Melittin-induced membrane disruption in hepatocellular carcinoma involves mtROS/GSDME-mediated pyroptosis

Abstract

Although melittin is classically regarded as a membrane-lytic peptide, it remains unclear whether its membrane-disruptive effects in tumour cells result exclusively from direct peptide-lipid interactions or are partly generated by pyroptosis. Here, we show that melittin markedly suppresses the viability and clonogenic growth of human Huh7 and murine Hepa1-6 HCC cells and rapidly induces characteristic features of pyroptosis, including cell swelling, balloon-like membrane protrusions, plasma-membrane permeabilization and lactate dehydrogenase release. In Hepa1-6 cells, melittin induced prominent GSDME cleavage and accumulation of its pore-forming N-terminal fragment, whereas GSDMD activation was not detected. Quantitative proteomics and ultrastructural analyses further revealed pronounced mitochondrial perturbation after melittin treatment, accompanied by loss of mitochondrial membrane potential and increased mitochondrial reactive oxygen species (mtROS). Importantly, scavenging mitochondrial oxidants with Mito-TEMPO markedly reduced mtROS accumulation, attenuated pyroptotic membrane damage and partially restored cell viability. Collectively, our findings provide, to our knowledge, the first evidence that melittin induces pyroptotic cell death in HCC cells through a mechanism involving mitochondrial oxidative stress and GSDME processing. These results extend the conventional view of melittin as a direct membrane-lytic peptide and uncover an mtROS/GSDME-associated pyroptotic mechanism underlying its antitumour activity in HCC.