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.