Procell Life Science & Technology Co., Ltd. provided the EGFR-mutant HCC827 cell line (human lung adenocarcinoma origin, harboring the classic EGFR Exon19del E746-A750 deletion mutation; cat. no. CL-0094) and the EGFR wild-type A549 cell line (cat. no. CL-0016). These cell lines were grown in an incubator at 37°C with 5% CO₂. HCC827 cells were cultured in RPMI 1640 medium (HyClone; Cytiva) supplemented with 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) and 10% fetal bovine serum (HyClone; Cytiva). A549 cells were grown in Ham's F-12K medium (Procell) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum.

A total of 3,000 A549 and HCC827 cells/well were plated into 96-well plates. After overnight incubation, various concentrations of Sch A (0.5, 1, 2, 4, 8, 16, 32, 64, 128 and 256 µg/ml; MilliporeSigma) were added to each well, with three replicates for each concentration. Drug-free medium served as a blank control for 48 h at 37°C. Subsequently, 10 µl CCK-8 reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added for 2-h incubation. Next, the absorbance was measured at 450 nm and the IC₅₀ values were calculated using GraphPad Prism 10.0 (Dotmatics).

The following inhibitors were co-incubated with Sch A: Z-VAD-FMK (pan-caspase inhibitor; HY-16658B; final concentration: 20 µM); 3-Methyladenine (3-MA) (autophagy inhibitor, HY-19312; final concentration: 5 mM); Necrostatin-1 (Nec-1) (necroptosis inhibitor; MCE; HY-15760, 10 µM); Ferrostatin-1 (Fer-1) (ferroptosis inhibitor; MCE; HY-100579, 1 µM); Deferoxamine (DFO) (iron chelator/ferroptosis inhibitor; HY-B1625; all MCE; final concentration: 100 µM). Vehicle controls received matching volumes of DMSO (≤0.1% v/v). Cells (A549 and HCC827) were pretreated with inhibitors at 37°C for 2 h, and then co-incubated with 6 µg/ml Sch A in A549 cells and 16 µg/ml Sch A in HCC827 cells together with the same inhibitors for an additional 24 h at 37°C.

In 6-well plates, A549 and HCC827 cells were plated at a density of 5×10⁵ cells/well and were incubated overnight. The cells were divided into Sch A-treated and control groups, with A549 cells treated with 6 µg/ml Sch A and HCC827 cells treated with 16 µg/ml Sch A for 48 h at 37°C. The cells were collected using EDTA-free trypsin and then washed with 100 µl 1X binding buffer (Annexin V-PE/7-AAD Apoptosis Kit; E-CK-A216, Wuhan Elabscience Biotechnology Co., Ltd). A total of 5 µl each of Annexin V-APC and 7-AAD were used to stain the cells. Following 15 min of staining at room temperature in the dark, the cells in quadrant 3 were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, Inc.) and FlowJo v10 software (BD Biosciences).

Ferroptosis- and NSCLC-associated genes were retrieved from GeneCards (genecards.org;). Venn diagram were generated using Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/), yielding the overlapping gene set for downstream analyses.

The overlapping genes were imported into the STRING database (v11.x; string-db.org) to build the PPI) network. Network topology (node degree) was used to prioritize key regulators; YAP1 emerged as a highly connected transcription factor within the network.

In 6-well plates, A549 and HCC827 cells were plated at a density of 5×10⁵ cells/well and incubated overnight at 37°C. The cells were divided into Sch A-treated and control groups, with A549 cells treated with 6 µg/ml Sch A and HCC827 cells treated with 16 µg/ml Sch A for 48 h at 37°C. For 12 h, A549 and HCC827 cells were harvested and washed once with PBS, adjusted to 1×10⁶ cells/ml, and fixed with ice-cold 70% ethanol at 4°C for 12 h. Fixed cells were washed with PBS, then incubated with 100 µl RNase A solution (DNA Content Quantitation Assay; Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 30 min. A total of 400 µl PI staining solution was added and samples were incubated for 30 min at 4°C before acquisition (Ex 488 nm). Next, flow cytometry (CytoFLEX S) was used to assess cell cycle progression, and FlowJo software was used to analyze the data.

A549 and HCC827 cells were seeded in 6-well plates at a density of 1×10⁶ cells/well, and the cells were grown for 24 h. After A549 and HCC827 cells were treated with 5, 10, 20, 40 µg/ml Sch A for 48 h at 37°C, they were lysed in RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), and the supernatant was centrifuged at 11,000 g for 15 min at 4°C to extract all the cellular protein. With a BCA kit, protein quantification was carried out (Beijing Solarbio Science & Technology Co., Ltd.). After being separated by SDS-PAGE on 12% gels, 20 µg proteins were transferred to a PVDF membrane (Thermo Fisher Scientific, Inc.). After three washes with 0.1% PBS-Tween (PBST), the membranes were blocked with 8% skimmed milk for 2 h at room temperature, after which the primary antibodies were added at 4°C overnight. The primary antibodies used were against YAP (cat. no. 14074; 1:1,000; Cell Signaling Technology, Inc.), ACSL4 (cat. no. T510198, 1:1,000; Abmart Pharmaceutical Technology Co., Ltd.), TfR (T56618; 1:1,000; Abmart Pharmaceutical Technology Co., Ltd.) and GAPDH (2118, 1:5,000; Cell Signaling Technology, Inc.). The membranes were then incubated with an ActivAb Goat Anti-Rabbit IgG/HRP (1:4,000 dilution, Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 h following three PBST washes (5 min each). The blots were subsequently visualized with ECL western blotting substrate (Beijing Solarbio Science & Technology Co., Ltd.) for detection. GAPDH served as the internal reference and ImageJ2 (National Institutes of Health) was used to evaluate the density of the protein bands.

After A549 and HCC827 cells were centrifuged at 10,000 × g for 10 min at 4°C, the supernatant was collected. According to the manufacturer's instructions, the total intracellular levels of MDA, Fe²⁺ and GSH in 50 µl supernatant were determined via the MDA Content Assay Kit (cat. no. BC0025), Ferrous Ion Content Assay Kit (cat. no. BC5415) and Reduced GSH Content Assay Kit (BC1175; all Beijing Solarbio Science & Technology Co., Ltd.), respectively. The source of all the assay kits was Beijing Solarbio Science & Technology Co., Ltd.

Shanghai GenePharma Co., Ltd. constructed both negative control (NC) siRNA (forward: 5′-UUCUCCGAACGUGUCACGUTT-3′; reverse: 5′-ACGUGACACGUUCGGAGAATT-3′) and YAP-targeting siRNA (si-YAP; forward: 5′-AUGUGAUUUAAGAAGUAUCUC-3′; reverse: 5′-GAGAUACUUCUUAAAUCACAU-3′). In 6-well plates, A549 and HCC827 cells were plated at a density of 1×10⁵ cells/well and cultured overnight. Transfections were performed with the siRNA-mate plus transfection kit (GenePharma; cat. no. G04026) following the manufacturer's instructions. Briefly, 15 pmol siRNA was mixed with Buffer (8.5 µl) to prepare the siRNA premix (final ~1.5 µM), combined with 3 µl siRNA-mate plus to form complexes, and added directly to each 24-well (30 nM siRNA in 0.5 ml complete medium). Cells were incubated with the transfection complexes at 37°C in a humidified incubator with 5% CO₂ for 48 h without changing the medium. Subsequent experiments were performed immediately.

Using the Total RNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd.), total RNA was extracted from each set of cells. A NanoDrop 2000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc.) was used to measure the concentration and purity of the RNA. For each sample, RT 1 µg total RNA into cDNA was performed with the HiScript II One Step RT-PCR Kit (Vazyme Biotech Co., Ltd.). The mRNA expression levels of prostaglandin-endoperoxide synthase 2 (ptgs2) and GSH-specific γ-glutamylcyclotransferase 1 (Chac1) were measured via AceQ qPCR SYBR Green Master Mix (without ROX) (Vazyme Biotech Co., Ltd.), with GAPDH serving as the internal reference gene. Thermocycling conditions were as follows: initial denaturation at 95°C for 5 min; followed by 40 cycles of denaturation at 95°C for 10 sec and annealing/extension at 60°C for 30 sec. The 2^−ΔΔCq method was used to determine the relative expression levels of target genes (16). The primers are listed in Table I.

After A549 and HCC827 cells were washed with serum-free medium, 500 µl 1 µM FerroOrange working solution (MedChemExpress) was added. The cells were then incubated at 37°C in a 5% CO₂ incubator for 30 min. A fluorescence microscope (Olympus Corporation) was used to measure the fluorescence intensity.

After A549 and HCC827 cells were treated with 6 µg/ml Sch A and HCC827 cells treated with 16 µg/ml Sch A for 48 h at 37°C, 100 µl JC-1 fluorescence staining solution [mitochondrial membrane potential (MMP) assay kit with JC-1; Beijing Solarbio Science & Technology Co., Ltd.] was added to each well at 37°C for 20 min, and the A549 and HCC827 cells were incubated in the dark for 20 min. Subsequently, the cells were inspected under a fluorescence microscope (Olympus Corporation). Green fluorescence (excitation, 485 nm; emission, 535 nm; mono JC-1) was used to identify apoptotic or necrotic cells, whereas red fluorescence (excitation, 550 nm; emission, 600 nm; poly JC-1) was used to identify healthy cells.

Following centrifugation and trypsin digestion, 5×10¹⁰/l serum-free DMEM (HyClone; Cytiva) was used to resuspend A549 cells at 300 × g for 5 min at 4°C. All BALB/c nude mice were maintained at 22±2°C, 50±10% relative humidity, under a 12:12-h light/dark cycle, with free access to autoclaved chow and water. Mice were acclimated for at least 7 days prior to inoculation. The right axilla of male BALB/c nude mice (n=10; age, 6-weeks; weight, 16–18 g; Beijing Vital River Laboratory Animal Technology Co., Ltd.; Charles River Laboratories, Inc.) was then aseptically injected with a 0.1-ml aliquot of the cell mixture (each mouse received an injection of 5×10⁶ A549 cells). According to relevant studies on lung cancer models (17,18), sex has a minimal impact on experimental results; therefore, sex was not considered a variable in the present study. After ~14 days, the tumor model was considered successfully established once the maximum tumor diameter was >5 mm or the tumor volume was >50 mm³.

Humane endpoints included maximum tumor diameter ≥15 mm, tumor volume ≥1,500 mm³, ulceration/necrosis/infection, ≥20% body weight loss, or failure to eat/drink. No mice reached humane endpoints before planned termination. The mice with successfully established tumors were randomly assigned to two groups: A control group and a Sch A treatment group. During the 14-day treatment period, the Sch A group of mice were given Sch A orally at a dose of 10 mg/kg/day, whereas the control group was given an equivalent volume of saline. Following treatment, the long and short diameters of the xenograft tumors were measured, and their volumes were computed using the following formula: Tumor volume=(long diameter × short diameter²)/2. In the present study, the maximum tumor diameter observed was 8.2 mm, and the maximum tumor volume was 107 mm³. Isoflurane anesthesia was administered at a concentration of 4% for induction and 1.5% for maintenance, with an oxygen flow rate of 1.5 l/min, during subcutaneous tumor cell injection and tumor measurement procedures. For oral gavage, anesthesia was not employed, as this procedure is generally considered minimally invasive and does not typically require anesthesia. The mice were anesthetized with 4% isoflurane 1 h after the final administration and euthanized by decapitation. The tumors were then removed and weighed for analysis.

The present study was conducted in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines and the Guide for the Care and Use of Laboratory Animals (US National Research Council) (19). All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Guangzhou University of Chinese Medicine (Shenzhen, China; approval no. 2024055R).

SPSS 22.0 (IBM Corp.) was used to analyze the experimental data, and the results are presented as the mean ± SD of ≥3 independent experimental repeats. Pairwise comparisons were performed using the unpaired Student's t-test, and group comparisons were performed with one-way ANOVA followed by Tukey's post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

The viability of A549 and HCC827 cells was evaluated following exposure to a range of Sch A concentrations. The CCK-8 assay revealed a concentration-dependent decrease in viability in both cell lines (Fig. 1A and B). The IC₅₀ values of Sch A were determined to be 6.53 µg/ml for A549 cells and 16.38 µg/ml for HCC827 cells. Based on these results, concentrations of 6 and 16 µg/ml were selected for subsequent experiments in A549 and HCC827 cells, respectively. Furthermore, the inhibitory effect of Sch A on the viability of both cell lines increased significantly with prolonged treatment (Fig. 1C and D).

Compared with the control, treatment with Sch A significantly increased late apoptosis in both A549 and HCC827 cells (Fig. 2A and B). Additionally, cell cycle analysis revealed a substantial accumulation of A549 and HCC827 cells in the G₁ phase, accompanied by a notable reduction in the percentage of cells in the S and G₂ phases (Fig. 2C and D). Upon preincubation for 1 h with a range of inhibitors, including the apoptosis inhibitor Z-VAD-FMK, the autophagy inhibitor 3-methyladenine, the necrosis inhibitor necrostatin-1 and the ferroptosis inhibitors Fer-1 and DFO, only the latter two inhibitors partially reversed the Sch A-induced reduction in cell viability. By contrast, apoptosis, autophagy and necrosis inhibitors had no significant effect on Sch A-induced cell death, highlighting the specific involvement of ferroptosis in this process (Fig. 2E and F).

Changes were also observed in the MMP, where Sch A treatment led to a decrease in JC-1 aggregation and an increase in monomeric JC-1 levels, indicating a reduction in the MMP compared with that in the control group (Fig. 3A and B). Preincubation with the ferroptosis inhibitors Fer-1 and DFO resulted in a reduction in monomeric JC-1 levels and an increase in polymeric JC-1 levels, suggesting the restoration of the MMP. Furthermore, Sch A treatment led to elevated MDA levels in both A549 and HCC827 cells, an effect that was reversed by pre-treatment with Fer-1 and DFO (Fig. 3C and D). Sch A also caused a decrease in GSH levels in both cell lines, which was reversed by pre-treatment with Fer-1 and DFO (Fig. 3E and F). Additionally, an increase in the expression of the ferroptosis markers Chac1 and ptgs2 was observed following Sch A treatment, with Fer-1 and DFO effectively reversing the Sch A-induced upregulation of these markers (Fig. 3G and H).

Using GeneCards, an intersection analysis of ferroptosis-related and NSCLC-associated target genes revealed 739 overlapping genes, suggesting that these genes may serve an important role in regulating ferroptosis within NSCLC (Fig. 4A). Among these 739 overlapping genes, transcription factors were further analyzed to identify key node genes, with YAP emerging as a critical regulator within this intersecting gene network, highlighting its potential role in modulating ferroptosis in NSCLC (Fig. 4B). RT-qPCR analysis revealed that Sch A treatment of A549 and HCC827 cells led to a significant increase in YAP1 mRNA expression, indicating that Sch A can upregulate YAP1 in NSCLC cell lines (Fig. 4C and D). Given the multifaceted role of the Hippo pathway in regulating tumor cell behavior, the phosphorylation status of YAP, a key downstream effector of this pathway, was further examined. Although no changes in YAP phosphorylation levels were observed (data not shown), a marked increase in YAP protein levels was detected (Fig. 4E and F), suggesting that YAP may participate in ferroptosis through mechanisms independent of phosphorylation. To further investigate this finding, it was hypothesized that YAP might regulate ferroptosis by transcriptionally activating ferroptosis-related proteins, specifically TfR and ACSL4. Western blot analysis confirmed the involvement of YAP in regulating ferroptosis, as Sch A treatment increased the expression of the ferroptosis-related proteins TfR and ACSL4.

To determine whether YAP activation has a role in Sch A-induced ferroptosis, the present study employed siRNAs targeting YAP. Western blot analysis demonstrated that transfection with si-YAP resulted in a marked reduction in the protein levels of YAP in both A549 and HCC827 cell lines when compared with cells transfected with the NC siRNA (Fig. 5A and B). The results, presented in Fig. 5A and B, demonstrated a significant reduction in YAP expression in both A549 and HCC827 cells following siRNA transfection, indicating efficient knockdown of YAP. Along with the knockdown of YAP, the downstream targets ACSL4 and TfR were also significantly suppressed by si-YAP. Following Sch A treatment conditions, si-YAP was still able to efficiently knock down YAP expression as well as the downstream targets ACSL4 and TfR in both cell lines. FerroOrange staining revealed that Sch A treatment led to notable Fe²⁺ accumulation in both A549 and HCC827 cells. However, upon YAP silencing, this increase in Fe²⁺ was effectively reversed, further indicating that YAP serves an important role in regulating iron metabolism during Sch A-induced ferroptosis (Fig. 5C and D). Additionally, quantification of Fe²⁺ and MDA levels in A549 and HCC827 cells revealed that Sch A treatment significantly elevated both Fe²⁺ and MDA levels compared with those in the control group, whereas YAP silencing effectively alleviated these Sch A-induced increases (Fig. 5E and F).

To assess the effect of Sch A on tumor growth in vivo, a nude mouse xenograft model was used. Compared with the control treatment, Sch A treatment resulted in a significant reduction in both tumor mass and volume, demonstrating its potent inhibitory effect on tumor growth in this animal model (Fig. 6A and B). Furthermore, Sch A treatment led to a marked increase in MDA levels and Fe²⁺ accumulation within the tumors, indicating the promotion of ferroptosis-related oxidative stress and iron overload in the TME (Fig. 6C and D). This increase was accompanied by a significant decrease in GSH levels, suggesting a depletion of antioxidant defenses in the tumor tissues (Fig. 6E). Additionally, Sch A treatment upregulated the protein expression levels of YAP, ACSL4 and TfR in tumor tissues, which was consistent with the in vitro findings, further supporting the involvement of these proteins in Sch A-induced ferroptosis and tumor suppression (Fig. 6F).