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.