Human NSCLC cell lines A549 (cat. no. CM-0016) and H1299 (cat. no. CM-0165) were purchased from Procell Life Science & Technology Co., Ltd. and cultured at 37°C in 5% CO₂ in DMEM (Gibco; Thermo Fisher Scientific, Inc.) or RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.). ZTO injection (Zhejiang Tianrui Pharmaceutical Co., Ltd.; national medical product approval/lot no. H20064312; cat. no. 423020101), F12K medium (cat. no. 21127030; Thermo Fisher Scientific, Inc.), FBS (cat. no. C04001-500; Shanghai XP Biomed Ltd.) and trypsin (cat. no. PB180225; Procell Biotechnology Co., Ltd.) were used. The BeyoClick™ EdU Cell Proliferation Kit with AF488 (cat. no. C0071S; Beyotime Biotechnology), 4% paraformaldehyde (cat. no. P0099; Beyotime Biotechnology), Acridine Orange Staining Kit (cat. no. C0233S; Beyotime Biotechnology), Annexin V-FITC Apoptosis Detection Kit (cat. no. BA00101; Beijing Biosynthesis Biotechnology Co., Ltd.), TRIzol^® Reagent (cat. no. 15596026CN; Invitrogen; Thermo Fisher Scientific, Inc.) and Reverse Transcription Kit and Real-Time Quantitative PCR Kit (cat. nos. AG0304-B and AH0104-B; Shandong Sparkjade Biotechnology Co., Ltd.) were used. Instruments used included the Spark™ 10M microplate reader (Tecan Group Ltd.), Z32HK high-speed refrigerated centrifuge (HERMLE Labortechnik GmbH) and Mastercycler^® Realplex4 Real-Time PCR System (Eppendorf SE).

A549 cells and H1299 cells in the logarithmic growth phase were seeded into 96-well plates and cultured for 24 h. Subsequently, the A549 cells were treated with ZTO at final concentrations of 0, 100, 200, 300 and 400 µg/ml at 37°C for 48 h and the H1299 cells were treated with ZTO at final concentrations of 0, 20, 40, 60, 80, 100, 120 and 140 µM at 37°C for 48 h. After incubation, 10 µl MTT solution (0.5 µg/µl) was added to each well and incubated at 37°C for 4 h. Subsequently, 150 µl DMSO was added to each well and allowed to stand for 10 min. The absorbance was measured at 490 nm using a multimode microplate reader.

After A549 and H1299 cells were treated with 100 µg/ml and 50 µM ZTO for 48 h, A549 and H1299 cells were resuspended at a density of 100 cells/ml and 1 ml cell suspension was seeded into 12-well plates. Cells were cultured in a 37°C, 5% CO₂ incubator for 7–10 days until ~50 cell colonies formed per well. The cultures were then terminated, washed twice with PBS and fixed with methanol at room temperature for 30 min. The cells were then stained with 0.1% crystal violet at room temperature for 1 h, gently rinsed under running water, air-dried and photographed. The number of colonies was quantified using ImageJ software (version 1.54k; National Institutes of Health). A colony was defined as a cluster of ≥50 cells; ImageJ counts were manually verified to ensure consistency with this definition.

A549 and H1299 cells were seeded in 6-well plates at a density of 5×105 cells/ml (2 ml/well) and cultured for 12 h. A549 cells were then treated with 100 µg/ml ZTO and H1299 cells were treated with 50 µM ZTO for 48 h at 37°C. Cell proliferation was assessed using the EdU Cell Proliferation Detection Kit (Beyotime Biotechnology), according to the manufacturer's instructions. The percentage of EdU-positive cells was calculated from five randomly selected images using ImageJ software (version 1.54k).

The A549 and H1299 cells were cultured until they reached 80–90% confluence, after which, they were treated with 100 µg/ml and 50 µM ZTO for 48 h at 37°C, respectively. Subsequently, the A549 and H1299 cells were scratched using a sterile 200-µl pipette tip. The detached cells were gently washed away with PBS, and serum-free medium was added. Wound closure was monitored and images were captured at 0, 6 and 24 h post-scratching. The migration rate was quantified by measuring the wound area at each time point using ImageJ software (version 1.54k).

After A549 and H1299 cells were treated with 100 µg/ml and 50 µM ZTO for 48 h at 37°C, respectively, they were trypsinized, resuspended in serum-free medium and adjusted to 1×10⁵ cells/ml. Then, a 300-µl cell suspension was added to the upper chamber of a Transwell insert (12-well plate; 8-µm pore size; Corning, Inc.), and 500 µl medium supplemented with 20% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation at 37°C with 5% CO₂, the cells that migrated to the lower membrane surface were fixed with 4% paraformaldehyde for 0.5 h, stained with 0.1% crystal violet for 1 h at room temperature and rinsed with distilled water. The inserts were air-dried, mounted on glass slides with neutral resin, and images were captured under a light microscope (Olympus IX72; Olympus Corporation). Migrated cells were counted in five randomly selected fields per well using ImageJ software (version 1.54k).

A549 and H1299 cells were seeded onto glass coverslips in 6-well plates and cultured for 12 h. A549 cells were then treated with 100 µg/ml ZTO and H1299 cells were treated with 50 µM ZTO for 48 h. The culture medium was removed and the cells were washed twice with PBS. Then, cells were fixed with methanol for 30 min at room temperature. After two additional PBS washes, cells were stained with acridine orange for 5 min at room temperature in the dark, then rinsed three times with PBS to remove excess dye. The coverslips were carefully inverted onto microscope slides freshly coated with anti-fade mounting medium (applied 1–2 min before mounting) at room temperature and images were immediately captured using a laser scanning confocal microscope (LSM 880; Zeiss AG) with appropriate filter sets for acridine orange detection (excitation/emission: 488/525 nm).

After A549 and H1299 cells were treated with 100 µg/ml and 50 µM ZTO for 48 h, respectively, they were trypsinized (using EDTA-free trypsin), transferred to centrifuge tubes and pelleted by centrifugation at 1,000 × g for 5 min at 4°C. The supernatant was discarded and cells were resuspended in PBS, then centrifuged (1,000 × g, 5 min, 4°C). Cell pellets were stained with 5 µl Annexin V-FITC and 10 µl PI solution, followed by incubation at 37°C in the dark for 30 min with gentle mixing. The cell suspensions were then filtered through a 400-mesh nylon sieve and analyzed using a Cytomics FC500 Flow Cytometer (Beckman Coulter, Inc.) within 1 h. All samples were analyzed with CXP Software ver.2.3 (Beckman Coulter, Inc.). Apoptotic populations were quantified using Annexin V-FITC+/PI-(early apoptosis), Annexin V-FITC+/PI+ (late apoptosis/necrosis) and Annexin V-FITC-/PI-(viable cells).

Specific platforms, software and databases used for network pharmacology analysis are shown in Table I.

Primary chemical constituents of ZTO were systematically retrieved from the Traditional Chinese Medicine Systems Pharmacology database (TCMSP) and supplemented with evidence from the literature (7,11,12). The chemical structures of the identified compounds were analyzed using the PubChem database, while putative therapeutic targets were predicted using the Swiss Target Prediction database. Targets with a probability >0 were selected as the target proteins. Probability indicates the confidence level of the predicted interaction between the compound and the target.

Potential therapeutic targets associated with NSCLC were identified by searching the Online Mendelian Inheritance in Man (OMIM) database, GeneCards database and Therapeutic Target Database (TTD) using the keyword ‘non-small cell lung cancer’. The retrieved targets were combined and duplicate entries were eliminated. High-confidence targets were then prioritized based on relevance scores and functional annotations.

Overlapping targets between ZTO active components and NSCLC were identified and the ‘ZTO-NSCLC Compound-Target Network’ was constructed using Cytoscape (version 3.10.1) (15). Node degree values were analyzed and the top five active components with the highest degree values were selected to visualize their pharmacological associations.

Standard targets of ZTO for lung cancer treatment were imported into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (version 12.0), with the species limited to Homo sapiens and a confidence score threshold of >0.900. Disconnected nodes were excluded to generate the PPI network. Network topology analysis was conducted using Cytoscape (version 3.10.1) and the degree values of each target were calculated to identify hub genes.

Overlapping target genes between ZTO and NSCLC were imported into the DAVID database for GO and KEGG pathway enrichment analysis. Visualization of the enrichment results, including bubble plots and bar charts, was performed using the Bioinformatics Online Analysis Platform (https://www.bioinformatics.com.cn/).

SDF files of the primary active components of ZTO were retrieved from the PubChem database, while the 3D structures of key target proteins were collected from the Protein Data Bank (PDB; http://www.rcsb.org/). The protein structures were optimized by removing water molecules using PyMOL (version 2.1.0; Schrödinger, Inc.), followed by hydrogenation and format conversion to PDBQT using AutoDock Tools (version 1.5.6; Molecular Graphics Laboratory, The Scripps Research Institute). Molecular docking was performed using the AutoDock Vina (version 2.0) module embedded in PyRx software (version 0.8; http://pyrx.sourceforge.io/) to calculate binding energies and generate output files.

After treatment with 100 µg/ml ZTO for 48 h, the mRNA expression levels of ESR1 and EGFR in A549 cells were analyzed using RT-qPCR. Total RNA was extracted and reverse-transcribed into cDNA using the ReverTra Ace™ qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.), as described by Zhang et al (16). qPCR was performed with TB Green Premix Ex Taq (Tli RNaseH Plus; cat. no. RR420L; Takara Bio, Inc.) by cycling at 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. All reactions were run in triplicate and relative gene expression was calculated using the 2^−ΔΔCq method (17). The primer sequences used, including that of the reference gene β-actin, are listed in Table II.

Whole-cell lysate was prepared using RIPA lysis buffer (cat. no. P0013B; Beyotime Biotechnology). The lysate was centrifuged at 13,000 × g at 4°C for 15 min. The supernatant was collected and protein concentration was measured with a BCA protein assay kit (Beyotime Biotechnology). Equal amounts of protein (12 µg per lane) were loaded onto 10% SDS-PAGE gels, separated and then transferred to PVDF membranes. The membranes were blocked with BeyoFC™ Fc Receptor Blocking Solution (Human) (Beyotime Biotechnology) at room temperature for 15 min, then incubated overnight at 4°C with primary antibodies against ESR1 (1:300; cat. no. WL00940; Shenyang Wanli Biotechnology Co., Ltd.) and EGFR (1:300; cat. no. CY5064; Shanghai Abways Biotechnology Co., Ltd.). Afterward, the membranes were incubated with IRDye^® 800CW goat anti-rabbit IgG secondary antibody (1:15,000; cat. no. 926–32211; LICORbio) or IRDye^® 800CW goat anti-mouse secondary antibody (1:15,000; cat. no. 926-32210; LICORbio) at room temperature for 1 h, then washed with 1X PBS-0.1% (v/v) Tween-20 (pH 7.4). The western blots were visualized using the Odyssey^® Infrared imaging system (LI-COR Biosciences) and the bands were analyzed and normalized to mouse β-actin (1:5,000; cat. no. ab8227; Abcam). using ImageJ software.

A 2–3 day-old cell culture medium was used as the sample for Mycoplasma testing. A 30 µl PCR reaction was assembled by adding 15 µl TransDetect^® PCR Mycoplasma SuperMix II (2×; TransGen Biotech Co., Ltd.), 2 µl Myco Primer Mix II, 8 µl MycoFree Water and 5 µl template in that order. Both a negative control (MycoFree Water) and a positive control (MycoPositive Control Template II) were included. The following program was then run: initial denaturation at 95°C for 4 min; 35 cycles of 95°C for 10 sec, 62°C for 25 sec and 72°C for 10 sec; final extension at 72°C for 5 min. A total of 10 µl product was loaded on a 1.5% agarose gel. Following electrophoresis, the gel was stained with ethidium bromide and visualized under UV transillumination; a ~254 bp band indicated a positive result. The samples were compared with controls to determine Mycoplasma contamination.

Statistical analyses were performed using SPSS (version 25.0; IBM Corp.) and GraphPad Prism (version 8.0; Dotmatics) software. Intergroup differences were evaluated using unpaired Student's t-test. Data are expressed as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of different concentrations of ZTO on the proliferation of human lung cancer cell lines A549 and H1299, the inhibitory effects were measured using an MTT assay. The results demonstrated that ZTO inhibited the proliferation of both cell lines in a concentration-dependent manner (Fig. 1A). The IC₅₀ values were determined to be 96.73 µg/ml for A549 cells and 46.82 µM for H1299 cells. Therefore, ZTO concentrations of 100 µg/ml and 50 µM were selected for subsequent experiments. The colony-forming efficiency and percentage of EdU-positive cells in both A549 and H1299 cells were found to be significantly decreased compared with the control group (Fig. 1B and C).

Effects of ZTO on the migratory capacity of A549 and H1299 cells were evaluated by wound-healing and Transwell assays. Compared with the control group, the wound healing rate and number of migrated cells were markedly reduced in the ZTO-treated groups (Fig. 2). These results indicated that ZTO suppressed the migration of lung adenocarcinoma cells.

As shown in Fig. 3A, acridine orange staining revealed that untreated A549 and H1299 cells exhibited high density, intact cellular structure and nearly round nuclei. By contrast, ZTO-treated cells displayed marked morphological changes, including cell shrinkage, chromatin condensation in the nuclei and early apoptotic alterations. The Annexin V-FITC/PI dual staining results demonstrated that, compared with the control group, ZTO treatment significantly increased the early apoptosis rate of A549 and H1299 cells (Fig. 3B). These findings collectively indicated that ZTO promoted apoptosis in A549 cells.

Through the TCMSP database and supplementary literature review, >100 components from ZTO were identified. Based on preliminary experiments and screening criteria from the literature, 27 major active components of ZTO were identified (Table III). Subsequently, target prediction was performed for these active components. After entering the components into the database and applying filtering parameters (probability >0), 301 target genes were obtained for further analysis.

Using ‘non-small cell lung cancer’ as the keyword, 156 targets were retrieved from the OMIM database, 1,317 targets (with relevance score >4 times the median value) from the GeneCards database and 100 targets from the TTD database. After merging and deduplication, 1,483 NSCLC-associated targets were identified. A cross-analysis of 301 ZTO targets and 1,483 NSCLC targets identified 96 common drug-disease targets (Table SI). A ZTO-NSCLC compound-target interaction network diagram was then constructed (Fig. 4A), whereby the number of edges emitted by active compounds reflects their potential regulatory scope. The top five active compounds with the highest target connectivity were curcumenone, zedoarondiol, 13-hydroxygermacrone, neocurdione and curcumol, indicating their key roles in NSCLC intervention.

The 96 overlapping targets were further analyzed using the STRING database to build a PPI network (Fig. 4B), comprising 96 nodes and 202 edges. Visualization and topological analysis using Cytoscape (Fig. 4C and Table IV) revealed that node size and color intensity were associated with degree values. The top five core targets ranked by degree values were ESR1, EGFR, PIK3CA, PIK3CB and PTPN11, indicating their central roles in mediating the anti-NSCLC effects of ZTO.

GO functional enrichment analysis of the 96 overlapping targets was performed using the DAVID database, yielding 526 significant entries (Fig. 5A). These included 391 biological process terms primarily associated with ‘Signal transduction’, ‘Phosphorylation’ and ‘Response to xenobiotic stimulus’, 52 cellular component terms enriched in the ‘Cytoplasm’, ‘Nucleus’ and ‘Membrane raft’ as well as 83 molecular function terms mainly associated with ‘Protein tyrosine kinase activity’, ‘Transcription coactivator binding’ and ‘Protein kinase binding’. KEGG pathway enrichment analysis identified 526 significant pathways (Fig. 5B), predominantly associated with ‘EGFR tyrosine kinase inhibitor resistance’, ‘PI3K-Akt signaling pathway’, ‘Pathways in cancer’, ‘HIF-1 signaling pathway’ and ‘Proteoglycans in cancer’. These results suggested that ZTO exerted therapeutic effects on NSCLC through multi-target modulation of oncogenic signaling pathways and regulation of cellular proliferation.

Molecular docking was performed to validate the interactions between the top five core components of ZTO (curcumenone, curcumadiol, 13-hydroxygermacrone, neocurdione and curcumol) and the key targets (ESR1, EGFR, PIK3CA, PIK3CB and PTPN11) identified for NSCLC treatment. The results demonstrated that the binding energies of all active components with the core targets were <-5.0 kcal/mol, indicating spontaneous and stable binding (Table V). The binding mode analyses, focusing on ESR1 and EGFR as representative cases, are illustrated in Fig. 6. Notably, ESR1 exhibited powerful interactions with the core components, with binding energies consistently <-7.0 kcal/mol, suggesting high structural stability (18).

To determine whether ZTO exerts its effects through ESR1 and EGFR, RT-qPCR and western blotting analyses were performed to assess changes in the expression levels of these two genes following ZTO treatment. The results demonstrated that, compared with the control group, ZTO significantly downregulated ESR1 and EGFR mRNA levels by 46.56 and 32.61%, respectively (Fig. 7A). At the protein level, ESR1 and EGFR expression decreased by 35.77 and 28.99%, respectively (Fig. 7B). These findings suggested that ZTO inhibited proliferation and induces apoptosis in A549 lung cancer cells by downregulating ESR1 and EGFR expression.