Lenvatinib induces ferroptosis-related changes in osteosarcoma cells involving the p-STAT3/p53/xCT axis

Lenvatinib is an effective antitumor agent for several solid tumors and has been used in pediatric and adolescent patients with refractory or recurrent osteosarcoma. However, the mechanism by which lenvatinib suppresses osteosarcoma progression remains incompletely defined. This study investigated whether lenvatinib induces ferroptosis-related changes in osteosarcoma cells and explored its effects on malignant biological behavior and the underlying molecular mechanism.

Human osteoblast hFOB1.19 cells and osteosarcoma MG63, 143B, and U2OS cells were treated with lenvatinib. Cell viability and IC50 values were determined using CCK-8 assays. Ferroptosis-related molecular changes in 143B and U2OS cells were evaluated by qRT-PCR, Western blotting, flow cytometry, and biochemical assays measuring intracellular Fe²⁺, reduced glutathione, reactive oxygen species, and lipid peroxidation. Mitochondrial ultrastructure was examined by transmission electron microscopy, and p53 and xCT expression patterns were assessed by immunofluorescence staining. Ferroptosis inhibition assays with ferrostatin-1 were performed, and stable xCT-overexpressing osteosarcoma cell lines were generated by lentiviral transduction.

Lenvatinib inhibited the proliferation of 143B and U2OS cells in a concentration-dependent manner, whereas its inhibitory effect was weaker in MG63 cells and limited in hFOB1.19 osteoblasts. Transmission electron microscopy revealed ferroptosis-associated mitochondrial alterations, including mitochondrial shrinkage, loss or reduction of cristae, and increased membrane density. Lenvatinib increased intracellular Fe²⁺, reactive oxygen species, and lipid peroxidation levels while decreasing reduced glutathione, and these effects were reversed by ferrostatin-1. qRT-PCR, Western blotting, and immunofluorescence analyses showed that lenvatinib treatment was associated with increased p53 expression, reduced xCT expression, and modulation of ferroptosis-related genes and proteins. Western blotting further indicated that lenvatinib inhibited STAT3 phosphorylation, whereas ferrostatin-1 attenuated lenvatinib-induced changes in p-STAT3 and p53. Functionally, xCT upregulation enhanced osteosarcoma cell proliferation, migration, and invasion and attenuated lenvatinib-induced ferroptosis-related changes.

These findings suggest that lenvatinib suppresses osteosarcoma cell proliferation, migration, and invasion, at least in part, by inducing ferroptosis-related changes associated with modulation of the p-STAT3/p53/xCT axis. This study reveals a previously underrecognized mechanism of lenvatinib action and supports further investigation of ferroptosis-targeted strategies in osteosarcoma.