Vanadates inhibit non-small cell lung cancer through modulation of ferroptosis mediated by the CBS-CPS1 axis

Abstract

Lung cancer remains the leading cause of cancer mortality, where intrinsic and acquired resistance to cisplatin-based chemotherapy constitutes a major therapeutic challenge in non-small cell lung cancer (NSCLC). Previous studies suggested that vanadium might represent a promising platinum-alternative therapeutic strategy for NSCLC through a unique chemical reaction pattern, though the regulatory networks remain incompletely characterized. Integrated pharmacological screening with cell death pathway inhibitors and transcriptomic profiling revealed that vanadium compounds suppress NSCLC progression by triggering ferroptosis, a process governed by cystathionine β-synthetase (CBS)-mediated regulatory mechanisms. CBS knockdown promoted intracellular Fe²⁺ accumulation, elevated lipid ROS levels, and increased MDA content, ultimately leading to the inhibition of cell growth and proliferation both in vitro and in vivo. Conversely, ICP-MS analysis revealed that CBS overexpression substantially decreased cellular iron uptake. The CBS-specific inhibitor AOAA demonstrated potent synergistic effects with vanadates in suppressing NSCLC cell growth. Furthermore, CoIP-MS identified CPS1 as a potential direct interacting protein of CBS. Remarkably, CPS1 complementation in shCBS NSCLC cells attenuated the key indicators of ferroptosis while restoring cell growth and proliferation. Collectively, our study systematically elucidated the underlying mechanisms by which vanadates inhibit NSCLC progression and defined the functional significance of the CBS-CPS1 axis, providing novel therapeutic insights for advancing vanadium-based metallodrugs in clinical oncology.

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Introduction

Lung cancer has emerged as the malignancy with the most rapidly escalating global incidence over the past three decades, representing the leading contributor to cancer-associated mortality worldwide.¹ Non-small cell lung cancer (NSCLC), the predominant histological subtype, accounts for 80–85% of all primary lung cancers.² Metallodrugs play a pivotal role in the prevention and management of diverse diseases, with platinum-based regimens forming the cornerstone of current oncology chemotherapeutic strategies.^3,4 While cisplatin-based adjuvant chemotherapy demonstrates survival benefits in NSCLC, its clinical implementation is limited by intrinsic/acquired resistance and dose-limiting toxicities. This therapeutic impasse has stimulated interest in alternative metallodrugs, particularly those containing transition metals with distinct bioinorganic properties like vanadium(V), whose therapeutic potential is now entering the forefront of next-generation drug discovery.⁵

Vanadium, a redox-active transition metal with biologically relevant valence states (−1 to +5), exists predominantly as extracellular pentavalent species (VO₃⁻) and intracellular tetravalent species (VO²⁺), with facile interconversion between V^IV and V^V states across biological media, and its cytotoxicity is positively correlated with its oxidation states.^6,7 Vanadium compounds exhibit therapeutic potential spanning both disease intervention and homeostatic regulation, particularly in antidiabetic, anticancer and antiparasitic activities, and tissue regeneration.^8,9 Notably, bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium(IV) (METVAN) demonstrated platinum-alternative efficacy in cisplatin-resistant malignancies, like ovarian cancer and testicular cancer cell lines.¹⁰ Additionally, oxovanadium(IV) complexes of ferrocenyl derivatives enabled photodynamic tumor eradication via light-activated ROS generation (400–700 nm).¹¹ Current NSCLC research predominantly focuses on the design, synthesis, and phenotypic screening of vanadium complexes coupled with preliminary mechanistic evaluations, while systematic insights into their anticancer mechanisms remain critically underexplored.

Emerging evidence has revealed that vanadates, including vanadyl sulfate (VOSO₄) and sodium metavanadate (NaVO₃), exert anticancer effects in NSCLC through non-apoptotic pathways, primarily via ROS-mediated cellular damage rather than classical caspase activation in A549 cells.¹² Beyond provoking oxidative stress, vanadium-based metallodrugs inhibited NSCLC through dose-dependent G2/S cell cycle arrest, epithelial to mesenchymal transition (EMT) blockage, and RAS signaling pathway suppression.^13–16 Ferroptosis, an iron-dependent regulated cell death pathway characterized by lipid peroxidation cascades, has emerged as a promising therapeutic target, particularly for targeting cancer cells that demand high iron supply.¹⁷ As an essential catalytic cofactor in redox biology, iron coordinates the electron transfer processes governing lipid peroxidation dynamics through the Fenton reaction.¹⁸ Iron overload disrupts redox homeostasis, leading to ferroptosis characterized by GPX4/GSH axis inactivation and lipid peroxide accumulation.^19,20 Previous studies demonstrated that diffuse large B cell lymphomas and renal cell carcinomas were susceptible to GPX4-regulated ferroptosis, while dioscin induced ferroptosis through ROS accumulation and iron upregulation in melanoma cells.^21,22 Notably, both natural compounds (e.g., curcumin) and synthetic agents (e.g., auranofin) induced ferroptosis in NSCLC via multi-target mechanisms, primarily by repressing the GSH-dependent GPX4 signaling pathway.²³ However, the mechanistic interplay between vanadium-based complexes and ferroptosis induction in NSCLC remains unexplored.

Our study demonstrates that vanadium compounds including VOSO₄(IV) and NaVO₃(V) suppress NSCLC progression both in vitro and in vivo through ferroptosis induction, as evidenced by characteristic lipid peroxidation, ferrous ion accumulation, glutathione depletion, and mitochondrial morphology alterations. Mechanistically, transcriptomic analysis identifies cystathionine β-synthetase (CBS), a key enzyme in the trans-sulfuration pathway, as the critical molecular target whose suppression drives vanadium-induced ferroptosis in NSCLC. The CETSA assay confirms a direct binding interaction between vanadium and CBS, which induces dysregulation of iron homeostasis and disrupts trans-sulfuration metabolic flux, as quantitatively validated through ICP-MS and untargeted metabolomics. Notably, CoIP-MS and colocalization studies uncover a novel CBS-CPS1 regulatory axis in NSCLC cells, where carbamoyl phosphate synthetase 1 (CPS1) functionally compensates for CBS depletion to regulate ferroptosis susceptibility during vanadate treatment. These findings establish the CBS-CPS1 axis as a critical regulator of NSCLC tumorigenesis and provide a theoretical basis for expanding the clinical applications of vanadium-based agents.

Results and discussion

Vanadium significantly inhibited the proliferation of NSCLC cells

The CCK8 assay was performed to detect the cytotoxicity of VOSO₄(IV) and NaVO₃(V) in a variety of NSCLC cell lines, including A549, H460 and PC9. As shown in Table 1 and Fig. S1,† vanadium compounds exhibited high cytotoxicity against NSCLC cells. The IC₅₀ value of NaVO₃ on A549 cells was 2.0 μM, which was superior to that of VOSO₄ (12.3 μM) and cisplatin (6.7 μM), whereas its toxicity on H460 and PC9 cells was comparable to that of cisplatin. We then chose the normal human lung epithelial cells (BEAS-2B) to determine the selectivity of the compounds against normal cells. The results showed that vanadium compounds had lower cytotoxicity compared to cisplatin towards BEAS-2B cells, as evidenced by about fourfold elevation of IC₅₀ values. The IC₅₀ values confirmed that vanadium compounds could effectively inhibit the growth and proliferation of NSCLC cells at low concentrations, and the cytotoxicity was positively correlated with the valence state.

Table 1 IC₅₀ value (μM) of the cisplatin and vanadium compounds^a

	NaVO3	VOSO4	Cisplatin
a Cell viability was measured by MTT assay after 72 h incubation with different compounds (dose–response curves are shown in Fig. S1†).
A549	2.0 ± 0.2	12.3 ± 0.5	6.7 ± 1.0
H460	3.8 ± 0.3	2.4 ± 0.2	2.7 ± 0.8
PC9	2.8 ± 0.6	5.6 ± 0.1	2.0 ± 0.4
BEAS-2B	26.8 ± 0.4	29.8 ± 0.2	7.2 ± 0.7

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Vanadium exerted anticancer activity by inducing ferroptosis

To explore the mode of cell death induced by vanadium in NSCLC cells, the CCK8 assay was utilized to detect changes in cell viabilities after treatment with different classical cell death pathway inhibitors Z-VAD-FMK (Z-VAD) for caspase activation-induced apoptosis, necrostatin (Nec-1) for necrosis, 3-methyladenine (3-MA) for autophagy, ferrostatin-1 (Fer-1) for ferroptosis targeting lipid metabolism and deferoxamine (DFO) for ferroptosis targeting iron metabolism combined with VOSO₄ and NaVO₃ compared to vanadium alone. The results showed that only DFO restored the cell viability suppressed by vanadium, implying that vanadium compounds might inhibit NSCLC through ferroptosis (Fig. 1A). Meanwhile, the results of the EdU fluorescent labeling assay showed that vanadium in combination with DFO increased the cell proliferation level compared to vanadium alone (Fig. 1B and S2A†). Ferroptosis was commonly caused by the accumulation of ferrous ions and ROS-dependent lipid peroxide. Detection of lipid ROS levels by flow cytometry suggested that VOSO₄ significantly increased intracellular lipid ROS in A549 and PC9 cells, and NaVO₃ had a more pronounced effect on H460 cells (Fig. 1C). The FerroOrange fluorescent probe was used to specifically detect ferrous ions. The images shown in Fig. 1D and S2B† confirmed that vanadium compounds stimulated the production of ferrous ions and the phenomenon could be alleviated by exogenous DFO. Moreover, the mRNA transcription and protein expression level of GPX4, a critical regulator of ferroptosis, was significantly inhibited by vanadium compounds (Fig. 1E). Then, we verified the content of GSH in NSCLC cells treated with vanadium, and both VOSO₄ and NaVO₃ decreased the GSH concentration (Fig. 1F). The above results indicated that the phenotype of NSCLC cells induced by vanadium was consistent with the characteristics of ferroptosis. Considering the key role played by iron in ferroptosis, ICP-MS was performed to determine the uptake of iron and vanadium in NSCLC cells treated with VOSO₄ and NaVO₃. Intracellular iron content increased with vanadium uptake and NaVO₃ promoted the absorption of iron and vanadium more markedly than VOSO₄. DFO chelated cellular iron and this resulted in a decrease in the vanadium content in vanadium-treated cells (Fig. 1G). Altogether, these findings suggested that vanadium interfered with the homeostasis of iron metabolism and redox by augmenting iron uptake, ultimately leading to ferroptosis.

Fig. 1 Investigation of cell death types of NSCLC cells induced by vanadium compounds. (A) The cell viabilities of A549, H460 and PC9 cells exposed to VOSO₄ or NaVO₃ alone or in combination with different cell death inhibitors (Z-VAD-FMK/Z-VAD, necrostatin-1/Nec-1, 3-methyladenine/3-MA, ferrostatin-1/Fer-1 and deferoxamine/DFO). (B) The proliferation rates of NSCLC cells treated with VOSO₄ or NaVO₃ alone or in combination with 20 μM DFO were detected by EdU assay. (C) Lipid ROS levels in NSCLC cells treated with VOSO₄, NaVO₃, and VOSO₄/NaVO₃ + 20μM DFO with PBS as the control, determined by BODIPY-C11 staining via flow cytometry. (D) The FerroOrange fluorescent probe was used to determine the ferrous ion levels in NSCLC cells treated with VOSO₄, NaVO₃, or in combination with DFO. (E) The mRNA transcription and protein expression levels of GPX4 in vanadium-treated NSCLC cells were evaluated by qRT-PCR and western blot assay, respectively. (F) Detection of reduced glutathione content in NSCLC cells under the action of vanadium compounds compared with the control group. (G) Changes in intracellular levels of vanadium and iron in NSCLC cells after incubation with VOSO₄, NaVO₃, or in combination with DFO were measured by ICP-MS. Values are expressed as the mean ± SEM (n = 3). Statistical significance was evaluated by t-test or one-way ANOVA; ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.

CBS acted as a potential target for NSCLC inhibition by vanadium compounds

Transcriptomics was performed to explore the underlying mechanisms of ferroptosis induced by vanadium compounds. Cluster analysis showed that there were significant differences in the overall gene transcription patterns between the control groups and the VOSO₄ and NaVO₃-treated groups, while the expression patterns in the vanadium and DFO combined-treatment groups were more similar to the control groups (Fig. 2A). We identified 519 significantly downregulated genes and 783 significantly upregulated genes in A549 cells treated with VOSO₄, whereas a total of 62 decreased DEGs and 120 increased DEGs were observed in the VOSO₄ and DFO combination group. Meanwhile, NaVO₃ induced significant downregulation of 579 genes and upregulation of 1680 genes, and 788 genes were significantly downregulated and 392 genes were significantly upregulated in combination with DFO (Fig. 2B). The above results implied that NaVO₃ caused a wider range of gene transcriptional changes than VOSO₄ and that the addition of DFO attenuated the transcriptional variation. DEGs identified in vanadium-treated groups versus control groups were screened for the intersection of genes with opposite expression induced by the combined treatment versus treatment with vanadium compounds alone (Fig. 2C). Both vanadium sulfate and NaVO₃ induced significantly upregulated gene transcription levels of LINC02177, CCDC187, SLC6A3, and ANKRD26P1, which were reduced by exogenous DFO. In contrast, the expression levels of 13 genes, such as CBS, SLC16A5 and Sh3rf2, were significantly decreased in the vanadium action groups and restored in the vanadium and DFO co-treatment groups.

Fig. 2 Transcriptome profiles of NSCLC cells treated with VOSO₄, NaVO₃, or in combination with DFO. (A) The heatmap for clustering analysis of gene expression patterns in A549 cells under treatment of VOSO₄ or VOSO₄ + DFO (above) and H460 cells treated with NaVO₃ or VOSO₄ + DFO (below). (B) Volcano plots showing significantly differential genes that were selected by p-value < 0.05 and log₂FoldChange > 1 (blue: downregulated; red: upregulated). (C) Venn diagrams showing the number of intersecting genes including upregulated DEGs in the vanadium-treated group and downregulated DEGs in the vanadium and DFO combined treatment group (above), and downregulated DEGs in the vanadium-treated group and upregulated DEGs in vanadium and DFO combined treatment group (below). (D) The biological process subontology of GO analysis of upregulated DEGs and KEGG pathway analysis of downregulated DEGs induced by vanadium. (E) Heatmap analysis of CBS transcription levels in different groups. (F) The protein expression levels of CBS in A549 and H460 cells exposed to vanadium compounds alone or combined with DFO. (G) Kaplan–Meier plot showing the effect of CBS on the survival curves of NSCLC patients.

Notably, the upregulated DEGs induced by vanadium mainly affected the biological process terms in Gene Ontology (GO) analysis, i.e., negative regulation of cell migration, cellular response to hypoxia, and positive regulation of the fibroblast apoptotic process. On the other hand, downregulated DEGs were associated with glutathione metabolism, central carbon metabolism in cancer, and metabolic pathways according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment annotation (Fig. 2D). The transcription level of CBS, which plays a vital role in the negative regulation of ferroptosis, was markedly inhibited by vanadium and reversed by the addition of DFO (Fig. 2E). Meanwhile, western blotting analysis further confirmed that the CBS protein expression levels were consistent with transcriptome profiles (Fig. 2F). Overall survival rates of NSCLC patients with high expression of CBS were significantly exacerbated when compared to patients with low CBS expression (Fig. 2G and S3A†). These data in bulk RNA-seq studies indicated that CBS is a pivotal target of vanadium action, intimately related to vanadium-mediated ferroptosis in NSCLC cells.

Vanadium suppressed NSCLC by inducing CBS-mediated ferroptosis in vivo

To comprehensively assess the antitumor effects of vanadium compounds in vivo, six-week-old Balb/c nude mice inoculated with H460 cells were administered 60 mg kg⁻¹ VOSO₄, 20 mg kg⁻¹ NaVO₃ or both in combination with 200 mg kg⁻¹ DFO. Notably, the size, volume, and weight of tumor tissues were markedly inhibited by vanadium compounds, with this effect being alleviated by DFO co-administration (Fig. 3A–D). Hematoxylin–eosin (HE) staining of the stripped tumor tissues revealed intact, tightly arranged nuclei and cytoplasm in the control group. In contrast, vanadium compounds reduced tumor tissue densification, inducing liquefactive necrosis. The cellular alterations could be reversed by combined treatment of DFO, restoring normal cell morphology. Immunohistochemistry (IHC) experiments indicated that vanadium compounds downregulated the Ki67 and CBS expression in xenograft tumor tissues. Moreover, Ki67 and CBS expression levels in vanadium and DFO combined-treatment groups were elevated compared to vanadium alone, implying CBS involvement in vanadium-induced inhibition of NSCLC proliferation in vivo (Fig. 3E). Then, we estimated the influence of vanadium compounds on mitochondrial morphology. As shown in Fig. 3F, VOSO₄ and NaVO₃ treatment resulted in shrunken mitochondria and mitochondrial cristae collapse in tumor tissue cells when compared to the control group as determined by transmission electron microscopy (TEM) imaging, and DFO addition normalized the mitochondrial morphology. Collectively, vanadium-induced ferroptosis, as mediated by CBS, led to the suppression of tumor growth and proliferation of NSCLC in vivo.

Fig. 3 Detection of biological activity of vanadium compounds in vivo. (A) Tumor growth in mice after treatment with vanadium compounds alone or in combination with DFO. (B) Representative images of tumors in different treatment groups. (C) The tumor growth curves of mice on vanadium treatment alone or combined with DFO treatment (n = 6). (D) The final masses of the subcutaneous tumors. (E) HE staining of tumors and representative images of Ki67 and CBS IHC staining. Image magnification: 200× (upper panel) and 400× (lower panel). (F) TEM images of mitochondria in subcutaneous tumor tissue cells treated with VOSO₄, NaVO₃, or in combination with DFO. Statistical significance was evaluated by t-test or one-way ANOVA; ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.

CBS negatively regulated ferroptosis in NSCLC cells

We further explored the role of CBS in the development of NSCLC cells. The concentrations of aminooxyacetic acid (AOAA), a common inhibitor of CBS, that did not affect cell growth were screened (Fig. S3B†). Treatment with AOAA in combination with VOSO₄ or NaVO₃ was administered to H460 cells to assess cell proliferation using the CCK8 assay. The results showed that the combined treatment markedly decreased cell viability compared to vanadium treatment alone (Fig. 4A). We established the stable CBS KD and OE A549 and H460 cells by transfecting them with shCBS and CBS overexpression plasmids, respectively. The transfection efficiency was determined using qRT-PCR and western blotting assays (Fig. S3C and S3D†).

Fig. 4 Knockdown of CBS-facilitated ferroptosis in A549 and H460 cells. (A) The growth curves of H460 cells treated with vanadates, or in combination with AOAA were detected by the CCK8 assay. The proliferation and migration of NSCLC cells transfected with shRNA and overexpression plasmid were examined by the colony formation assay (B) and wound healing assay (C). (D) The contents of ferrous ions in CBS KD and OE NSCLC cells were quantified through the FerroOrange fluorescent probe. (E) Lipid ROS levels were measured in CBS KD and OE A549 cells after VOSO₄ treatment for 72 h by BODIPY 581/591-C11 staining and flow cytometry analysis. (F) MDA content detection in CBS knockdown and overexpression in NSCLC cells under treatment of VOSO₄ and NaVO₃. (G) The concentrations of Fe and V in A549 cells transfected with shCBS and CBS-OE plasmids, following treatment with VOSO₄, were detected by ICP-MS. Statistical significance was evaluated by t-test or one-way ANOVA; ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.

Knockdown of CBS inhibited the proliferation and migration of NSCLC cells, whereas CBS overexpression promoted cell growth (Fig. 4B, C and S4†).

Additionally, we investigated whether CBS regulated ferroptosis of NSCLC cells. As shown in Fig. 4D, CBS depletion induced the accumulation of cellular ferrous ions, while elevating CBS reduced the fluorescence intensity caused by ferrous ions. Meanwhile, flow cytometry analysis indicated that CBS KD and VOSO₄ or NaVO₃ synergistically increased the accumulation of lipid peroxidation, and CBS overexpression markedly decreased lipid ROS in A549 and H460 cells (Fig. 4E and S5†). Suppression of CBS significantly increased the content of malondialdehyde (MDA), an indicator of membrane lipid peroxidation, whereas CBS overexpression reduced the MDA content in NSCLC cells pretreated with vanadium compounds (Fig. 4F). To test whether CBS directly affected ferroptosis via modulating cellular uptake of vanadium and iron, the accumulation of metal in A549 cells after CBS knockdown and overexpression in the presence of VOSO₄ was examined by ICP-MS. Intriguingly, CBS depletion had no effect on the absorption of vanadium and iron, but the overexpression of CBS significantly reduced the metal accumulation in A549 cells (Fig. 4G). This might suggest incomplete CBS knockdown, with potential compensatory mechanisms from other metabolic pathways. Therefore, CBS reduced cytotoxicity by decreasing the cellular uptake of vanadium and iron and negatively regulated ferroptosis.

Vanadium interfered with the metabolism via the transsulfuration pathway involving CBS

Vanadium reduced CBS expression, with the Cellular Thermal Shift Assay (CETSA) revealing direct binding of vanadium to CBS, which enhanced its stability, thereby confirming CBS as a target of vanadium compounds in inhibiting NSCLC cells (Fig. 5A and B). CBS was involved in the first step of the transsulfuration pathway of homocysteine and was closely related to downstream GSH synthesis (Fig. 5C). The abundance of metabolites in the transsulfuration pathway and glutamine metabolism of NSCLC cells treated with vanadium compounds were determined through LC-MS/MS-based untargeted metabolomics. Metabolite expression patterns between the control group and the vanadium-treated group were observed and significantly differential metabolites were screened using the OPLS-DA model (Fig. S6†). Intracellular amino acids including methionine, serine, cysteine, glycine, glutamate, and glutamine were both significantly upregulated by vanadium stimulation, implying that more amino acids were compensatorily produced under vanadium stimulus to overcome oxidative stress (Fig. 5D). However, transcript levels of genes encoding key proteins in the relevant metabolic pathways, such as CBS, CTH, GPX4 and glutathione S-transferase Mu 4 (GSTM4), were downregulated in NSCLC cells under vanadium treatment, leading to the accumulation of lipid ROS. Together, vanadium inhibited NSCLC by modulating the CBS-involved transsulfuration pathway.

Fig. 5 Vanadium compounds modulated the metabolite abundances in the CBS-mediated transsulfuration pathway. CETSA assay for detecting the binding efficiency of VOSO₄ (A) or NaVO₃ (B) to CBS. (C) The diagram of protein and metabolite expression in the transsulfuration pathway (green: downregulated; red: upregulated). (D) Abundance of representative metabolites detected through untargeted metabolomics. (E) Tumor growth curves in a xenograft model of H460 cells transfected with the shNC and shCBS vector (n = 6). (F) The final masses of the subcutaneous tumors were determined. (G) IHC staining of Ki67 in CBS KD tissues. (H) The verification of CBS expression in stripped tumor tissues inoculated with CBS KD H460 cells by western blotting assay. Statistical significance was evaluated by t-test or one-way ANOVA; ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.

We further explored the role played by CBS in NSCLC tumorigenesis in vivo using a xenograft mouse model inoculated with CBS KD and control H460 cells. Compared with the control mice, mice xenografted with shCBS cells exhibited marked reductions in tumor size, volume and weight (Fig. 5E, 5F and S7A†). IHC staining revealed that the Ki67 protein level was downregulated by CBS knockdown (Fig. 5G). The knockdown efficiency of CBS in stripped tumor tissues was further confirmed using western blotting analysis (Fig. 5H). These results indicated that CBS ablation inhibited tumorigenesis and development in vivo.

CPS1 was identified as a potential target of CBS

To investigate the potential target of CBS involved in ferroptosis in NSCLC, we conducted the CoIP-MS analysis combined with the protein–protein interaction (PPI) network. Immunoprecipitation of proteins associated with Flag-tagged recombinant proteins was followed by mass spectrometry-based proteomics to identify the interacting proteins of CBS (Fig. 6A and S7B†). According to the results of CoIP-MS, a total of 95 proteins specifically interacting with CBS were screened (Fig. 6B). The integration of protein interaction data from the STRING database suggested that CPS1 might serve as a pivotal target for CBS (Fig. 6C). This hypothesis was corroborated by qRT-PCR analysis, which demonstrated a concordant transcriptional pattern between CPS1 and CBS (Fig. 6D). Further insights into their interaction were gained through western blot analysis of co-immunoprecipitated proteins, confirming the interaction between CBS and CPS1 (Fig. 6E). Additionally, colocalization studies revealed that both CBS and CPS1 reside within the same spatial compartment in NSCLC cells (Fig. 6F and S7C†).

Fig. 6 CPS1 was identified as a potential target of CBS-mediated ferroptosis. (A) CBS interacting protein bands obtained in CBS-VEC and CBS-OE cells were separated by SDS-Tris-glycine gel and stained by the Ag staining method. (B) Screening for proteins specifically interacting with CBS using CoIP-MS. (C) CBS-specific interaction protein network constructed by STRING database. (D) The transcription levels of CPS1 in CBS KD and OE A549 cells were determined by qRT-PCR. (E) A549 cells were transfected with Flag-CBS and HA-CPS1. Cell lysates were analyzed by immunoblotting with Flag and HA antibodies. (F) The representative images of colocalization of CBS with CPS1 in A549 and H460 cells. (G) Western blotting analysis of CBS and CPS1 expression in A549 and H460 cells pretreated with shCPS1. (H) The growth curves of CPS1 KD A549 cells were detected by the CCK8 assay at 0 h, 24 h, 48 h, 72 h and 96 h. (I) Colony formation assay of A549 and H460 cells transfected with shCtrl or shCPS1. Statistical significance was evaluated by t-test or one-way ANOVA; ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.

To explore the functional role played by the CBS-CPS1 signaling axis in ferroptosis, we successfully constructed NSCLC cells with CPS1 depletion (Fig. S7D†). Notably, CPS1 knockdown led to a marked decrease in protein expression levels of CBS in both A549 and H460 cells (Fig. 6G). The reduced CPS1 expression resulted in the inhibition of cell growth and proliferation (Fig. 6H and I). Together, these results demonstrated that CPS1 functioned as a target for CBS and that the CBS-CPS1 signaling axis regulated tumor progression in NSCLC.

CBS-CPS1 axis is involved in the regulation of ferroptosis in NSCLC

CPS1 catalyzed the rate-limiting step of the urea cycle (Fig. 7A). As shown in the previous study, CPS1 depletion in HCC cells produced extra ammonia leading to ROS accumulation.²⁴ To explore the association between CPS1 and ROS production in NSCLC cells, we examined the total ROS levels in shCPS1 cells by flow cytometry. Upon comparison with shCtrl cells, CPS1 knockdown promoted ROS production. Notably, a more significant elevation in the ROS level was observed in shCPS1 cells treated with VOSO₄ (Fig. 7B). To further determine the regulatory role played by the CBS-CPS1 signaling axis in cell response to ferroptosis, we established stably transfected shCBS NSCLC cells with CPS1 complementation (Fig. S7E†). The results of lipid ROS levels showed that rescue with CPS1 significantly attenuated the accumulation of lipid ROS induced by CBS knockdown (Fig. 7C). Under NaVO₃ treatment, the increased MDA content induced by CBS knockdown was reversed by the CPS1 complement (Fig. 7D). Moreover, CPS1 complementation reduced the elevated ferrous ion levels caused by CBS knockdown in NSCLC cells (Fig. 7E).

Fig. 7 CBS-CPS1 axis-regulated ferroptosis in NSCLC cells. (A) The diagram of the urea cycle pathway involving CPS1. (B) The intracellular ROS levels in A549 and H460 cells transfected with shCtrl and shCPS1 under treatment of VOSO₄ were detected by flow cytometry. (C) A549 cells pretreated with shCBS were stably transfected with CPS1, and then lipid ROS levels were examined by BODIPY 581/591 C11 staining and flow cytometry analysis. (D) MDA content detection in H460 cells transfected with shCtrl, shCBS, and shCBS + CPS1 under vanadium treatment. (E) The fluorescence of Fe²⁺ in NSCLC cells transfected with shCBS and shCBS + CPS1. (F) The expression levels of ferroptosis-related proteins regulated by the CBS/CPS1 axis in NSCLC cells were detected by western blotting assay. (G) The schematic diagram illustrating the underlying mechanisms through which the CBS-CPS1 axis inhibited NSCLC by regulating ferroptosis. Statistical significance was evaluated by t-test or one-way ANOVA; ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.

To investigate the underlying mechanism through which the CBS-CPS1 signaling axis modulated ferroptosis, we compared the expression of core ferroptosis-related proteins between CBS depletion cells and CPS1-reconstituted shCBS cells using western blotting assay (Fig. 7F). The results showed that CBS knockdown inhibited the expression of redox-related proteins, including CPS1, GPX4, cystine/glutamate transporter (SLC7A11) and nuclear factor erythroid 2-related factor 2 (NRF2). Notably, CPS1 complementation in shCBS cells not only elevated CBS expression levels but also rescued GPX4, SLC7A11, and NRF2 expression, while suppressing NADPH oxidase 4 (NOX4) expression. The above findings demonstrated that vanadium, functioning as a multi-target mode-of-action modulator, induced ferroptosis in NSCLC through synergistic mechanisms, including disruption of iron homeostasis via enhancing cellular iron uptake and suppression of the transsulfuration pathway and the xCT/GPX4 antioxidant pathway mediated by the CBS-CPS1 signaling axis, collectively leading to lethal lipid peroxidation (Fig. 7G).

Discussion

Although vanadium compounds were advocated for the treatment of syphilis and leishmaniasis during the 1920s and 1930s, medicinal applications of vanadium preparations have remained uncommon.⁶ Bis(ethylmaltolato)oxavanadium (BEOV), the first vanadium-based antidiabetic complex to enter Phase IIa clinical trials for reducing blood glucose levels in patients with Type 2 diabetes, was discontinued due to observed toxic side effects.²⁵ Vanadium exhibits insulin-mimetic properties as a phosphate analog interfering with phosphate-dependent physiological functions by integrating phosphate channels in cell membranes and interacting with protein tyrosine phosphatases, thereby inhibiting tyrosine dephosphorylation on insulin receptors.⁹ Apart from their superior antidiabetic activity, vanadium complexes also serve as promising alternatives to platinum-based chemotherapies for broad-spectrum anticancer activity and favorable pharmacodynamics.^10,26,27 The currently known anticancer mechanisms of vanadium complexes involve DNA cleavage through forming DNA crosslinks, oxidative stress via ROS promotion or GSH/GSSG ratio reduction, cell cycle arrest by modulating related proteins/kinases, programmed cell death (apoptosis, necrosis, and autophagy), inhibition of cell migration and invasion by EMT suppression, and disruption of metabolic processes like glucose uptake and glycolysis in tumor cells.²⁸ Our study innovatively elucidates a novel cell death mode in NSCLC cells, specifically ferroptosis induced by vanadates independently of the classical apoptotic signaling pathway, with the efficiency of this cell death being contingent upon the valence state of vanadium, aligning with prior research.¹² Given the broad biological activities of vanadium complexes, designing and synthesizing novel ones for the diagnosis and treatment of clinical diseases remain pivotal in bioinorganic chemistry. Identifying new proteins or signaling pathways associated with the underlying mechanism of vanadium complexes is crucial for rational drug design and cancer therapy.

Ferroptosis is a form of necrotic cell death catalyzed by iron ions. The initiation of ferroptosis involves pathways including system Xc⁻, the canonical GPX4-regulated pathway, iron metabolism pathway, and lipid metabolism pathway.^20,29 In the present study, vanadates significantly inhibited the growth and proliferation of NSCLC cells, with NaVO₃(V) being more cytotoxic than VOSO₄(IV), and the inhibitory effect was attenuated by DFO, a small molecule with high affinity for highly charged metal ions that chelates vanadium(IV) or vanadium(V) and inhibits ferroptosis by binding free iron.³⁰ Simultaneously, vanadate treatment led to elevated lipid ROS and ferrous ions, GSH depletion, and downregulated GPX4 expression in NSCLC cells. In xenograft tumors of mice, vanadates caused a reduction in the mitochondrial volume, disruption of the outer mitochondrial membrane, and decreased mitochondrial cristae counts, aligning with the hallmarks of ferroptosis. Ferroptosis due to lipid peroxidation facilitated by ferrous ions via the Fenton reaction, which requires iron participation, might be directly related to enhanced cellular iron ions in NSCLC cells resulting from exogenous vanadium uptake confirmed by ICP-MS. In previous studies, vanadium(IV/V) selectively binds to the empty Fe(III) binding sites of transferrin (Tf) without displacing Tf-bound Fe(III), driving the release of vanadium-coordinated organic ligands that act as ionic carriers to transport excess Fe(III) and Cu(II) into cells, potentially augmenting the cytotoxicity of vanadium complexes.^31–33 These observations are consistent with our findings. Consequently, the internalization of vanadium interfered with the processes of uptake, transport, and release of intracellular iron ions, ultimately leading to ferroptosis of NSCLC by disturbing the balance of iron metabolism and inducing lipid peroxidation.

Transcriptomic analysis revealed CBS as a potential key target for the inhibitory effects of vanadium in NSCLC. CBS is the initial and rate-limiting enzyme in the transsulfuration pathway, critical for cysteine synthesis.³⁴ Cysteine, a key substrate involved in antioxidant systems such as GSH to defend tumor cells against oxidative damage, can be synthesized via the transsulfuration pathway using excess methionine in addition to the system Xc⁻ amino acid transporter.³⁵ It is noteworthy that CBS was identified as a new negative regulator for ferroptosis in diverse tumor cell lines.^36,37 High expression of CBS is associated with the poor prognosis of lung squamous cell carcinoma (LUSC).³⁸ To date, the role of CBS in regulating ferroptosis in NSCLC remains unknown. The results of the CETSA assay indicated that vanadium directly bonded to and regulated CBS expression, further inhibiting cell proliferation in synergy with AOAA, a specific inhibitor of CBS. CBS knockdown suppressed NSCLC proliferation and migration in vitro and in vivo, and promoted intracellular levels of ferrous ions, lipid ROS and MDA, whereas CBS overexpression reduced the above ferroptosis indicators as well as cellular uptake of vanadium and iron. The above data suggest that CBS remains a negative regulator of ferroptosis in NSCLC, consistent with previous findings in other tumor species. To clarify the effect of vanadium on the transsulfuration pathway, we detected changes in the abundance of related metabolites through LC-MS/MS-based untargeted metabolomics. The results revealed that methionine, serine, cysteine, glycine, and glutamine levels were significantly upregulated in NSCLC cells under vanadium treatment. Cysteine synthesis via the transsulfuration pathway is crucial for tumor cell growth and proliferation, and vanadium inhibits cell development and downregulates CBS expression;³⁹ meanwhile, CBS knockdown concurrently decreases SLC7A11 expression, suggesting the disruption of the cysteine metabolic pathway. However, the increased metabolite levels caused by vanadium might be attributed to cellular stress responses, necessitating further investigation into the dynamic protein regulatory mechanisms involved.

CPS1 was screened as a potential interacting protein of CBS using CoIP-MS in combination with the STRING database and verified by immunoblotting and colocalization assays. Urea cycle enzyme CPS1 catalyzes the formation of carbamoyl phosphate (CP) from ammonia and bicarbonate. In A549 and H460 cell lines with characteristics of KRAS mutation and LKB1 loss (KL), CPS1 knockdown induces cell death via pyrimidine depletion and suppresses lung cancer cell growth.^40,41 In hepatocellular carcinoma with CPS1 deficiency, urea cycle dysregulation resulted in the deceleration of the tricarboxylic acid (TCA) cycle, while excess ammonia induced by CPS1 deficiency activated fatty acid β-oxidation (FAO) through p-AMPK.²⁴ Reduced CPS1 expression promotes FAO dependence in tumor cells, and we hypothesize that the resulting accumulation of lipid ROS may be directly related to cellular ferroptosis. In our study, vanadium decreased CPS1 expression, which in turn inhibited NSCLC cell proliferation and led to the accumulation of ferrous ions and ROS levels in shCPS1 cells. Complementation of CPS1 in NSCLC cells with CBS depletion restored cell growth and proliferation while attenuating the relevant indicators inducing ferroptosis. Thus, the CBS-CPS1 signaling axis may function as a critical target for clinical treatment of NSCLC.

Conclusions

In summary, we identified the CBS-CPS1 signaling axis toward ferroptosis in NSCLC under vanadate treatment. Vanadium ultimately leads to ferroptosis by disrupting iron metabolism and inducing lipid peroxidation in NSCLC cells via the CBS/CPS1 signaling axis. Our study elucidates the novel underlying mechanisms by which vanadium inhibits NSCLC, offering insights into potential therapeutic targets for NSCLC and the development and application of vanadium-based metallodrugs.

Author contributions

Tianxiang Su: data acquisition, validation, and visualization. Xiaofen Zhang: formal analysis and investigation. Yuanyuan Sun: validation and writing – review & editing. Xing Chen: methodology and validation. Meiling Tian: data acquisition and validation. Dan Yan: validation and visualization. Yi Zhao: resources and visualization. Bingjie Han: conceptualization, supervision, project administration, funding acquisition, writing – original draft, and writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data that support the findings of this study are available in the ESI† or on request from the corresponding author. The RNA-Seq datasets generated during the current study are available in the NCBI repository with BioProject ID: PRJNA1230352.

Acknowledgements

Financial support was provided by the National Natural Science Foundation of China (22007085), the Science and Technology Foundation of Henan Province (242102310151), the Key Scientific Research Project of Colleges and Universities in Henan Province (24A320028 and 24A320017), the Joint Construction Project for Medical Science and Technology Research of Henan Province (LHGJ20230168) and the Medical Science and Technology Research Project of Henan Province (SBGJ202302045).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5qi01088h

‡ Equal Contribution.

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