Asaraldehyde suppresses non-small cell lung cancer progression via ferroptosis induction and inhibition of PI3K-AKT signaling

Abstract

Non-small cell lung cancer (NSCLC) is an aggressive malignancy characterized by poor therapeutic outcomes, and its progression is closely linked to dysregulated PI3K-AKT signaling and resistance to ferroptosis. This study aimed to investigate the anti-tumor effects of Asaraldehyde (Asa) on NSCLC, elucidate its underlying mechanisms, and explore its therapeutic potential by targeting both ferroptosis and the PI3K-AKT pathway. It was revealed that Asa treatment significantly suppressed NSCLC cell proliferation, migration, and epithelial-mesenchymal transition (EMT) while inducing ferroptosis, as evidenced by increased lipid peroxidation, reactive oxygen species (ROS) accumulation, and glutathione (GSH) depletion. Additionally, Asa promoted ubiquitin-mediated degradation of AKT, leading to inhibition of the PI3K-AKT signaling pathway, and this effect was partially attenuated by Ferrostatin-1, a ferroptosis inhibitor. In vivo, Asa administration significantly reduced tumor growth in a xenograft mouse model, accompanied by decreased expression of Ki67, AKT, and ferroptosis-related proteins. These findings demonstrate that Asa exerts potent anti-NSCLC activity through ferroptosis induction and PI3K-AKT pathway suppression, and suggest that targeting these pathways with Asa could provide a novel therapeutic strategy for NSCLC treatment.

Introduction

Lung cancer continues to pose a major public health burden globally, standing as the foremost contributor to cancer-associated deaths. Non-small cell lung cancer (NSCLC), the predominant histological subtype, comprises roughly 85% of lung cancer diagnoses and is frequently associated with unfavorable clinical outcomes [1]. Despite notable advances in disease management, therapeutic interventions for NSCLC are still inadequate in many cases. Surgery remains the cornerstone of curative treatment, yet it is viable only for individuals with early-stage, non-metastatic disease [2]. For patients diagnosed with advanced or metastatic NSCLC, molecularly targeted therapies-especially epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs)-play a pivotal role. These drugs, spanning first-generation (gefitinib, erlotinib), second-generation (afatinib), and third-generation (osimertinib) variants, function by inhibiting hyperactive EGFR signaling, a key driver of tumor growth in specific NSCLC subsets [3]. Immunotherapy has also emerged as a transformative approach in recent years, primarily through immune checkpoint blockade targeting the PD-1/PD-L1 axis [4]. Agents such as pembrolizumab and nivolumab have demonstrated substantial survival benefits by reactivating the immune response against tumor cells [5,6]. Nevertheless, the clinical utility of these treatments is often constrained by acquired resistance, treatment-related toxicities, and heterogeneous patient responses, highlighting the urgent need for novel treatment strategies.

Ferroptosis represents a unique mode of regulated cell death distinguished by iron-mediated lipid peroxidation, culminating in irreversible membrane disruption and cellular destruction. In contrast to apoptotic or necrotic pathways, this process is mediated through excessive reactive oxygen species (ROS) generation coupled with diminished glutathione peroxidase 4 (GPX4) activity, a critical antioxidant defense enzyme [7]. Growing evidence underscores the tumor-suppressive potential of ferroptosis. Research indicates that therapy-resistant mesenchymal-state malignancies exhibit particular reliance on GPX4-regulated lipid peroxidase mechanisms to evade ferroptotic cell death [8]. Notably, androgen receptor degradation via ubiquitination has been demonstrated to downregulate GPX4, overcoming temozolomide resistance in glioma through ferroptosis induction [9]. The therapeutic targeting of ferroptosis has gained considerable attention in oncology. Sulfasalazine promotes ferroptotic cell death in glioblastoma by disrupting cystine uptake via the XC system, subsequently inhibiting PI3K/AKT signaling [10]. Similarly, artesunate has been identified as a selective ferroptosis activator in pancreatic carcinoma models [11]. Importantly, many chemotherapy-resistant malignancies retain vulnerability to ferroptosis, positioning it as a viable approach for eradicating treatment-refractory tumors. For instance, Erastin has been shown to enhance cisplatin sensitivity in pulmonary malignancies through ferroptosis activation [12]. Synergistic administration of aspirin and sorafenib induces XC system suppression, glutathione exhaustion, and subsequent ferroptosis in therapy-resistant head and neck carcinomas, thereby potentiating cisplatin efficacy [13]. These findings collectively highlight ferroptosis as a critical tumor-suppressive mechanism. Consequently, the discovery of novel ferroptosis-inducing agents may yield innovative therapeutic approaches for NSCLC treatment.

The PI3K-AKT cascade serves as a critical regulator of malignant cell survival, proliferation, and therapeutic resistance. Activation of this pathway occurs through multiple growth factor receptors and oncogenic stimuli, resulting in increased cellular growth, metabolic reprogramming, and apoptotic avoidance [14]. In NSCLC, hyperactivation of PI3K-AKT signaling commonly arises from genetic alterations, including amplification of receptor tyrosine kinases (RTKs) and loss of phosphatase and tensin homolog (PTEN) [15,16]. Such constitutive pathway activation has been strongly associated with disease advancement and the development of osimertinib resistance [17,18]. Consequently, pharmacological inhibition of this signaling network represents a viable approach for circumventing treatment resistance in NSCLC patients.

Asaraldehyde, a natural phenylpropanoid compound isolated from Asarum plants, has demonstrated significant antimicrobial activity against fungal pathogens [19,20]. However, its potential anticancer properties remain poorly characterized. Notably, no systematic investigation has elucidated its capacity to initiate ferroptotic cell death or influence critical oncogenic signaling networks, particularly the PI3K-AKT axis. Comprehensive examination of these pharmacological mechanisms could reveal novel therapeutic applications for this compound in NSCLC management.

Materials and methods

Cell culture

NSCLC cell lines (A549, H1299, H292, HCC827) were purchased from the cell library of the Chinese Academy of Sciences, and grown in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS). The cells were maintained in an environment with 5% CO₂ at 37°C and a controlled humidity level. Authentication of the cell lines was confirmed through short tandem repeat (STR) analysis performed by Biowing Applied Biotechnology in Shanghai. Furthermore, all cell lines were verified to be free of mycoplasma contamination.

Cell Counting Kit-8 (CCK8) assay

Cells were plated in 96-well plates at a density of 5×10³ cells per well. Following the specified treatment, 10 µL of the Cell Counting Kit-8 reagent (AbMole BioScience, USA) was added to each well, and the plate was incubated at 37°C. Absorbance was measured at 450 nm using a microplate reader (PerkinElmer EnSpire, Shanghai, China).

Colony formation assay

Cells were seeded into 12-well plates and subjected to transfection with the specified plasmids or siRNAs. Post-transfection, cells were incubated for a period of 10-15 days, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet solution. Photographs of the stained wells were taken, and colony numbers in each well were quantified.

Wound healing assay

Cells were seeded into 6-well plates and cultured to 80%-90% confluence. A straight line was drawn across the cell layer using a 200 μL pipet tip. Cells were washed three times with PBS and cultured with RPMI 1640 medium supplemented with 1% FBS. At the indicated times, the samples were photographed under a light phase-contrast microscope.

Calcein AM/PI staining

After indicated treatment, cells were washed with PBS twice and then incubated with 6 µM PI and 1.5 μM Calcein-AM (#CA1630, Servicebio Co., Ltd., China) for 15 min at 37°C in dark. The images were captured using an inverted fluorescence microscope (IX71, OLYMPUS, Japan).

Immunoprecipitation (IP) and western blot

For the IP assay, cells were lysed in NP-40 lysis buffer, and the lysate was centrifuged at 12,000 g for 20 minutes at 4°C. The co-immunoprecipitation mixture, containing 1 μg antibody and 30 μL of protein A/G agarose beads, was incubated on a Rotator Shaker at 4°C for 8 hours. After incubation, the beads were washed three times with lysis buffer and boiled for 10 minutes at 100°C in 1× loading buffer.

For Western blot analysis, proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% fat-free milk and incubated overnight at 4°C with the following primary antibodies: E-Cadherin (ZenBio, #201283), N-Cadherin (Proteintech, #22018-1-AP), Snail (ZenBio, #340942), PI3K (Proteintech, #67071-1-Ig), AKT (ZenBio, #342529), p-AKT (Ser473) (Proteintech, #66444-1-Ig), ERK (ZenBio, #201245-4A4), p-ERK (Thr202/Tyr204) (Proteintech, #28733-1-AP), MEK (ZenBio, #380797), p-MEK (Thr386) (Proteintech, #81304-1-RR), ubiquitin (Proteintech, #10201-2-AP), SLC7A11 (Proteintech, #26864-1-AP), GPX4 (Cell Signaling Technology, #52455S), FTH1 (Proteintech, #83428-1-RR), and GAPDH (Proteintech, #10494-1-AP). The membranes were washed three times with Tris-buffered saline containing 0.1% Tween-20 before being incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using a chemiluminescence detection kit and images were captured with a BioRad ChemiDoc MP digital gel image analysis system.

Transcriptomic (RNA-seq) analysis

RNA-sequencing analysis was performed by Wuhan IGeneBook Biotechnology CO., LTD.

Immunofluorescence assay

To assess reactive oxygen species (ROS) levels, cells were incubated with 5 μM Dihydroethidium (DHE; MedChemExpress, #HY-D0079) for 30 minutes at 37°C in the dark, then washed twice with PBS. To assess labile iron levels, cells were incubated with 1 μM FerroOrange (Cell Signaling Technology, #36104) for 30 minutes at 37°C in the dark, then washed twice with PBS. Fluorescent images were taken using an inverted fluorescence microscope (Nikon Eclipse Ti2-U).

Measurement of intracellular glutathione (GSH), lipid peroxidation (LPO) and malondialdehyde (MDA) levels

The levels of intracellular GSH, LPO, and MDA were quantified utilizing the Reduced Glutathione Content Assay Kit (Solarbio, #BC1175), the Lipid Peroxide Content Assay Kit (Solarbio, #BC5245), and the Malondialdehyde Content Assay Kit (Solarbio, #BC0025), following the protocols outlined by the manufacturer.

Xenograft tumor model

Four-week-old male BALB/c-nude mice were obtained from SPF Biotechnology Co., Ltd. (Beijing) and were randomly assigned to groups. The mice were housed in a controlled, pathogen-free environment with free access to food and water. Each group of mice was anesthetized with isoflurane and subcutaneously injected with 3×10⁶ cells. Asa was administered daily via intraperitoneal injection. At the designated time points, euthanasia was carried out using carbon dioxide, and the xenografted tumors were collected. The researcher was blinded to the group allocations throughout the experiment. No animals were excluded from the analysis. The study was conducted under the approval of the Ethics Committee of the First Affiliated Hospital of Nanchang University (protocol code CDYFY-IACUC-202501GR043). All experiments were performed in accordance with the approved protocol and guidelines.

Immunohistochemistry (IHC) staining

IHC staining of xenografted tumors were performed by Wuhan ServiceBio Technology Co., Ltd.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical analysis of the data obtained from experiments was performed using GraphPad Prism 10 Software. Statistical significance between groups was determined using either a Student’s t-test or one-way analysis of variance (ANOVA), followed by Fisher’s LSD test. A p-value of less than 0.05 was considered statistically significant.

Results

Asaraldehyde suppresses the malignant phenotypes of NSCLC cells

To evaluate the effects of Asa on lung cancer cell viability, four NSCLC cell lines (A549, HCC827, H292, and H1299) were treated with varying concentrations of Asa, and cell viability was assessed using CCK-8 assays. Asa demonstrated a dose-dependent inhibitory effect on all cell lines, with IC50 values of 86.1 μM, 95.6 μM, 127.2 μM, and 133.4 μM for A549, HCC827, H292, and H1299, respectively (Figure 1A-D). Among these, A549 cells exhibited the greatest sensitivity to Asa. On the contrary, Asa exhibited low cytotoxicity on BEAS-2B, a human bronchial epithelial cell line (Figure 1E). These results indicate that Asa effectively reduces cell viability across different lung cancer cell types. The effect of Asa on the colony-forming ability of lung cancer cells was examined through colony formation assays. Treatment with increasing concentrations of Asa significantly reduced the number of colonies formed by A549 and H1299 cells compared to untreated controls (Figure 1F, 1G). To assess whether Asa affects cell migration, wound-healing assays were performed on A549 and H1299 cells. Asa treatment significantly inhibited cell migration in both cell lines (Figure 1H, 1I). Results of Calcein AM/PI staining indicate that Asa treatment significantly increased the ratio of dead cells (Figure 1J, 1K). Additionally, the effect of Asa on epithelial-to-mesenchymal transition (EMT) was evaluated by assessing the expression levels of EMT markers via Western blot analysis. Asa treatment led to a dose-dependent increase in E-Cadherin (epithelial marker) and a decrease in N-Cadherin and Snail (mesenchymal markers) in both A549 and H1299 cells (Figure 1L). These results demonstrate that Asa inhibits lung cancer cell viability, colony formation, and migration while reversing EMT.

Figure 1.

Asaraldehyde suppresses the malignant phenotypes of NSCLC cells. A-E. IC50 values of Asaraldehyde for the A549, HCC827, H292 and H1299 and BEAS-2B cells were examined by CCK-8 assay. F, G. A549 and H1299 cells were treated with various concentrations of Asaraldehyde, and colony formation were examined by crystal violet staining (magnification, ×1, Scale bar = 5 mm). H, I. A549 and H1299 cells were treated with various concentrations of Asaraldehyde, and cell migration ability was examined by wound healing assay (magnification, ×50, Scale bar = 200 µm). J, K. A549 and H1299 cells were treated with various concentrations of Asaraldehyde, and ratio of dead cells was examined by Calcein AM-PI staining. The green fluorescence indicates viable cells, and red fluorescence indicates dead cell (magnification, ×200, Scale bar = 100 µm). L. A549 and H1299 cells were treated with various concentrations of Asaraldehyde for 48 h, and protein expression was detected by western blot. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Asaraldehyde suppresses the progression of NSCLC through inhibition of PI3K-AKT signaling

To elucidate the corresponding mechanism underlying the anti-tumor effect of Asa, RNA-seq analysis was performed. The results revealed a significant alteration in the transcriptome of Asa-treated cells compared to controls. Specifically, 1113 genes were downregulated, and 456 were upregulated (Figure 2A). Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) highlighted a strong association with cell cycle and migration regulation, encompassing processes such as mitotic cell cycle, chromosome organization, and microtubule-based processes (Figure 2B). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment in PI3K-AKT signaling (Figure 2C). These findings confirm that the treatment exerts a substantial influence on the malignant phenotypes of NSCLC cells.

Figure 2.

RNA-seq analysis of differentially expressed genes. A. Volcano plot showing differentially expressed genes (DEGs) between treated and control groups. Red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent non-significant DEGs. |log2(FC)| > 1, FDR < 0.05. B. Gene Ontology (GO) enrichment analysis of upregulated DEGs. The x-axis represents the gene ratio, indicating the proportion of DEGs in a specific GO term. The y-axis represents the -log10 (p-value) for each GO term. C. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs. The x-axis represents the gene ratio, indicating the proportion of DEGs in a specific pathway. The y-axis represents the -log10 (p-value) for each pathway.

In align with previous studies, we showed that the expression of AKT and p-AKT was significantly upregulated in NSCLC cells compared with those in BEAS-2B cells (Figure 3A). To assess the impact of Asa on key signaling pathways, we performed western blot analysis on A549 and H1299 lung cancer cell lines treated with increasing concentrations of Asa (Figure 3B). Our results demonstrate that Asa treatment significantly inhibited the phosphorylation of AKT and MEK, two crucial proteins involved in cell proliferation and survival. Additionally, we observed a dose-dependent decrease in the phosphorylation of ERK, a downstream kinase of MEK. Intriguingly, while the expression of PI3K, MEK and ERK remains largely unchanged following Asa treatment, the expression of AKT decreased significantly. To further investigate the mechanism of Asa-induced downregulation of AKT, we performed experiments using cycloheximide (CHX) to block protein synthesis. We found that Asa treatment potentiated the turnover of AKT (Figure 3C). Furthermore, we examined the role of protein degradation in Asa-induced AKT inhibition by treating cells with MG132, a proteasome inhibitor and CQ, a lysosome inhibitor. We observed that MG132 partially rescued downregulation of AKT in Asa-treated cells (Figure 3D), Additionally, Additionally, we observed a significant increase in ubiquitinated AKT in Asa-treated cells (Figure 3E), suggesting that Asa may promote AKT degradation through the ubiquitin-proteasome pathway.

Figure 3.

Asaraldehyde facilitates ubiquitin degradation of AKT and inhibition of PI3K/AKT signaling. A. Expression of AKT and p-AKT was upregulated in NSCLC cells. Total protein in BEAS-2B, A549, H1299, HCC827 and H292 cells was extracted, and the expression of AKT and p-AKT was detected by western blot. B. A549 and H1299 cells were treated with various concentrations of Asaraldehyde for 48 h, and protein expression was detected by western blot. C. A549 cells were treated with 50 μg/mL Cycloheximide in the presence or absence of 100 μM Asaraldehyde for the indicated times. AKT expression was detected by western blot. D. A549 cells were treated with 100 µM Asaraldehyde in the presence or absence of 10 μM MG132 or 10 μM chloroquine. AKT expression was detected by western blot. E. A549 cells were treated with 0, 50, 100 µM Asaraldehyde for 48 h. AKT ubiquitination level was detected by immunoprecipitation and western blot. Data were presented as mean ± SD. ns P > 0.05, ***P < 0.001, ****P < 0.0001.

To investigate whether AKT participates in Asa-induced effects, lung cancer cell lines (A549, HCC827, H292, and H1299) were treated with Asa alone or in combination with AKT overexpression (Asa + AKT). As shown in Figure 4A-D, Asa significantly decreased cell viability across all cell lines, while AKT overexpression partially restored viability compared to Asa treatment alone. Similarly, colony formation assays demonstrated that Asa treatment significantly inhibited the colony-forming ability of A549 and H1299 cells, whereas AKT overexpression partially rescued this effect (Figure 4E, 4F). Wound-healing assays further revealed that Asa markedly suppressed migration in A549 and H1299 cells (Figure 4G, 4H). However, AKT overexpression alleviated this inhibition, resulting in a higher relative wound closure rate compared to cells treated with Asa alone. These findings indicate that activation of AKT signaling mitigates the anti-proliferative and anti-migratory effects of Asa, highlighting the involvement of the PI3K-AKT pathway in Asa-mediated cancer suppression.

Figure 4.

AKT overexpression ameliorates the antitumor effects of Asaraldehyde on NSCLC cells. A549, HCC827, H292 and H1299 cells were transfected with vector or Flag-AKT plasmid, and treated with 100 or 150 µM Asaraldehyde. A-D. Cell viability was examined by CCK-8 assay. E, F. Colony formation was examined by crystal violet staining (magnification, ×1, Scale bar = 5 mm). G, H. Cell migration ability was examined by wound healing assay (magnification, ×50, Scale bar = 200 µm). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Asaraldehyde inhibits malignant phenotypes of NSCLC through ferroptosis induction

Notably, KEGG pathway analysis indicated that Asa treatment might activate ferroptosis pathway. To investigate the potential role of ferroptosis in the anti-cancer effects of Asa, we performed Western blot analysis on A549 and H1299 lung cancer cells treated with increasing concentrations of Asa. Our results demonstrate that Asa treatment significantly downregulated the expression of GPX4, a crucial enzyme for lipid hydroperoxide reduction, and FTH1, a key transporter involved in iron storage (Figure 5A). To further confirm the induction of ferroptosis by Asa, we performed immunofluorescence DHE and FerroOrange staining to detect ROS production and intracellular labile iron levels. We observed a significant increase in both ROS production and intracellular iron levels in Asa-treated cells compared to control cells (Figure 5B, 5C). Additionally, we also observed decreased GSH level following Asa treatment (Figure 5D). To quantify the extent of ferroptosis, we measured the levels of MDA and LPO using biochemical assays. Our results showed that Asa treatment significantly increased both MDA and LPO levels in a dose-dependent manner (Figure 5E, 5F). Taken together, these findings suggest that Asa induces ferroptosis in lung cancer cells by downregulating GPX4 and FTH1, leading to increased lipid peroxidation and iron-dependent cell death.

Figure 5.

Asaraldehyde inhibits malignant phenotypes of NSCLC through ferroptosis induction. A549 and H1299 cells were treated with various concentrations of Asaraldehyde for 48 h. A. Expression of ferroptosis-related proteins was detected by western blot. B. Intracellular ROS level was detected by DHE staining (magnification, ×100, Scale bar = 50 μm). C. Intracellular labile iron level was detected by FerroOrange staining. (magnification, ×630, Scale bar = 10 μm). D-F. Intracellular GSH, LPO and MDA level were detected by commercial kits. A549, HCC827, H292 and H1299 cells were treated with Asaraldehyde in the presence or absence of 10 µM Ferrostatin-1. G-J. Cell viability was examined by CCK-8 assay. K, L. Colony formation was examined by crystal violet staining. (magnification, ×1, Scale bar = 5 mm). M, N. Cell migration ability was examined by wound healing assay (magnification, ×50, Scale bar = 200 µm). Data were presented as mean ± SD. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

To determine whether ferroptosis contributes to Asa’s anti-cancer effects, we employed Fer-1, a ferroptosis inhibitor. We found that Fer-1 significantly attenuated Asa-induced inhibition on cell viability (Figure 5G-J), suggesting that ferroptosis plays a crucial role in the anti-cancer effects of Asa. Additionally, the addition of Fer-1 also reversed the inhibitory effects of Asa on colony formation and wound healing (Figure 5K-N). Taken together, these findings demonstrate that Asa induces ferroptosis in lung cancer cells, leading to inhibition of tumor cell proliferation, colony formation and migration.

Asaraldehyde-induced ferroptosis facilitates AKT degradation and inhibition of PI3K/AKT signaling

Subsequently, we explored the role of ferroptosis in AKT inhibition. We first treated A549 and H1299 lung cancer cells with Asa and Fer-1 (a ferroptosis inhibitor) and analyzed the levels of total and phosphorylated AKT, ERK, and MEK by Western blot. Our results demonstrated that Asa significantly inhibited the expression of AKT and phosphorylation of AKT, ERK, and MEK in both cell lines. However, co-treatment with Fer-1 partially rescued phosphorylation of these proteins and the expression of AKT, suggesting that ferroptosis contributes to the inhibition of the AKT pathway by Asa (Figure 6A). To confirm our hypothesis, cycloheximide chase assay was performed. We found that Fer-1 treatment alleviated Asa-induced AKT turnover (Figure 6B). Additionally, we observed that Fer-1 partially rescued AKT ubiquitination in Asa-treated cells (Figure 6C). Taken together, these findings demonstrate that Asa inhibits the AKT pathway through ferroptosis induction.

Figure 6.

Ferrostatin-1 alleviates Asaraldehyde-induced AKT degradation and inhibition on PI3K/AKT signaling. A. A549 and H1299 cells were treated with Asaraldehyde in the presence or absence of 10 µM Ferrostatin-1. Protein expression was detected by western blot. B. A549 cells were treated with 100 µM Asaraldehyde and 50 μg/mL Cycloheximide in the presence or absence of 10 μM Ferrostatin-1 for the indicated times. AKT expression was detected by western blot. C. A549 cells were treated with 100 µM Asaraldehyde in the presence or absence of 10 µM Ferrostatin-1. AKT ubiquitination level was detected by immunoprecipitation and western blot. Data were presented as mean ± SD. * ns P > 0.05, ***P < 0.001, ****P < 0.0001.

Asaraldehyde suppresses growth of NSCLC in vivo

To evaluate the efficacy of Asaraldehyde in vivo, we established a A549 xenograft model in BALB/c nude mice. As shown in Figure 7A, four-week-old mice were subcutaneously injected with A549 cells and then treated with intraperitoneal injections of Asaraldehyde at doses of 5 mg/kg and 10 mg/kg, respectively. At the indicated time, mice were sacrificed and tumors were dissected. As shown in Figure 7B-D, treatment with Asaraldehyde significantly inhibited tumor growth in a dose-dependent manner, with the 10 mg/kg dose showing the most potent effect. To further investigate the underlying mechanism of action, we performed histological analysis on tumor tissues. IHC staining showed that Asaraldehyde treatment significantly downregulated the expression of Ki67, AKT, GPX4 and FTH1 (Figure 7E). These results confirm that Asaraldehyde exerts its anti-tumor effects by inducing ferroptosis and inhibiting the PI3K/AKT signaling pathway.

Figure 7.

Asaraldehyde suppresses growth of NSCLC in vivo. A. Schematic of tumor xenograft model and Asaraldehyde administration. B. Photographs of the excised tumors in different groups. C, D. Tumor volumes and weight in different groups. E. IHC staining of tumor sections (magnification, ×200, Scale bar < 100 μm). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Discussion

NSCLC is the most prevalent form of lung cancer, driven by various genetic alterations that influence its pathogenesis and therapeutic response [21]. In this study, we demonstrated that Asaraldehyde effectively suppresses the progression of NSCLC through ferroptosis induction and inhibition of the PI3K-AKT signaling pathway. Our findings reveal that treatment with Asaraldehyde significantly reduces NSCLC cell viability and proliferation, accompanied by hallmark features of ferroptosis, including increased lipid peroxidation and depletion of GSH. Furthermore, molecular analysis indicates that Asaraldehyde disrupts the PI3K-AKT signaling axis, a crucial pathway implicated in cancer cell survival and resistance to therapy. These results suggest that Asaraldehyde exerts potent anticancer effects by triggering ferroptotic cell death and impairing key pro-survival signaling, thereby offering a promising therapeutic strategy for NSCLC.

Recent advances in oncology have identified ferroptosis as a promising therapeutic alternative to conventional apoptosis-targeted treatments [22]. Erastin, a prototype of ferroptosis inducer, effectively depletes GSH stores and suppressing proliferation in both cervical and ovarian carcinoma models [23]. Similarly, RSL3 directly inactivates GPX4 through the alkylation of selenocysteine, resulting in potent growth inhibition of fibrosarcoma [24]. Consistent with these findings, our study underscores the critical role of ferroptosis in suppressing NSCLC. Mechanistically, our study suggests that Asaraldehyde induces ferroptosis in NSCLC cells by promoting lipid peroxidation and disrupting antioxidant defenses. The observed increase in MDA, LPO levels and ROS accumulation indicates that Asaraldehyde enhances oxidative stress, a key driver of ferroptotic cell death. Additionally, the reduction in GSH levels and suppression of GPX4 activity further corroborate the role of Asaraldehyde in impairing cellular antioxidant capacity, rendering NSCLC cells more susceptible to ferroptosis.

Aberrant activation of the PI3K-AKT cascade represents a fundamental characteristic of numerous malignancies, driving tumor initiation, advancement, and therapeutic refractoriness. In prostate carcinoma, AKT activation confers protection against TNFα-mediated apoptotic cell death [25], while in breast cancer models, this pathway has been implicated in resistance to endocrine therapies [26]. In this study, we also reveal that the level of AKT phosphorylation is significantly upregulated in NSCLC cells. The central involvement of PI3K/AKT in oncogenic processes has spurred extensive investigation into pharmacological inhibitors. As a pan-class I PI3K inhibitor, GDC-0941 was the first inhibitor to enter clinical trials [27]. Preclinical investigations have further demonstrated the efficacy of BKM120, another pan-class I inhibitor, in suppressing intracranial growth of U-87MG glioblastoma xenografts [28]. Beyond ferroptosis induction, our findings also reveal that Asaraldehyde effectively inhibits the PI3K-AKT signaling pathway, as evidenced by the downregulation of phosphorylated AKT, ERK and MEK. The compound facilitates proteasomal degradation of AKT through ubiquitin-mediated targeting, with functional studies confirming that AKT overexpression mitigates Asaraldehyde’s antitumor effects. NEDD4, a known E3 ligase for AKT, has been reported to be activated by oxidative stress [29,30]. It is possible that the oxidative stress induced by ferroptosis may activate NEDD4, thereby promoting AKT ubiquitination and degradation. Notably, pharmacological inhibition of ferroptosis partially restores PI3K-AKT signaling, suggesting interplay between these mechanisms. These mechanisms highlight the therapeutic potential of Asaraldehyde in targeting NSCLC through a combination of oxidative stress induction and pro-survival pathway inhibition.

In conclusion, this investigation establishes Asaraldehyde as a dual-function agent against NSCLC, operating through coordinated induction of ferroptotic cell death and PI3K-AKT pathway blockade. Although additional studies are required to determine optimal therapeutic parameters, these findings provide a robust preclinical rationale for further development of Asaraldehyde as a potential therapeutic candidate.

Disclosure of conflict of interest

None.