Aloin suppresses cancer cell growth via autophagy-promoted NEDD8 de-NEDDylation to target GPX4 for ferroptosis induction.

Aloin (ALO), an anthraquinone derived from Aloe vera, exhibits antitumor activity; however, its precise mechanisms of action remain unclear. In this study, in silico molecular docking analysis first revealed that Aloin (ALO) bound effectively to ferroptosis-related proteins (SLC7A11, GPX4, ACSL4, and TFR1). Subsequently, in vitro ALO treatment triggered ferroptosis hallmarks in human cervical cancer cell line Hela and mouse colon cancer cell line MC38 at both transcriptional and protein levels-downregulating SLC7A11/GPX4, upregulating ACSL4/TFR1, with Fe2+/ROS accumulation, GSH depletion, and ferroptosis-specific mitochondrial cristae loss. Furtherly, ALO inhibited cancer cell proliferation, migration, and invasion, effects that were reversed by the ferroptosis inhibitor Ferrostatin-1. Concurrently, ALO induced autophagy, as evidenced by increased levels of LC3, LaminB1, and ULK1, decreased levels of P62, and TEM-visualized autophagosomes. Notably, the autophagy inhibitor chloroquine reversed ALO-induced ferroptosis, NEDD8 downregulation, and NEDP1 upregulation, linking ALO-induced autophagy to NEDD8 de-NEDDylation. Genetic and pharmacological perturbation of the NEDD8 pathway confirmed this: NEDD8 inhibition enhanced, while NEDP1 knockdown attenuated, ALO-induced ferroptosis. Co-immunoprecipitation and laser-scanning confocal microscope confirmed a direct NEDD8-GPX4 interaction diminished by ALO, positioning GPX4 as a key effector. In in vivo study, ALO effectively inhibited cancer cell growth in a murine colon carcinoma MC38 xenograft models, while exhibiting no obvious toxicity or side effects in mice. Moreover, ALO exhibited the same regulatory effects and trends on ferroptosis-related proteins in vivo as those in vitro. In summary, this study reveals a novel mechanism that ALO-induced autophagy promotes NEDD8 de-NEDDylation, driving ferroptosis via the NEDD8-GPX4 axis to suppress cancer cell growth.