G-4 inhibits triple negative breast cancer by inducing cell apoptosis and promoting LCN2-dependent ferroptosis.

ABSTRACT

Compound G-4 is a derivate of cyclin-dependent kinase inhibitor Rocovitine and showed strong sensitivity to triple negative breast cancer (TNBC) cells. In this study, the antitumor activity, mechanism and possible targets of G-4 in TNBC were investigated. Flow cytometry and immunoblotting showed that G-4 not only arrested the S phase of the cell cycle, but also induced apoptosis in TNBC cells via the mitochondrial pathway through inhibiting epidermal growth factor receptor (EGFR), AKT and MAPK pathways. In addition, G-4 induced the iron-mutagenesis process in TNBC cells and down-regulated differentially expressed gene lipid carrier protein 2 (LCN2) by RNA-seq. Moreover, G-4 elevated levels of cytosolic reactive oxygen species (ROS), lipid ROS, Fe and malondialdehyde (MDA), but decreased levels of superoxide dismutase (SOD) and glutathione (GSH), consistent with the effects of iron-mutagenic agonists Erastin and RSL3, which were inhibited by the iron inhibitor ferrostatin-1 (Fer-1). Furthermore, a LCN2 knockdown cell model was established by siRNA transfection, the IC50 of G-4 was increased nearly 100-fold, accompanied by a trend of no ferroptosis characteristic index. The results indicated that G-4 suppressed the malignant phenotype of TNBC, induced apoptosis by inhibiting EGFR pathway and promoted LCN2-dependent ferroptosis.