Natural exosome-like nanovesicles from Smilax China rhizome induce mitophagy-dependent ferroptosis in hepatocellular carcinoma via GPX4/ACSL4 axis.

[BACKGROUND] Hepatocellular carcinoma (HCC) remains challenging due to limited therapies and resistance. Mitophagy-associated ferroptosis, an iron-dependent cell death mechanism, emerges as a novel strategy, while plant-derived extracellular nanovesicles offer the possibility for nanotherapeutics.

[METHODS] Natural exosome-like nanovesicles from Smilax china Rhizome (SCRENs) were isolated via sucrose density gradient ultracentrifugation and characterized (TEM, NTA, SDS-PAGE). Anti-HCC effects were assessed in vitro (EdU, colony formation, migration assays). Ferroptosis and mitophagy mechanisms were studied using inhibitors (Fer-1, 3-MA), inducers (CCCP, Erastin), and biochemical assays (ROS, Fe, GSH, MDA). Mitochondrial dysfunction was evaluated via JC-1, mtDNA, morphology, and protein analysis (ACSL4, SLC7A11, GPX4).

[RESULTS] SCRENs (139.7 nm diameter) inhibited HCC proliferation, migration, and induced apoptosis, with HepG2 cells most sensitive. SCRENs triggered ferroptosis (Fe, ROS, MDA, GSH) and altered ferroptosis markers (ACSL4, SLC7A11, GPX4). Mitophagy was evidenced by mitochondrial depolarization, mtDNA loss, and ultrastructural damage. Pharmacological inhibition confirmed mitophagy-ferroptosis crosstalk. SCRENs mediated dual GPX4/ACSL4 regulation, GPX4 suppression impaired antioxidant capacity, while ACSL4 upregulation promoted lipid peroxidation. Ferrostatin-1 reversed SCRENs' effects, whereas GPX4 activation failed to rescue viability, highlighting ACSL4/mitophagy-driven iron release as critical.

[CONCLUSIONS] Our study successfully isolated SCRENs from Smilax china rhizomes and demonstrated their inhibitory effects on HCC cell proliferation, migration, and invasion through in vitro cellular experiments. Mechanistic investigations revealed that SCRENs mediate their anti-tumor effects primarily through modulation of mitophagy-associated ferroptosis via GPX4/ACSL4 axis.