The cell lines used in this study [NCI-H460 (H460) and NCI-H1299 (H1299)] were obtained from the American Type Culture Collection [ATCC] (Manassas, VA, USA) and cultured in RPMI-1640 (Shanghai BasalMedia Technologies Co., Ltd, Shanghai, China) supplemented with 10% FBS (BIOVISTECH PTY. LTD., Sydney, Australia) in a 5% CO₂ cell culture incubator at 37°C. Erianin was obtained from Shanghaiyuanye Bio-Technology Co., Ltd. (Shanghai, China). Uridine and rapamycin were sourced from Shanghai Zeye Biotechnology Co., Ltd. (Shanghai, China). L-aspartic acid was obtained from Shanghai Canspec Scientific Instruments Co., Ltd. (Shanghai, China) and 3BDO (Shanghai, China).

Cell viability was determined using a CCK-8 kit (Sigma-Aldrich, St. Louis, MO, USA). H460 and H1299 cells were seeded in 96-well plates at a density of 2 × 10⁴ cells per well. After a 24-h incubation to allow for cell adhesion and growth, erianin was added to the wells at concentrations of 200, 100, 50, 25, 12.5, and 0 nM with the latter serving as the control representing no erianin treatment. Following a 24-h incubation, the culture medium (RPMI-1640) was replaced with fresh serum-free RPMI-1640 containing 10% CCK-8 solution. After an additional 4-h incubation with the CCK-8 solution, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader.

The Annexin V-FITC (Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China) was utilized for apoptosis detection. Cell cycle experiments used the Cell cycle analysis kit [MULTISCIENCES (LIANKE) BIOTECH, CO., LTD., Hangzhou, China], which facilitated analysis of the cell cycle distribution. Following treatment, the cells were prepared and stained with the provided reagents from the kits per the manufacturer’s instructions. Flow cytometry analysis was then performed on the stained cells using a flow cytometer.

RNA-seq analysis was carried out by Lianchuan Biological Information Technology Co., Ltd. (Hangzhou, China). RNA was extracted from both the erianin-treated and control groups after 24 h and transcriptome sequencing was performed on the samples. Metabolomics analysis was performed by Wuhan Maiteville Biotechnology Co., Ltd. (Wuhan, China). The metabolome profiling utilized UPLC-Q-TOF-MS technology to analyze the differential metabolites between erianin-treated and untreated samples. Integrative analysis of the transcriptome sequencing and metabolomics results was performed using the MetaboAnalyst online tool (https://www.metaboanalyst.ca/).

The putative targets of erianin were identified using the SwissTarget online tool (http://swisstargetprediction.ch/) based on the chemical structure of the compound, while potential therapeutic targets associated with lung cancer were sourced from the OMIM database. Pan-cancer analysis of mTOR expression was conducted using the Oncomine database. The UALCAN database was utilized to determine mTOR expression across different stages of cancer development. The Human Protein Atlas (HPA) database provided validation of the levels of mTOR protein expression in normal and lung cancer tissues. Molecular docking and dynamics simulations were performed to validate mTOR as a target of erianin. Crystal structures of mTOR were obtained from the Protein Data Bank (PDB) and utilized as initial configurations for the docking studies. The structure of erianin was obtained from PubChem. Protein and ligand preparation for molecular docking were conducted using the induced fit docking method.

CAD knockdown in lung cancer cells was accomplished through transient transfection with shRNA-CAD using Lipofectamine 2,000 (Invitrogen, Waltham, MA, USA), a widely used transfection reagent. The specific shRNA sequence utilized in this study was 5′-GCATATACGATACAAGGCTGTTAGAG-3′. Conversely, mTOR was overexpressed in lung cancer cells using an mTOR plasmid and Lipofectamine 2,000.

Total protein was extracted from tissues or cells using a protein extraction buffer and the protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. The proteins were subsequently separated by SDS-PAGE on a 10% polyacrylamide gel. The separated proteins were then transferred onto a PVDF membrane, which was subsequently incubated overnight with specific primary antibodies targeting the protein of interest. After washing to remove unbound antibodies, the membrane was incubated with a secondary antibody conjugated to an enzyme or fluorophore for 1.5 h. Protein bands on the PVDF membrane were visualized using an enhanced chemiluminescence reaget (New Cell & Molecular Biotech Co., Ltd., Suzhou, China) and captured with an exposure meter equipped with a chemiluminescence imaging system (BIO-RAD, California, USA).

The cells were seeded at a density of 4,000 cells/6 cm dish and treated with erianin by replacing the culture medium with erianin-containing medium every 2 days for approximately 2 weeks. After colony formation the cells were fixed with 4% fixative solution and stained with 0.1% crystal violet solution. The colonies, visible as distinct violet-colored structures, were photographed using an appropriate camera.

H460 and H1299 cells were treated with DMSO or 100 nM erianin for 2 h. Following incubation, the samples underwent thermal cycling on a PCR instrument with a temperature gradient ranging from 55°C–80°C for 5 min. Thermal cycling was followed by five cycles of freeze-thawing in liquid nitrogen. After centrifugation, the supernatant was collected and mixed with SDS loading buffer, then heated at 100°C for 5 min in a metal bath. The prepared samples were stored at −80°C and subsequently analyzed by western blotting.

All animal experiments were performed in accordance with the guidelines of the Use and Care of Animals Committee at Hangzhou Normal University (HSD-20241203-01). Initially, 4 × 10⁶ H460 cells were subcutaneously inoculated into mice to establish a lung cancer-bearing nude mice model. Erianin, dissolved in 50% dimethyl sulfoxide, was intraperitoneally injected into the mice at 50 mg/kg, 100 mg/kg, and 200 mg/kg doses during the pre-experiment phase. The mice in the erianin group were administered erianin at a concentration of 100 mg/kg and uridine (33.4 g/kg) and 3BDO (0.05 g/kg) in corollary experiments. The tumor size was regularly monitored every 2^nd day to track changes in tumor growth. The mice were sacrificed after 18 days of treatment and the tumors were harvested for further analysis.

The data were prepared and analyzed using GraphPad Prism (10.1.2). Cell line-based experiments were performed with three biological replicates per condition and the animal experiments included five replicates per group. Following assessment of normality, differences between the two groups with normal distribution were assessed using Student’s t-test. For comparisons involving more than two groups with a normal distribution, an initial ANOVA analysis was performed followed by post-hoc pairwise comparisons using Tukey’s multiple comparisons test. Statistical significance was set at a P < 0.05.

Erianin has been shown to exert significant anticancer effects on lung cancer cells by reducing cell viability, inducing cell cycle arrest, and promoting apoptosis. In the current study the anticancer effects of erianin were validated using two lung cancer cell lines (H460 and H1299). The CCK-8 kit and flow cytometry were used to assess cell viability, cell cycle distribution, and apoptosis. To evaluate the effects of erianin on lung cancer cell viability, varying concentrations of erianin were administrated to H460 and H1299 cells and cell viability was measured using the CCK-8 assay after 24 h. Erianin significantly reduced the survival rate of both cell lines with IC₅₀ values calculated at 61.33 nM for H460 cells (Figure 1A) and 21.89 nM for H1299 cells (Figure 1B). Furthermore, apoptosis analysis revealed that erianin induced apoptosis in lung cancer cells in a dose-dependent manner in both cell lines (Figure 1C-E), indicating the ability of erianin to trigger programmed cell death. As we reported in a previous study¹², erianin has also been shown to induce ferroptosis in lung cancer cells, indicating that apoptosis is not the sole mechanism contributing to erianin-induced cell death. Natural compounds like erianin often exert antitumor effects through multiple, potentially interconnected mechanisms, which may be influenced by factors, such as the compound mode of action, tumor cell type, and the surrounding microenvironment. Additionally, erianin significantly induced G₂/M phase cell cycle arrest in H460 and H1299 cells (Figure 1F-H). These findings collectively underscore the potent anticancer effects of erianin on lung cancer cells, highlighting the potential of erianin as a promising therapeutic candidate for lung cancer.

Our previous study¹² in combination with the analyses presented herein have confirmed that erianin significantly reduces the viability of lung cancer cells, induces cell cycle arrest, and triggers apoptosis. However, the precise mechanisms underlying the anti-tumor effects of erianin are not completely understood. To address this knowledge gap and elucidate the potential mechanisms of action underlying erianin in lung cancer, H460 cells were treated with 100 nM erianin for 24 h, followed by transcriptomic sequencing and metabolomics analyses. As depicted in Figure 2A, B, these analyses revealed 12,627 differentially expressed genes and 59 metabolites in H460 cells when comparing the erianin-treated group to the untreated group. Further integrated analysis of these differentially expressed genes and metabolites using the MetaboAnalyst 6.0 online tool indicated that erianin significantly impacts nucleotide metabolic pathways in lung cancer cells, especially those pathways involved in purine and pyrimidine metabolism (Figure 2C). Nucleotides are critical for DNA and RNA synthesis and are essential for tumor cell growth and proliferation. These findings suggest that erianin may interfere with critical biochemical pathways involved in nucleotide synthesis and utilization, thereby inhibiting the growth and proliferation of lung cancer cells.

To further elucidate the pathway by which erianin affects nucleotide metabolism of lung cancer cells, an analysis of potential erianin targets in lung cancer was performed using network pharmacology. The SwissTarget online tool predicted 100 potential targets for erianin, while 200 potential therapeutic targets for lung cancer were identified through the OMIM database. These targets were subsequently analyzed using Venn cross-analysis, revealing three potential targets for erianin in lung cancer (EPHB2, mTOR, and KDM1A; Figure 2D). To further validate these targets, molecular docking and kinetic simulations were performed to assess the interactions between erianin and EPHB2, mTOR, and KDM1A. Initial protein configurations (EPHB2 PDB ID: 3ZFM; KDM1A PDB ID: 5LHI; and mTOR PDB ID: 3FAP) were obtained from the PDB and the erianin structure was sourced from PubChem. The induced fit docking (IFD) method was used to account for receptor flexibility, which improved binding interaction predictions. The optimal binding conformations, which were based on affinity and energy evaluations, were selected to represent binding efficacy. Due to structural similarities with known mTOR inhibitors, the crystal structure of the mTOR FRB domain complexed with FKBP12 (PDB ID: 3FAP) was used as the receptor for erianin docking. Molecular dynamics simulations provided insights into the stability and dynamics of erianin-receptor complexes over time, which were monitored through root-mean-square deviation (RMSD) values. Binding free energies (∆Gbind) were predicted using the MM/GBSA approach combined with energy components, including van der Waals, electrostatic, polar, and non-polar interactions. This approach aimed to elucidate binding modes, stability, and energetic attributes of erianin interactions with EPHB2, KDM1A, and mTOR. The binding conformations and the binding conformation pockets were visually represented (Figure 2E), accompanied by binding energy computations. EPHB2 and mTOR had the highest docking scores (-8.238 kcal/mol and −8.233 kcal/mol, respectively), followed by KDM1A with a score of −7.479 kcal/mol (Table 1). To substantiate these findings, extended five nanosecond molecular dynamics simulations²⁹ were executed, which refined energy levels and permitting evaluation of interaction energies (Figure 2F). The interaction energies of different targets were calculated after the molecular dynamic simulations. A lower ΔGcal value (ΔGcal = ΔEvdw + ΔEele + ΔGpol + ΔGnonpol) is indicative of more favorable binding with a ΔGcal of −29.71, −29.51, and −19.72 for EPHB2, mTOR, and KDM1A, respectively (Table 2), suggesting the high potential of EPHB2 and mTOR as targets of erianin in lung cancer cells. Furthermore, the results of the cellular thermal shift assays (CETSA) demonstrated that erianin enhanced the thermotolerance of mTOR (Figure 2G), suggesting the potential interaction between erianin and mTOR. Previous studies have demonstrated that mTOR-mediated phosphorylation activates S6K, leading to the activation of CAD and subsequent promotion of de novo pyrimidine synthesis^17,18. Given the significant disruption of pyrimidine metabolism by erianin observed in our combined transcriptome and metabolomics analyses, mTOR emerged as a compelling candidate for erianin intervention in lung cancer cells. We therefore concentrated on mTOR in the subsequent analysis. Additionally, data from the Oncomine database revealed a significant upregulation of mTOR in lung adenocarcinoma (P = 6.91E-4) and lung squamous cell carcinoma (P = 0.042; Figure 2H). Further examination of mTOR expression across different stages of lung adenocarcinoma and squamous cell carcinoma development using the UALCAN database demonstrated consistent upregulation of mTOR expression throughout various stages of cancer progression (Figure 2I). To validate these findings at the protein level, mTOR protein expression was analyzed in normal and cancerous lung tissues using the HPA database, which provided immunohistochemistry-based protein expression data. The results confirmed that mTOR is overexpressed in lung cancer tissues compared to adjacent normal tissues (Figure 2J), highlighting the potential role of mTOR in lung cancer progression and the potential as a therapeutic target. Furthermore, western blot analysis showed that erianin suppresses mTOR activation in lung cancer cells along with mTOR downstream effectors (S6K and CAD; Figure 2K). Erianin reduced the levels of p-S6K and p-CAD in H460 lung cancer cells. A similar suppression was also observed when cells were treated with the mTOR inhibitor, rapamycin.

Given the significant downregulation of mTOR phosphorylation by erianin in lung cancer cells, the role of mTOR expression in modulating the inhibitory effects of erianin on cell proliferation was investigated. Specifically, the effect of 3BDO, an activator of mTOR, on the erianin-induced inhibition of cell viability was determined using the CCK-8 assay. The addition of 3BDO partially antagonized the suppressive effect of erianin on cell viability (Figure S1), suggesting that mTOR has a role in mediating inhibition of cell viability induced by erianin in lung cancer cells. Furthermore, mTOR was overexpressed in H460 and H1299 cells using an mTOR plasmid (Figure 3A). To assess the impact of mTOR overexpression on cell proliferation and the inhibitory effects of erianin on cell growth, the cells were treated with 100 nM erianin for 24 h with or without mTOR overexpression. Microscopic observations indicated that mTOR overexpression reduced cell inhibition induced by erianin treatment (Figure 3B, Figure S2). Subsequent colony formation assays demonstrated that mTOR overexpression significantly enhanced cellular proliferation and conferred protection against erianin-induced inhibition of cell growth in H460 and H1299 cell lines (Figure 3C). Additionally, an apoptosis assay revealed that erianin treatment significantly increased the percentage of apoptotic cells in both H460 and H1299 cells compared to the control groups (Figure 3D, E). However, mTOR overexpression mitigated the apoptotic effect induced by erianin, leading to a reduction in the percentage of apoptotic cells in both cell lines (Figure 3D, E). Furthermore, cell cycle analysis showed that erianin treatment caused G₂/M phase cell cycle arrest with approximately 90% of H460 and H1299 cells arrested in the G₂/M phase. Overexpression of mTOR counteracted this effect, reducing the extent of G₂/M phase arrest in both cell lines (Figure 3F, G). These findings suggest that mTOR has a critical role in modulating erianin-induced growth inhibition in lung cancer cells.

To further elucidate the role of erianin-induced downregulation of mTOR activation and the mTOR downstream pivotal pyrimidine metabolism factor, CAD, in mediating erianin-induced cell growth inhibition in lung cancer cells, CAD knockdown experiments were performed. Western blot analysis confirmed CAD knockdown in protein expression (Figure 4A). Subsequent microscopic observations (Figure 4B) and colony formation assays (Figure 4C) revealed that CAD knockdown in H460 and H1299 cells attenuated cellular proliferation and reduced the number of colonies formed. Importantly, CAD knockdown enhanced the induction of cell death and amplified the inhibition of colony formation by erianin in H460 and H1299 cells (Figure 4B, C). Furthermore, CAD knockdown intensified the apoptosis induction and G₂/M phase cell cycle arrest effects of erianin in lung cancer cells. A significant increase in the percentage of apoptotic cells (Figure 4D, E) and a greater extent of G₂/M phase cell cycle arrest (Figure 4F, G) was observed in CAD knockdown H460 and H1299 cells compared to cells treated with erianin alone. Downregulation of CAD activation induced by erianin may impair de novo pyrimidine synthesis, resulting in growth inhibition in lung cancer cells. This disruption supports the observation of altered pyrimidine metabolism in erianin-treated lung cancer cells.

Given the significant perturbations in pyrimidine metabolism and downregulation of the mTOR-S6K-CAD signaling axis induced by erianin treatment in lung cancer cells, in vitro experiments were performed to determine if the growth inhibition caused by erianin could be rescued by supplementing exogenous pyrimidines and the synthesis substrates. Remarkably, the addition of exogenous uridine was shown to reduce erianin-induced apoptosis in lung cancer cells (Figure 5A, B). This finding provides compelling evidence that disruptions in pyrimidine metabolism are integral to the erianin’ anticancer mechanism. Additionally, supplementation with exogenous aspartate, a key precursor in pyrimidine biosynthesis, produced effects, especially in H460 cells (Figure 5C, D). This finding suggests that the anticancer activity of erianin may, at least in part, be attributed to the ability to impede de novo pyrimidine synthesis. Experiments to assess the impact of uridine and aspartate supplementation on the cell cycle arrest induced by erianin were performed in these cell lines. The addition of uridine partially antagonized the effect of erianin on cell cycle distribution, while supplementation with aspartate did not show a significant effect on cell cycle arrest (Figure 5E, F). It is possible that the exogenous addition of uridine typically exerts effects within a shorter time frame because uridine directly participates in the synthesis of pyrimidine nucleotides and influences RNA synthesis within the cell. In contrast, aspartate, as a precursor in pyrimidine biosynthesis, requires conversion through metabolic pathways to form carbamoylglutamate, which subsequently contributes to the synthesis of uridine monophosphate (UMP) and other pyrimidine nucleotides. Therefore, the exogenous supplementation of aspartate necessitates a more prolonged metabolic process to be converted into active pyrimidine nucleotides. Consequently, the effects of uridine supplementation may work more rapidly than aspartate.

To evaluate the inhibitory effects of erianin on lung cancer through downregulation of mTOR activation and disruption of pyrimidine metabolism in vivo, a subcutaneous xenograft model was established using H460 cells. Once the xenografts reached approximately 70 mm³ in size, the mice were randomly divided into the following 4 groups: low-dose erianin (50 mg/kg); medium-dose erianin (100 mg/kg); high-dose erianin (200 mg/kg); and a control group. Parameters, such as body weight and tumor volume, were measured every other day to assess treatment effects. As shown in Figure 6A-C, intraperitoneal administration of medium and high doses of erianin resulted in significant inhibition of tumor growth compared to the control group. There was no significant difference in the body weight among the groups (Figure 6D). Based on these observations, the medium dose of erianin (100 mg/kg) was selected for subsequent investigations.

To further confirm that erianin inhibits lung cancer cell growth by regulating pyrimidine metabolism and mTOR activation in vivo, mice with tumors were divided into the following four groups: erianin; erianin + uridine; erianin + 3BDO (an mTOR activator); and a control group. The erianin group received erianin (100 mg/kg) and the uridine and 3BDO groups were treated with uridine (33.4 g/kg) and 3BDO (0.05 g/kg) in addition to erianin (100 mg/kg). Tumor volume and weight were measured to evaluate the impact of each treatment on lung cancer cell growth. Tumor tissue in each group was photographed at the end of the experiment (Figure 6E). Remarkably, co-treatment with erianin and 3BDO or exogenous uridine resulted in increased tumor weight (Figure 6F) and volume (Figure 6G) compared to erianin treatment alone. This finding suggests that the addition of an mTOR activator or exogenous pyrimidines counteracts the tumor growth-inhibitory effects of erianin in vivo.

Furthermore, western blot analysis of tumor tissue revealed reduced levels of p-mTOR, p-S6K, and p-CAD expression in erianin-treated tumors. However, the administration of uridine or 3BDO antagonized the inhibitory effect of erianin on the mTOR-S6K-CAD axis (Figure 6H). These results support the notion that erianin effectively inhibits the phosphorylation and activation of mTOR and mTOR downstream factors (S6K and CAD) in vivo. This finding further validates the relevance of the mTOR-related signaling pathway and pyrimidine metabolism in the erianin anticancer effects, suggesting that the therapeutic potential of erianin in lung cancer may be, at least in part, mediated through modulation of pyrimidine metabolism via suppression of mTOR activation.