Artesunate overcomes lenvatinib resistance by inhibiting the STAT3/SLC25A27 pathway and promoting ferroptosis in hepatocellular carcinoma

Lenvatinib is the first-line drug used in the systemic treatment of advanced hepatocellular carcinoma (HCC), although its therapeutic efficacy is limited by emerging resistance mechanisms. Our study aims to investigate the molecular pathways underlying lenvatinib resistance in HCC to inform circumvention strategies for therapeutic resistance. Our findings demonstrated that solute carrier family 25 member 27 (SLC25A27) is upregulated in lenvatinib-resistant (Lenva-R) HCC cells by RNA sequencing (RNA-seq). Similarly, SLC25A27 expression is higher in Lenva-R than lenvatinib-sensitive (Lenva-S) HCC cells and tissues via Quantitative real-time PCR (qRT-PCR), western blot, and immunohistochemistry (IHC) in vitro and vivo. Cell Counting Kit-8 (CCK-8) assay was performed to determine cell viability. Colony formation assay was performed to determine cell proliferation capacity. DCFH-DA staining was used to detect the levels of intracellular reactive oxygen species (ROS). Observation of mitochondrial morphology by using transmission electron microscopy (TEM). The corresponding assay kits were used to analyze the levels of malondialdehyde (MDA), glutathione (GSH)/ oxidized glutathione disulfide (GSSG) ratio, and mitochondrial membrane potential (MMP). Gain- and loss-of-function experiments demonstrated that SLC25A27 promotes survival, proliferation and lenvatinib resistance in vitro. Mechanistic studies showed that SLC25A27 inhibits ferroptosis pathway via activating solute carrier family 7 member A11 (SLC7A11)/glutathione peroxidase 4 (GPX4). Dual-luciferase reporter experiments confirmed signal transducer and activator of transcription 3 (STAT3) significantly enhances SLC25A27 transcription. Moreover, artesunate promotes ferroptosis by inhibiting the STAT3/SLC25A27/ SLC7A11/GPX4 axis in vivo and in vitro. Overall, our study confirmed the combination of artesunate and lenvatinib significantly enhances the anti-HCC effect of lenvatinib, which promising therapeutic strategy to circumvent resistance in HCC.