 °C for 1 h. Cell viability was assessed by measuring the absorbance at 450 nm using a microplate reader (Multiskan-GO, Thermo Fisher Scientific, China).
2.7 |Western blotting and Real-time PCR assay
Cells were washed three times with cold PBS and lysed in RIPA buffer, and then centrifuged for 15 min to collect supernatant. The protein concentration was measured using a BCA assay kit. The protein levels were assessed by western blotting.
The total RNA was extracted from PCa cells, or xenograft tumor tissues following the RNAiso Plus manufacturer’s protocol (Takara Bio, Beijing, China). The concentration and quality of RNA samples were determined, and then reverse-transcribed to cDNA. The mRNA levels of genes were measured by real-time PCR system according to the manufacturer’s instructions. Primers for RT-PCR were: UHRF1 : 5’-AGGTGGTCATGCTCAACTACA-3’ (forward), 5’-CACGTTGGCGTAGAGTTCCC-3’ (reverse). p16 : 5’-CGGTCGGAGGCCGATCCAG-3’ (forward), 5’-GCGCCGTGGAGCAGCAGCAGCT-3’ (reverse). p21 : 5’-ATGGAACTTCGACTTTGTCACC-3’ (forward), 5’-AGGCACAAGGGTACAAGACAGT-3’ (reverse). LXN : 5’-ACAAGCCAGCATGGAGGATA-3’ (forward), 5’-TCAGCTGTGCAGTTCACCTT-3’ (reverse).
2.8 | In vivo ubiquitination assay
Cells were transfected with the indicated plasmids, and treated with DSG for 48 h and followed by the treatment of 50 µM MG132 for additional 6 h. The cells were lysed in RIPA buffer and boiled at 100 °C for 10 min. The cell lysates were centrifuged at 12,000 g for 15 min. UHRF1 protein was immunoprecipitated with anti-His antibody on a rotator at 4 °C for 12 h, and the immune complexes were incubated with protein A/G-magnetic beads. After being washed three times, the immunocomplex was subjected to SDS-PAGE and the ubiquitination levels were assessed by western blotting.
2.9 | Dot blot analysis
Genomic DNA was extracted using a genomic DNA purification kit according to the manufacturer’s protocol (CoWin Biosciences, Beijing, China). The extracted DNA was denatured at 95 °C for 10 min, and then 100 ng DNA was blotted onto nitrocellulose membrane and fixed at 85 °C for 30 min. The cross-linked nylon membrane was incubated in blocking solution (5% BSA) for 1 h at room temperature, and hybridized with anti-5-mC overnight at 4 °C. The membrane was incubated with the secondary antibody at room temperature for 1 h, and detected by chemiluminescent detection reagents. Methylene blue intensity of DNA dots was used to determine the amount of genomic DNA methylation.
2.10 |Cellular senescence assay
Cellular senescence was assessed by Senescence-Associated β-Galactosidase Staining Kit (Beyotime Biotechnology, Beijing, China) according to the manufacturer’s instructions. Cells were treated with DSG for 3 days, the cells were washed twice with PBS, fixed at room temperature for 10-15 min, and incubated with fresh β-gal staining solution at 37 °C overnight. The β-gal-positive cells were monitored under a microscope.
2.11 | Cell cycle analysis
Cells were treated with DSG for 72 h, and were fixed in 70% ethanol in PBS at 4 °C for 24 h. The supernatant was discarded after centrifugation at 1,500 rpm for 10 minutes. For cell cycle analysis, cells were re-suspended in 1 mL PBS containing propidium iodide (PI) incubated at room temperature avoiding light for 30 min. Cell cycle distribution was analyzed by flow cytometer (Attune NxT, Thermo Fisher Scientific, China).
2.13 |In vivo animal study
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Institute of Laboratory Animal Science (Number:00287876), Guangdong Pharmaceutical University (Guangzhou, China), and conformed to the relevant regulatory standards. Nude mice (4-5weeks old, male) were kept for one week before the start of the experiment to adopt the conditions. All animals were housed in airconditioned rooms (22 ± 2 °C, 50% humidity and 12 h of dark or light cycles), and had free access to standard drinking water.
The tumor xenografts were induced by subcutaneously inoculating DU145 cells (5 × 106·100−1 µL) into the left flank region of mice. Mice were randomly divided into 3 groups; a control group, a low‐dose group (DSG 40 mg/kg), and a high‐dose group (DSG 80 mg/kg). The control group was gavaged with 0.5% CMC-Na. Tumor size was measured with calipers every three days. and the tumor volumes were calculated according to the following formula: V = (max diameter) × (min diameter)2·2-1. The tumor xenografts were isolated at the endpoint of experiment, and the tumors were then photographed and weighed. The liver and kidney tissues were fixed in 10% buffered formalin and embedded in paraffin for H&E staining.
2.13 | Statistical analysis
All statistical analyses were performed using GraphPad Prism8 (v8.0.2). The results are expressed as means ± standard deviation. The significance of differences among groups was assessed using one-way analysis of variance with post hoc Bonferroni test. Paired data were analyzed using the paired-samples t test. * p< 0.05, ** p< 0.01, *** p< 0.001, and**** p< 0.0001 denoted statistical significance. The Kaplan-Meier provides a method for estimating the survival curves, and the log-rank test provides a statistical comparison of two groups.