Alexidine dihydrochloride enhances the sensitivity of human hepatocellular carcinoma to disulfidptosis via ATF4-DDIT3 activation.

Disulfidptosis, an emerging regulated cell death modality characterized by pathological disulfide bond aggregation and subsequent actin cytoskeletal disintegration, presents promising therapeutic potential for apoptosis- and ferroptosis-resistant malignancies. However, the molecular initiators of disulfidptosis and their connection with mitochondrial dysregulation remain elusive. Herein, we identified alexidine dihydrochloride (AD) as a selective disulfidptosis enhancer in hepatocellular carcinoma (HCC), and the comprehensive multi-omics analysis and other functional validation indicated that ATF4-DDIT3 signaling could be significantly activated by AD. Mechanistically, AD triggers severe mitochondrial stress, manifested by ultrastructural disruption, mitochondrial reactive oxygen species (ROS) overproduction, and activation of the mitochondrial unfolded protein response (UPRmt). Crucially, these mitochondrial insults converge on the ATF4-DDIT3 signaling axis, which orchestrates disulfide stress amplification. This redox imbalance culminates in irreversible disulfide-mediated crosslinking of cytoskeletal proteins, the hallmark of disulfidptosis. Additionally, knockdown of ATF4 or DDIT3 abolishes AD-enhanced disulfidptosis, confirming their indispensable roles in this process. Totally, our findings not only characterize AD as a novel disulfidptosis enhancer but also establish a functional crosstalk between mitochondrial dysfunction and disulfidptosis execution, proposing a dual-target therapeutic strategy for drug resistance of HCC.