Ponicidin promotes ferroptosis to enhance treatment sensitivity in Lenvatinib-resistant hepatocellular carcinoma cells through regulation of KEAP1/NRF2.

[OBJECTIVE] This study explores the therapeutic potential of Ponicidin on Lenvatinib-resistant hepatocellular carcinoma (HCC), elucidates its mechanism in reversing Lenvatinib resistance, and provides experimental evidence for its clinical application in overcoming this resistance.

[METHODS] Huh7 and HCC-LM3 cells were used to construct Lenvatinib-resistant cell lines, Huh7-LR and HCC-LM3-LR. Changes in the ferroptosis pathway post-drug resistance were observed by measuring ferroptosis-related markers. The proliferation assay were assessed by CCK-8, while the migration and invasion were measured by scratch and Transwell invasion assays. In mechanistic study, chip analysis and immunoprecipitation with biotin-labeled Ponicidin, were conducted to explore how Ponicidin overcame drug resistance. Xenograft model in nude mice was established to examine Ponicidin's anti-HCC effects In vivo. Clinical specimens were used to assess the true status of patients in Lenvatinib-resistant HCC patients.

[RESULTS] Our study reveals for the first time that ferroptosis inhibition drives Lenvatinib resistance in HCC and identifies Ponicidin as a novel KEAP1-targeting agent to reverse this process. In vitro, ferroptosis pathway was suppressed in Lenvatinib-resistant cells. Ponicidin suppressed proliferation, clonogenicity, migration, and invasion in these cells. The combination of Ponicidin and Lenvatinib significantly inhibited proliferation and reversed drug resistance by activating the ferroptosis pathway. Preliminary mechanistic studies showed that Ponicidin binds to KEAP1, stabilizing the KEAP1/NRF2 interaction, inhibiting the nuclear translocation and activation of NRF2, and thereby inducing ferroptosis to overcome Lenvatinib resistance. In vivo, the combination of Ponicidin and Lenvatinib exhibited a synergistic effect, significantly delaying tumor growth. Clinically, p-NRF2 and GPX4 expression was higher in the Lenvatinib-insensitive group, suggesting that the ferroptosis pathway was inhibited in these patients. Thus, this study demonstrated that Ponicidin promotes ferroptosis to enhances treatment sensitivity in Lenvatinib-resistant HCC cells through KEAP1/NRF2.