Recombinant HALT-1 induces mitochondrial-associated apoptotic mechanism in HeLa cells

The study explored the apoptotic mechanism of Hydra actinoporin-like toxin-1 (HALT-1), an α-pore-forming toxin (α-PFT) produced by Hydra magnipapillata. α-PFT has been known to induce membrane pores in human cells upon contact, leading to the cell death. While previous research has covered HALT-1’s structural, membrane binding, cytolytic, and haemolytic aspects, the detailed information on apoptotic mechanism and cell signalling pathways remain unknown. Our study confirmed previous findings of rHALT-1's dose-dependent cytotoxicity, with a CC₅₀ of 15.4 µg/mL observed after 24 hours of treatment in our case. Hence, an rHALT-1 concentration below 15.4 µg/mL was selected to examine its apoptotic activity. Real-time Annexin V and DNA dye assays revealed dose- and time-dependent apoptotic patterns, with 12 µg/mL rHALT-1 inducing maximum apoptosis at 7 hours and minimal necrosis. Subsequently, flow cytometric analysis showed mitochondrial membrane potential depolarization without active caspase-3 throughout 6, 12, and 24-hour treatments. Western blot analysis indicated upregulation of apoptotic-inducing proteins (Bad, Bax, cytochrome c, caspase-9) and downregulation of antiapoptotic proteins (Bcl-2, Bcl-xL) at 12 µg/mL of rHALT-1. The absence of active caspases 3, 6, and 8 expressions suggests alternative cell death pathways. In conclusion, the study proposes, for the first time, that rHALT-1 induces apoptosis in HeLa cells by mediating the mitochondrial pathway, although active caspase-3 does not appear to be involved in the execution process. These findings provide a foundation for elucidating the mechanistic basis of rHALT-1 activity and highlight its potential utility in toxin-related research and biotechnological applications.