EGFR suppression mediates aurantio-obtusin-induced autophagy-dependent ferroptosis in breast cancer cells

Emerging evidence suggests that triggering ferroptosis could be a promising approach for combating breast cancer. Here, we investigated the role of aurantio-obtusin (AO), a key bioactive compound sourced from Cassiae semen, in the treatment of breast cancer. Cell viability and death were determined via a CCK-8 assay and propidium iodide (PI) staining. Ferroptosis was determined by the levels of Fe2+, lipid ROS, and GSH. Autophagy was assessed by immunofluorescence staining. An MDA-MB-231 cell transplantation tumor model was established in vivo. AO inhibited breast cancer cell growth in a concentration-dependent manner while promoting cell death. Ferroptotic changes, including blockage of the GSH–GPX4 antioxidation system, massive iron release, and lipid ROS accumulation, were evident in AO-treated breast cancer cells. Antioxidant N -acetylcysteine (NAC) treatment markedly restored GSH content and suppressed lipid ROS overproduction to relieve AO-triggered ferroptosis. The introduction of the iron scavenger deferoxamine or the ferroptosis inhibitor ferrostatin-1 weakened AO-induced ferroptosis in breast cancer cells. Moreover, AO treatment promoted autophagosome formation in breast cancer cells via the mTOR-ULK1 pathway. AO-induced autophagy and ferroptosis were both reversed by Atg7 knockdown or mTOR activator MHY1485 treatment. Network pharmacology and molecular docking investigations suggested that EGFR was a potential target of AO in breast cancer and that its protein expression was suppressed by AO. Selective EGFR inhibitor gefitinib exacerbated AO-mediated ferroptosis and autophagy, while genetic overexpression of EGFR completely reversed these AO-mediated phenotypes. In vivo assays confirmed the antitumor effects of AO. Our study revealed that AO facilitated autophagy-dependent ferroptosis in breast cancer cells through the downregulation of EGFR. This work provides a new perspective on the antitumor activity of AO, which is valuable for further investigations of the practical application of AO in breast cancer.