Predictive analysis of the drug-forming potential of AIL using the SwissADME database (http://www.swissadme.ch/) encompassed six parameters; physiochemical properties, lipid solubility, water solubility, pharmacokinetic properties, drug-like properties, and medicinal properties (Huang et al., 2022).

A comprehensive search for AIL target genes using TCMSP, HERB, Swiss Target Prediction, and PharmMapper databases was conducted. The keyword “ailanthone” served as the query for this search to screen relevant AIL targets. To ensure consistency and standardize target gene nomenclature (gene symbols), the UniProt database (https://sparql.uniprot.org/) (Han et al., 2023) was employed.

Acquisition of RNA sequencing data from 487 samples from the TCGA database was followed by the annotation of these data using the Perl programming language to convert the probe matrix into a gene matrix.

To identify genes with distinct expression patterns between the tumor and normal groups, a transcriptomic analysis was executed using the “LIMMA” package in the R programming language. The filter criteria of |LogFC| ≥ 1 and corrected p < 0.05 were used. Volcano plots and heatmaps were generated to visualize the DEGs using the “pheatmap” package in R language.

Target genes associated with AIL were intersected with DEGs observed in CRC. The “VennDiagram” package in R was employed to create Venn diagrams, which illustrated the intersecting genes between the two datasets.

For PPI network construction, the intersecting genes between AIL and CRC were identified. Network topology analysis was then conducted using the online platform STRING-DB (https://string-db.org/). The resulting “tsv” file was downloaded and imported into Cytoscape 7.2.1 using the “cytomubba” plugin for core gene screening. A PPI network diagram featuring central genes was generated. Additionally, the “string_interactions.tsv” file was used in R to create a histogram illustrating core gene interactions.

The “survival” and “survminer” packages in R (Liu et al., 2021) were used to perform an analysis of the variation in expression levels of these 10 core genes between normal and CRC tissues in a total of 446 CRC samples. Furthermore, in order to further analyse the prognostic significance of these core genes, we employed the Kaplan-Meier online platform (Song et al., 2023) to assess the impact of these core genes on overall survival in 1061 CRC samples.

To gain insights into the biological significance of the identified genes, functional enrichment analyses were performed incorporating GO terms (http://GeneOntology.org/) and KEGG pathways (https://www.kegg.jp/eg/) (Yu et al., 2023). R packages (Yu et al., 2023), including “clusterProfiler,” “org.Hs.eg.db,” “enrichplot,” “ggplot2,” and “DOSE,” were used with specified filter conditions: “p-value cutoff = 0.05” and “q value cutoff = 0.05.” GO enrichment analysis encompassing CC, BP, and MF outcomes were presented in a visually informative bubble chart. Similarly, the KEGG pathway enrichment analysis outcomes were depicted in a column chart.

Cytoscape7.2.1 was used to visualize the “AIL–CRC–intersection gene–KEGG” network and elucidate the AIL–CRC relationship as well as its corresponding pathways.

To further explore the key targets of the anti-CRC effect of AIL, molecular docking analysis was performed between AIL and potential core targets identified in the PPI analysis and key proteins PI3K and AKT in the PI3K/AKT signaling pathway screened by KEGG enrichment analysis. The molecular docking process involved several sequential steps (Trott and Olson, 2010; Eberhardt et al., 2021):

1). Acquisition of AIL Structure:

- The energy of the 3D structure was optimized, and the resulting structure was saved in the mol2 format.

- Finally, this optimized structure was imported into AutoDockTools-1.5.6 software and saved in the pdbqt format.

CRC cells (HCT116 and SW620 cells obtained from the Chinese Academy of Sciences, Beijing, China) were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin–streptomycin. They were incubated at 37°C in a 5% CO₂ environment. When cellular density reached 80%–90%, trypsin (0.25%) was used for cell digestion and passaging.

CRC cells (SW620 and HCT116) in their logarithmic growth phase were seeded into 96-well culture plates at a density of 1 × 10⁴ cells per well (100 μL/well). After an initial 24-h incubation period in a controlled incubator, the cells were exposed to various AIL concentrations (0.1, 0.3, 1, 3, 10, and 30 μmol/L) for an additional 24 h at 37°C under 5% CO₂. Each AIL concentration was tested in four replicate wells. After exposure, 10 μL of a 5 mg/mL MTT solution was added to each well, and the cells were incubated for 2 h. Following supernatant removal, 100 μL of dimethyl sulfoxide was added to each well, agitated in the dark for 5 min, and absorbance readings were collected at 490 nm. Cell viability (%) was determined by dividing the absorbance value of the test group by that of the control group and multiplying by 100. The experimental procedure was replicated thrice, and concentrations of 0.1 and 0.3 μmol/L were selected for subsequent experiments.

In 6-well culture plates, SW620 and HCT116 CRC cells in their logarithmic growth phase were inoculated at a density of 5 × 10⁵ cells per well and cultured for 24 h in a controlled incubator. Two groups, the control (fresh medium) and AIL treatment (0.1 and 0.3 μmol/L) groups, were established. Following 24 h of incubation, the cells were washed with phosphate-buffered saline (PBS), subjected to digestion, collected, and then washed twice with cold PBS.

For cell cycle analysis, the cells were first fixed in 75% cold ethanol at 4°C for 12 h, followed by two washes with cold PBS. Subsequently, they were stained with 10 µL of propidium iodide (PI) dye in the dark for 15 min. Finally, they were analyzed through flow cytometry.

To assess apoptosis, the cells were incubated with 5 µL Annexin V/FITC for 5 min at room temperature in the absence of light. Subsequently, 10 µL PI dye with 400 µL PBS was added, and flow-through was immediately analyzed.

SW620 and HCT116 CRC cells in the logarithmic growth phase were adjusted to an appropriate density for invasion and migration assays. A 0.2 mL suspension of serum-free cells was introduced into the upper chamber of a Transwell chamber with or without matrix gel in 24-well plates (cell density: 4 × 10⁴ cells per well). The lower chamber contained 600 μL culture medium containing 30% fetal bovine serum. Two groups, control and AIL treatment (0.1 and 0.3 μmol/L), were established. Transwell chambers were incubated for 24 h, after which the liquid within and outside the Transwell chamber was removed, and the chambers were rinsed thrice with PBS. Subsequently, cells were fixed with 1 mL of 4% paraformaldehyde for 15 min at room temperature. After three washes with PBS buffer, the cells were stained with 1 mL 0.1% crystal violet solution for 10 min at room temperature. After another three washes with PBS, any cells that had not invaded or migrated in the upper chamber of the Transwell were gently removed using cotton swabs. The chambers were subsequently examined under a microscope, following which they were transferred to a new 24-well plate. In each well, 800 μL 33% glacial acetic acid was added, and the plates were shaken for 5 min to achieve decolorization. Cell invasion and migration were quantified by measuring absorbance at 570 nm using an enzyme-linked immunoassay detector.

CRC cells (SW-620, HCT-116) in logarithmic growth phase were implanted in 24-well plates containing climbing tablets at a density of 2 × 10⁴ cells per well, cultured in 600 μL medium for 24 h, the supernatant was discarded, and the complete medium was replaced in the normal group. The drug group received complete medium containing AIL at 0.1 μmol/L and 0.3 μmol/L, respectively. 24 h after administration, the protein expression levels of PI3K, AKT and p-AKT were detected by immunofluorescence method. The detection methods are as follows: PBS wash for 3 min × 3 times, 4% paraformaldehyde fixed cells for 15 min, PBS wash for 3 min × 3 times; add TritonX100 to permeate for 10 min, dry and wash with PBS for 3 min × 3 times; 5% BSA occluded for 40 min without washing. Primary antibodies against specific proteins (PI3K, AKT, p-AKT, 1:200) were purchased from Abcam (MA, United States) and added to incubate overnight at 4°C, removed the next day and re-warmed for 1 h and washed with PBS for 5 min × 3 times. The cells were incubated with anti-light plus fluorescent secondary antibody (1:200) for 1 h at room temperature, washed with PBS for 5 min × 3 times, stained with DAPI for 8 min and washed for 5 min × 3 times, sealed with anti-fluorescence quencher, observed and photographed under a fluorescence microscope, and the results were calculated by optical density.

Statistical analysis was performed using SPSS version 26.0 software. Data are presented as means ± standard deviations (mean ± SD). Differences between groups were assed using either one-way analysis of variance or t-test, with p < 0.05 indicating statistical significance.

This study assessed the pharmacodynamic activity of AIL by integrating Lipinski’s rule, oral bioavailability (OB), and drug-likeness (DL). Lipinski’s rule comprises several criteria, including molecular weight (MW < 500), lipid–water partition coefficient (−2 < AlogP <5), hydrogen bond donors (Hdon <5), hydrogen bond acceptor count (Hacc <10), and rotational bonds (RBN <10). DL indicates robust clinical efficacy; a higher DL value suggests greater drug-generating potential and OB is a critical pharmacokinetic parameter for orally administered drugs. DL and OB were considered key indicators of drug effectiveness. Using the Traditional Chinese Medicine Systems Pharmacology.

(TCMSP) database, the following criteria were employed for drug screening: OB = 20%, DL = 0.1, and RBN <10. According to our evaluation, the AIL parameters were as follows: OB = 27.96%, DL = 0.74, and RBN = 0. These results indicate highly promising pharmacodynamic activity (refer to Table 1 for detailed ADME parameters).

By calculating gene expression differences between 41 normal samples and 446 CRC samples, we identified 2,551 differentially expressed genes (DEGs), including 1,125 upregulated genes and 1,426 downregulated genes (Supplementary Tables S1–S3). Figures 1A, B show the heatmap and volcano map illustrating DEGs, respectively.

Analysis of TCMSP, HERB, Swiss Target Prediction, and PharmMapper databases identified 137 target genes of AIL (Supplementary Table S4).

Venn diagrams were obtained by intersecting DEGs in CRC with AIL target genes (Figure 1C). In total, 38 genes were enriched in both the groups (Supplementary Table S5). The top 10 upregulated genes included FABP6, MET, AURKA, CHEK1, CDK4, HSP90AB1, CCNA2, CDK2, PGF, and GSTP1 (Figure 1D). The top 10 downregulated genes included ADH1C, FABP4, GSTA1, FGFR2, MAOA, KIT, PLA2G10, HMOX1, PLA2G2A, and AKR1C3 (Figure 1D).

Figure 2A presents the PPI analysis results of target proteins conducted using the STRING database. The top 20 differential genes within the network were arranged in a ranked manner (Figure 2B). A comprehensive analysis of DEGs derived from AIL-treated CRC was performed, following which these DEGs were evaluated and assigned scores using Cytoscape 7.2.1 software. A molecular network diagram was visualized using the “cytomubba” plugin (Figure 2C). For each target gene, the top 10 proteins with the highest number of neighboring genes were selected for statistical analysis (Figure 2D). Proteins with ≥30 connectivity included CDC6, CDK2, CHEK1, CDK4, and CCND1. Other proteins with high connectivity, including CDKN1B, CCNB1, CCNA1, MCM5, and CCNA2, were also obtained using PPI screening.

Expression analysis of the 10 core genes in the drug–target network, conducted in normal and tumor tissues, revealed significant differences (p < 0.001) in the expression levels of CCND1, CDK4, CHEK1, CDK2, CDC6, CCNA1, CCNA2, CCNB1, CDKN1B, and MCM5 (Figure 3A). In addition, the Kaplan-Meier online platform analysis indicated that the expression of these 10 core genes was significantly correlated with the survival of CRC patients (Figures 3B–K). Notably, a high expression of CCND1 and CCNA1 emerges as a prognostic indicator for unfavorable prognosis.

Enrichment analyses of GO gene functions and KEGG pathways were performed using the R programming language, and AIL targets in CRC were identified. Among the most enriched GO terms in Biological Process (BP) were animal organ regeneration, histone phosphorylation, regeneration, eicosanoid metabolic processes, and cellular responses to inorganic substances. The most enriched terms in Cellular Component (CC) and Molecular Function (MF) included the cyclin-dependent protein kinase holoenzyme complex and monocarboxylic acid binding, respectively (Figure 4A). In KEGG pathway analysis, the core genes in the network were primarily associated with cancer-related pathways, including the PI3K-AKT signaling pathway, Ras signaling pathway, drug metabolism (cytochrome P450), PPAR signaling pathway, and hepatocellular carcinoma (Figure 4B). Functional enrichment analysis results were visualized using Cytoscape 7.2.1. Thus, a regulatory network diagram termed as “AIL–CRC–Target Gene–KEGG” was established (Figure 4C), and the drug–disease relationships along with the pathways involved were analyzed.

The Protein Data Bank (PDB) was searched for protein crystal structures corresponding to the genes we need. It is generally believed that the binding energy between small molecules and proteins is ≤ −5.0 kJ/mol, meaning that both have good binding activity (Liao et al., 2023). Our docking results show that the binding energy of AIL to PI3K and AKT is the lowest, which is −8.6 kcal/mol and −7.5 kcal/mol, respectively (Supplementary Table S6), indicating that AIL has a strong binding affinity with PI3K and AKT. Hydrogen bonding plays a major role in the stability of compound-target binding (Iksen et al., 2023). As shown in Figure 5, AIL and AKT form three hydrogen bonds at ARG-243 and one hydrogen bond at THR-371. AIL and PI3K form two hydrogen bonds at VAL-202 and one hydrogen bond each at PRO-200, ASN-688, ARG-684, and ASP-653.

The effects of 24-h AIL treatment at varying concentrations (0.1, 0.3, 1, 3, 10, and 30 μmol/L) on SW620 and HCT116 CRC cell proliferation were assessed using the MTT assay. AIL exhibited a significant and dose-dependent inhibition of this proliferation in SW620 and HCT116, with half-maximal inhibitory concentration values of 9.16 ± 0.93 and 18.42 ± 1.77 μmol/L, respectively (Figure 6A). Notably, at 0.1 and 0.3 μmol/L AIL, the HCT116 cell inhibition rates were 7.8% and 16%, whereas SW620 cells exhibited inhibition rates of 6.4% and 12%, respectively; thus, subsequent investigations focused on the AIL concentrations of 0.1 and 0.3 μmol/L (Supplementary Table S7).

Flow cytometry analysis revealed a concentration-dependent increase in apoptosis in HCT116 and SW620 CRC cells following AIL treatment compared with that in the control group (Figure 6B). Treatment with AIL (0.1 and 0.3 μmol/L) significantly reduced HCT116 and SW620 CRC cells in the S phase coupled with a marked increase in cells in the G2 phase of the cell cycle compared with that in the control group (Figure 6C). This indicates that AIL effectively blocked the G2 phase in CRC cells, impacting cell proliferation.

Transwell chambers with or without matrix gel were used to assess the effect of AIL treatment (0.1 and 0.3 μmol/L) on the migration and invasion of SW620 and HCT116 CRC cells in the logarithmic growth phase. AIL exerted a concentration-dependent inhibitory effect on the migration and invasion abilities of both cell types compared to the control group (Figure 7).

KEGG enrichment analysis results indicated that PI3K/AKT may be a key signaling pathway in the AIL-mediated treatment of CRC. The key to activation of the PI3K/AKT pathway is phosphorylation of the AKT protein, and phosphorylation of the AKT protein at S473 indicates activation of the PI3K/AKT pathway (Chen et al., 2022). Therefore, we detected the expression level of key proteins in the PI3K/AKT pathway in AIL-treated cells by immunofluorescence. The results showed that compared with the control group, the expression levels of PI3K and AKT protein in CRC cells treated with AIL did not show any significant change (Supplementary Figures S2, S3), but the expression level of p-AKT^S473 protein was significantly decreased (p < 0.01), indicating that AIL could inhibit AKT phosphorylation, thereby inhibiting the activation of the PI3K/AKT signaling pathway and thus inhibiting the proliferation and metastasis of CRC cells, as shown in Figure 8.