Induction of apoptosis-like cell death by coelomocyte extracts from Eisenia andrei earthworms

- Coelomocyte lysate (CCL)-derived cytotoxicity evoked membrane damage.
- CCL exposed cells were Annexin V+ and TUNEL+ without caspase involvement.
- Mitochondrial membrane depolarization was pronounced in CCL treated cells.
- Anti-lysenin mAb pretreatment of CCL did not rescue target cells from cell death.

Earthworm's innate immunity is maintained by cellular and humoral components.Our objective was to characterize the cytotoxicity leading to target cell death caused by earthworm coelomocytes. Coelomocyte lysates induced strong cytotoxicity in tumor cell lines. Transmission electron microscopy revealed cell membrane and intracellular damage in cells treated with coelomocyte lysates. Using TUNEL-assay, within 5 min of incubation we detected DNA fragmentation. Moreover, we found phosphatidylserine translocation in target cell-membranes. Furthermore, we detected dose-dependent Ca2+ influx and decrease of mitochondrial membrane potential in coelomocyte lysate-treated cells. Interestingly, caspase 3/8 activation was undetectable in exposed tumor cells. One such cytotoxic molecule, lysenin identified in earthworms binds to sphingomyelin and causes target cell lysis in vertebrates. Pretreatment with our anti-lysenin monoclonal antibody rescued the majority but not all target cells from coelomocyte induced death. These data suggest that, not only lysenin but also other factors participate in the caspase-independent apoptosis induced by coelomocytes.