4 | DISCUSSION
Under
normal physiological conditions, UHRF1 is highly expressed only in
actively proliferating cells and tissues, and its level is modified with
cell cycle progression (Yamashita, et al., 2018, Mancini, et al., 2021).
UHRF1 protein steadily elevates in G1/S phase, and reaches the peak at
mid S-phase, when heterochromatic regions are replicated, and it is
down-regulated at the end of M phase (Mancini, et al., 2021). However,
in tumor cells, aberrant overexpression of UHRF1 results in the
dyregulation of cell cycle. UHRF1 has been regarded as a typical
oncogene, and significantly promotes tumorigenesis and cancer progress
(Ashraf, et al., 2017). In this present study, we found that UHRF1 was
highly expressed in PCa tissues, and the level increased with the
elevation of PCa grades, and was negatively correlated with the survival
of PCa patients (Fig 1a-b, and e), suggesting that UHRF1 as an oncogene
promotes the initiation and progress of PCa.
UHRF1 facilitates the establishment and maintenance of DNA methylation
patterns in mammalian cells, and induces the silencing of TSGs
expression in tumors (Kong, et al., 2019b). In the present manuscript,
knockdown of UHRF1 in PCa cells inhibited
cell clonogenicity (Fig 1c). The
results indicated that UHRF1 is a potential therapeutic target for PCa,
but no specific inhibitor of UHRF1 has been registered in clinical
trials. It has been reported that currently 65% of anticancer drugs on
the market derived from the nomomer structures of natural products
(Newman&Cragg, 2016). According to the Compendium of Materia Medica,
the proportion of botanical medicinal materials in TCM is approximately
70%. Since UHRF1 plays a critical role in the development and
progression of PCa, and TCM has a long history and rich experience in
the treatment of PCa, we speculate that some monomer components from
botanical medicinal materials in TCM probably make anti-PCa effect by
inhibiting UHRF1-mediated pathway. Therefore, we for the first time
screened a natural molecule bank for PCa treatment by using network
pharmacology together with molecular docking. Diosgenin (DSG) was
identified as a novel UHRF1 specific inhibitor from 36 traditional
Chinese medicines (Fig 2). DSG demonstrated significant anti-cancer
therapeutic efficacy in the In vitro cell lines and in
vivo tumor xenograft models (Fig 5 and Fig 7).
Further mechanism investigations clarified that DSG induced the protein
degradation of UHRF1 through the ubiquitination-proteasome pathway (Fig
4). DSG reduced genomic DNA methylation and reactivated the expression
of TSGs, resulting in cell cycle arrest and inducing cell senescence
(Fig 6). It has been reported that USP7 is a deubiquitinase removing
ubiquitin a 76 amino acid protein that is added onto lysines in UHRF1
protein, and sustaining its stability (Felle, et al., 2011, Turnbull, et
al., 2017). We investigated the molecular mechanism by which DSG induced
UHRF1 protein degradation. Beyond our expectations, compared to the
working doses of DSG for cytotoxicity, an extremely high concentration
(60uM) was required to destroy the protein interaction of UHRF1 and USP7
(Supplementary Fig 1). Therefore, the molecular mechanism by which DSG
induce UHRF1 protein degradation needs further exploration in our future
studies.
Altogether, we for the first time identified DSG as a novel UHRF1
specific inhibitor for PCa treatment after screening a natural molecule
bank by using network pharmacology together with molecular docking. We
then found that DSG induced UHRF1 protein degradation through the
ubiquitin-proteasome pathway. UHRF1 protein degradation reactivated the
expression of TSGs, resulting in cell cycle arrest and inducing cell
senescence. By using network pharmacology together with molecular
docking in this study, we will identify more specific small molecule
inhibitors targeting specific oncoproteins from natural products in TCM.
These molecule-targeted inhibitors have great potentials for new drug
development after structural modifications, and being tested the
therapeutic efficacy, safety and pharmaceutical characters.