Fosinopril mediates antitumor efficacy by inducing GSDME-dependent pyroptosis in NSCLC

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  Fig. 1. Fosinopril inhibited cell viability and colony formation and released LDH in human NSCLC cells. A The chemical structure of fosinopril. B A549 and H1299 cells were incubated with various concentrations of fosinopril (0, 45, 60, 75, 90, 105, 120, 135, and 150 µM) for 24 h. The effects of fosinopril on cell proliferation were assessed by CCK-8 assay. C Half-maximal inhibitory concentration (IC50) of fosinopril in inhibiting the proliferation of A549 and H1299 cells. D The effect of fosinopril on the colony-forming ability of A549 and H1299 cells was assessed using a colony formation assay. E The number of colonies in A549 and H1299 cells after treatment with fosinopril for 24 h at indicated concentrations (0, 45, 90, and 135 μM). F The number of A549 and H1299 cells after fosinopril treatment for 24 h at indicated concentrations (0, 45, 90, and 135 μM) was measured, with findings from quantitative analysis of cell counting shown. G Levels of lactate dehydrogenase (LDH) in cell cultures were measured after fosinopril treatment (0, 45, and 90 μM) for 24 h. H, I A549 and H1299 cells treated with fosinopril (0, 45, 90, and 135 μM for 24 h) were co-stained with PI and Annexin V and…

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  Fig. 2. Fosinopril induced GSDME-dependent pyroptosis in human NSCLC cells. A GO analysis of significantly differentially expressed proteins. B Morphological changes in A549 and H1299 cells induced by fosinopril (0, 45, and 90 μM). Scale bar: 200 µm. C Western blot analysis of GSDME-N in A549 cells after fosinopril treatment (0, 45, and 90 μM) and their corresponding statistical analysis. D Localization of total GSDME (GSDME-N and/or GSDME-FL) in A549 cells treated with fosinopril (0, 45, 90 μM for 24 h) visualized by immunofluorescence assays, with quantitative analysis of fluorescence intensity shown. Scale bar: 200 µm. E Cell viability following treatment with fosinopril (0, 45, or 90 μM) for 24 h, with or without the GSDME inhibitor (2-BP). Western blot analysis was performed to analyze the protein levels of GSDME-N, and statistical analysis was performed. F, G Cell viability following fosinopril treatment (0, 45, 90 μM) for 24 h, with or without the GSDME inhibitor (2-BP; 1 mM) determined by immunofluorescence assays, with quantitative analysis of fluorescence intensity. H Relative LDH activity in the culture medium of cells was measured. I, J Morphological changes and the number of…

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  Fig. 3. Network pharmacological analysis. A Venn diagram showing the overlap between 6793 potential NSCLC targets, 219 targets related to anxiety and insomnia, and 172 common targets. B “Herbal–ingredient–target” network of fosinopril in the treatment of NSCLC. C, D PPI network between fosinopril and the intersection targets of NSCLC. E GO enrichment analysis of the association networks between 27 key genes and their corresponding biological concepts. F GO enrichment analysis identifying the key biological processes of fosinopril in treating NSCLC.

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  Fig. 4. Fosinopril promoted tumor cell pyroptosis by enhancing Caspase-3 cleavage in human NSCLC cells. A A549 cells were treated with fosinopril (0, 45, and 90 μM) for 24 h. The Caspase-3 and Caspase-9 cleaved protein levels, with corresponding statistical analysis. B Western blot analysis of Caspase-3 and GSDME-N cleaved protein levels. C, D Cell viability after treatment with fosinopril (0, 45, 90 μM) for 24 h, with or without the Caspase-3 inhibitor (Ac-DEVD-CHO; 1 mM), as determined by immunofluorescence assays. Quantitative analysis of fluorescence intensity from the immunofluorescence assays is shown. E Relative LDH activities in the culture mediums of cells treated with fosinopril. F, G Morphological changes and the number of A549 cells after fosinopril treatment. Data are presented as mean ± SD for n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 5. Fosinopril enhanced ROS production, resulting in a decrease in MMP. ROS generation (A) and MMP (B) in A549 cells treated with fosinopril at indicated concentrations (0, 45, and 90 μM) for 24 h, as determined by immunofluorescence assays. Scale bar: 100 μm. Histograms of the corresponding mean fluorescence intensity (MFI) are shown. C Western blot analysis of Bax protein levels. D–F ROS generation and MMP of A549 cells exposed to fosinopril at 0, 45, and 90 μM for 24 h, with or without ROS inhibitor NAC (25 μM), were determined by immunofluorescence assays. Histograms of corresponding MFI are shown. Scale bar: 100 μm. G, H Western blot analysis of Cleaved-Caspase-3, Cleaved-Caspase-9, and GSDME-N cleaved protein levels. I Relative LDH activities in the culture mediums of cells treated with fosinopril. J, K Morphological alterations and the number of cells after treatment with fosinopril. Scale bar: 100 μm. Data are presented as mean ± SD for n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 6. Fosinopril induced pyroptosis and antitumor activity by activating ROS through MOMP in A549 cell xenografts in vivo. A Treatment schedule for mice with A549 xenograft tumors and fosinopril treatment. B–E Effect of fosinopril on tumor weight, volume weight, and body weight in A549 xenograft model (n = 5 per group). F Hematoxylin and eosin (HE) staining of tumor tissues (HE, original magnification, ×200; Scale bar = 50 µm). G Tumor cells co-stained with PI and Annexin V were visualized by immunofluorescence assays. Quantitative analysis of fluorescence intensity is shown. H Immunofluorescence analysis of GSDME localization in tumor cells. Quantitative analysis of fluorescence intensity is shown. I Protein expression levels of Bax, Cleaved-Caspase-3, Cleaved-Caspase-9, and GSDME-N in tumor tissues were detected by Western blot. J ROS levels in tumor cells were detected by immunofluorescence assays (n ≥ 3, ***P < 0.001 vs. fosinopril 0 mg/kg group).

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  Fig. 7. A novel pathway of fosinopril-induced GSDME-dependent pyroptosis in NSCLC cells. In NSCLC cells, fosinopril-induced ROS triggers a decrease in the Bax ratio, leading to an increase in mitochondrial membrane potential. This activation results in the activation of Caspase-9 and Caspase-3, ultimately causing the cleavage of GSDME and inducing pyroptotic cell death in NSCLC cells.