The chemical structure of Icaritin was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Potential targets of Icaritin were collected from SwissTargetPrediction (http://www.swisstargetprediction.ch/), PharmMapper (http://www.lilab-ecust.cn/pharmmapper/), and TargetNet databases (http://targetnet.scbdd.com/), focusing on Homo sapiens targets (Yao et al., 2016; Wang et al., 2017; Daina et al., 2019). After removing duplicates, the potential targets were imported into the UniProt database (https://www.uniprot.org/) and converted to UniProt IDs with corresponding gene names.

Hepatocellular carcinoma (HCC) related targets were gathered using the GeneCards database (https://www.genecards.org/), with “hepatocellular carcinoma” and “liver cancer” as keywords (Safran, 2022). Duplicates were removed, and target names were normalized using the UniProt database (https://www.uniprot.org/).

Intersection of Icaritin targets and liver cancer disease targets were performed using R software, yielding the anti-HCC targets of Icaritin. The anti-HCC targets of Icaritin were uploaded to the STRING database (https://string-db.org/) to obtain PPI information. A confidence score of ≥0.9 was set for interactions, and unconnected targets were discarded, resulting in a PPI network.

The core targets (hub genes) were screened using the cytoHubba plugin in Cytoscape (https://cytoscape.org/), utilizing the Maximal Clique Centrality (MCC) method. The top ten core targets were visualized, with darker colors indicating higher scores and importance within the network.

The DAVID tool was employed to do an enrichment study on the anti-HCC targets of Icaritin (Sherman et al., 2022). This included Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, with GO analysis covering cellular components (CC), molecular functions (MF), and biological processes (BP).

The X-ray crystal structures of core targets were obtained from the Protein Data Bank (PDB) (https://www.rcsb.org/), while Icaritin’s structure file was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and optimized using Chem3D. Protein receptor and ligand files were processed with AutoDock tools (http://autodock.scripps.edu/), followed by molecular docking of Icaritin to core targets using AutoDock Vina software (https://vina.scripps.edu/) (Morris et al., 2009; Trott et al., 2009). The lowest binding energy binding mode was selected for further analysis, and interactions were visualized as 2D plots using PyMOL software (https://pymol.org/2/).

Molecular dynamics simulations were carried out for 20 ns on selected protein-ligand complexes to determine binding affinity (Que et al., 2021). And utilizes the GROMACS 2021 software running on Linux Ubuntu 20.04, driven by an AMD R5 3600 CPUU (Hess et al., 2008). Generation of protein topology using Charmm36 force field and construction of ligand topology using CGenFF (Vanommeslaeghe et al., 2012; Vanommeslaeghe and MacKerell, 2012). The TIP3P water model was employed for solvation, and the system was neutralized with Na+ and Cl- (Hess, 2008). Energy minimization, heating, and equilibration steps were performed, followed by a 20 ns molecular dynamics production run. Trajectories were used for kinetic studies and protein-ligand free energy calculations with MM/PBSA (Paissoni et al., 2014).).

HepG2 human hepatocellular carcinoma cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) obtained from Gibco (catalog #11965-092), supplemented with 10% fetal bovine serum (FBS) from Hyclone (catalog #SH30396.03), 100 U/mL penicillin, and 100 μg/mL streptomycin from Invitrogen (catalog #15140-122). Cells were maintained in a humidified atmosphere containing 5% CO₂ at 37°C. For treatment, cells were seeded in 6-well plates at a density of 1 × 10^5 cells per well and allowed to adhere overnight. The cells were then treated with either vehicle (DMSO) or Icaritin (10 μM, Sigma, catalog SML0551) for 2 days. Each treatment group consisted of three replicates, including the control group.

For the colony formation assay, cells were seeded into 6-well plates at a density of 500 cells per well and allowed to adhere for 24 h. Following this, cells were treated with 10 µM Icaritin for 2 days. After the treatment, the medium was replaced with fresh drug-free medium, and the cells were allowed to grow for an additional 14 days to form colonies. The medium was changed every 3 days to maintain optimal growth conditions. Once the colonies were clearly visible, the cells were washed twice with cold PBS, fixed with 4% paraformaldehyde from Sigma-Aldrich (catalog #158127) for 15 min at room temperature, and stained with 0.1% crystal violet solution from Sigma-Aldrich (catalog #C3886) for 30 min. Excess stain was washed away with distilled water, and the plates were allowed to air-dry. Colonies consisting of 50 or more cells were counted, and the colony formation efficiency was calculated as the number of colonies divided by the number of cells seeded, expressed as a percentage. The experiment was performed in triplicate, and the results were averaged to obtain the final colony formation efficiency.

The Animal Ethics Committee of Fujian Medical University (Fuzhou, China) approved the animal experiment under IACUC FJMU 2022-0036. Six-week-old male BALB/c nude mice were utilized for this investigation. Prior to the study, the animals were housed in a specific pathogen-free facility, under regulated temperature and lighting conditions, with unrestricted access to food and water. HepG2 cells were prepared by harvesting and suspending them in sterile phosphate-buffered saline (PBS) from Gibco (catalog #10010-023) at a suitable concentration for injection. Mice were then inoculated subcutaneously with HepG2 cells in the right flank region. Following tumor establishment, the mice were randomly allocated into two groups: the Icaritin treatment group (n = 6) and the control group (n = 6). Mice in the Icaritin treatment group received intraperitoneal injections of Icaritin at a concentration of 0.2 mg/kg Icaritin, while those in the control group were given injections of physiological saline. Injections were administered for 8 weeks until the study’s completion. Tumor progression and body weight were periodically assessed. At the end of the experiment, mice were euthanized, and tumor tissues were excised for subsequent analysis.

For a proteomics study, tumor samples from the Icaritin therapy group and the control group were gathered. After being homogenized, the tissues were lysed in lysis solution that included protease inhibitors. The BCA protein assay was used to determine the protein concentrations. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to in-gel digestion. Each sample had the same quantity of proteins removed for reduction, alkylation, and trypsin digestion. Identification by tandem mass spectrometry (LC-MS/MS) analysis and quantify peptides using an Orbitrap mass spectrometer coupled to a nano-flow liquid chromatography system. On a reverse-phase C18 column, peptides were isolated and then analyzed using data-dependent acquisition mode. MaxQuant software was applied to analyze the raw mass spectrometry data for label-free protein identification and quantification. A false discovery rate (FDR) of 1% was applied to filter the identified proteins.

Total RNA from tumor tissues and cell lines was extracted using TRIzol reagent (Invitrogen, #15596026) according to the manufacturer’s instructions. The concentration and purity of the isolated RNA were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from the total RNA using a reverse transcription kit (Thermo Fisher Scientific, #K1622) following the manufacturer’s protocol. Quantitative real-time PCR was performed using a specific SYBR Green Gene Expression Assay (Applied Biosystems, #4309155) for the FYN gene and an endogenous control (ACTB) on a real-time PCR system. FYN: forward, 5′-CAG​TTG​ACT​CCA​TCC​AGG​CAG​A-3′, and reverse, 5′-CAC​GGA​TGG​AAA​GTG​AGT​AGG​C-3′.

PIK3R1: forward 5′-CGC​CTC​TTC​TTA​TCA​AGC​TCG​TG-3′, and reverse, 5′- GAA​GCT​GTC​GTA​ATT​CTG​CCA​GG-3′. ACTB: forward, 5′-CAT​TGC​TGA​CAG​GAT​GCA​GAA​GG-3′, and reverse, 5′-TGC​TGG​AAG​GTG​GAC​AGT​GAG​G-3′. Each reaction was run in triplicate. The relative expression levels of the FYN gene were calculated using the 2^(−ΔΔCt) method, where Ct represents the threshold cycle, and data were normalized to the endogenous control. The qPCR results presented in display the average RNA expression level of FYN and PIK3R1 across three mice.

Cell lysates were prepared by washing cells with cold PBS, followed by lysis in RIPA buffer (Cell Signaling Technology, #9806) containing protease and phosphatase inhibitors. Protein concentrations were determined to use the BCA Protein Assay Kit (Thermo Fisher Scientific, #23225). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, #IPVH00010). The membranes were blocked with 5% non-fat milk in TBS-Tween for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies against FYN (Cell Signaling Technology, #4023), PIK3R1 (Abcam, #ab32089) and GAPDH (Cell Signaling Technology, #5174) at the recommended dilutions. After washing, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, #34076) and quantified with densitometry analysis software.

Figure 7A illustrates that tumors treated with Icaritin were clearly small for the control group. A targeted proteomics analysis was performed on tumors from both groups. Figure 7B presents a heatmap of the predicted Icaritin-targeted protein expression, which demonstrates a significant upregulation of FYN protein in the Icaritin-treated group with excellent repeatability. Furthermore, qPCR results depicted in Figure 7C reveal a significant upregulation of the FYN gene in the Icaritin-treated group.

In vitro validation was carried out using HEPG2 cells. Figure 7D displays images of colony formation for Icaritin-treated and control groups, revealing that the number of colonies formed by Icaritin-treated cells was significantly lower compared to the control group. FYN protein expression differed considerably between the two groups, and FYN protein expression was significantly upregulated in the Icaritin group (Figure 7E).