Cyclophosphamide Induces Autophagy-Dependent Ferroptosis Through Promoting ETNK1 Expression in Breast Cancer Cells

Breast cancer is the most common cancer in females worldwide, and the incidence rate in China has been increasing in recent decades. The treatment of breast cancer with cyclophosphamide (CTX) is one of the cornerstones of combination chemotherapy. However, the mechanisms underlying the anti-tumor effect of CTX is not fully understood. CCK8 assay was employed to detect the IC50 value and viability of breast cancer cell lines (MCF-7 and 4T1). Cell morphology was observed. Tunel assay was carried out to determine cell apoptosis. The content of iron level (Fe²⁺), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) was assessed to measure the ferroptosis level. Western blot measured the expression of ETNK1, and autophagy-related proteins (Beclin1 and LC3). The mechanism in vivo was verified in the nude mice model transplanted with MCF7 cells. CTX inhibited cell proliferation, promoted cell apoptosis and ferroptosis in vitro. Inhibition of autophagy could suppress CTX-induced ferroptosis. CTX treatment could increase ETNK1 expression. Downregulation of ETNK1 could reverse the impacts of CTX on cell survival, ferroptosis, and autophagy both in vitro and in vivo. CTX-induced iron death dependent on autophagy in breast cancer cells by promoting the expression of ETNK1.