AG8 induces apoptosis and ferroptosis through regulating caspase 3 and SLC7A11/GPX4 signaling pathway in triple-negative breast cancer

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Background AG8 is a triterpenoid saponin isolated from Ardisia gigantifolia stapf., we previously found that AG8 demonstrated excellent antitumor activity against triple negative breast cancers (TNBCs) through reactive oxygen species (ROS) generation and mitochondrial apoptotic pathways triggering. Intracellular ROS plays a vital role in cell survival, it can activate both apoptotic and ferroptosis signaling pathways. However, the effects of AG8 on ferroptosis of TNBCs have not been investigated. Objective: In this study, we selected basal-like 1 (BL1) and luminal androgen receptor (LAR) subtypes of TNBC to further investigate the effects of AG8 on ferroptosis and apoptosis. Methods The cell viabilities were tested using MTT. Glutathione/ oxidized glutathione (GSH/GSSG) ratio, malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured by ELISA kits. C11-BODIPY 581/591and flow cytometry were used to tested lipid ROS. Ferrous ion was analyzed using FeRhoNox-1. Molecular docking was used to test the interactions of AG8 with caspase 3, solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4). The influences of AG8 on caspase 3, cleaved-caspase 3, SLC7A11 and GPX4 protein levels were measured using western blotting. In vivo antitumor effect of AG8 was tested by HCC-1937 tumor bearing nude mice. Results AG8 significantly suppressed cell proliferation and colony-formation in HCC-1937 (BL1) and MDA-MB-453(LAR) cells. Interestingly, Z-VAD-FMK (apoptosis inhibitor) and Ferrostatin-1(Fer-1, ferroptosis inhibitor) both rescued AG8-induced cell death, but MDA-MB-453 was more sensitive to Fer-1. AG8 induced ferroptosis of TNBC cells by the accumulation of ROS, increasing ferrous ion and MDA levels, and decreasing GSH/GSSG ratio and SOD levels. AG8 reduced caspase 3, SLC7A11 and GPX4 protein levels, but induced cleaved caspase 3 level in TNBC cells. The results of molecular docking showed that AG8 bound effectively to caspase 3, SLC7A11 and GPX4. Moreover, AG8 treatment significantly inhibited the tumor volumes in HCC-1937 tumor bearing nude mice. Conclusion Above all, AG8 induced apoptosis and ferroptosis through regulating caspase 3 and SLC7A11/GPX4 signaling pathway. Ferroptosis of LAR subtypes are easily to be induced by AG8, the underlying mechanism need to be further investigated.