Hydrogen peroxide induces apoptosis-like death in Entamoeba histolytica trophozoites

Programmed cell death (PCD) is an essential process in the growth and development of multicellular organisms. However, accumulating evidence indicates that unicellular eukaryotes can also undergo PCD with apoptosis-like features. This study demonstrates that after exposure to 0.8 mM H₂O₂ for 9 h Entamoeba histolytica presents morphological and biochemical evidence of apoptosis-like death. Morphological characteristics of apoptosis-like death including DNA fragmentation, increased vacuolization, nuclear condensation and cell rounding were observed for H₂O₂-exposed trophozoites with preservation of membrane integrity. Biochemical alteration in ion fluxes is also a key feature in PCD, and H₂O₂-exposed trophozoites showed overproduction of reactive oxygen species, increased cytosolic Ca ²⁺ and decreased intracellular pH. Phosphatidylserine was also found to be expressed in the outer leaflet of the plasma membrane of the H ₂O₂-treated trophozoites. Pretreatment with the cysteine protease inhibitor E-64d, the extracellular and intracellular Ca²⁺ chelators EGTA and BAPTA/AM, and the Ca²⁺ influx inhibitor verapamil prior to H₂O₂ exposure abolished DNA fragmentation. The oxidatively stressed trophozoites also showed an increased calpain activity, indicating involvement of Ca²⁺-dependent calpain-like cysteine proteases in PCD of E. histolytica. A homogeneous caspase assay showed no significant caspase activity, and administration of caspase 1 inhibitor also did not prevent the death phenotype for the oxidatively stressed trophozoites, indicating a caspase-independent apoptosis-like death. Our observations clearly demonstrate that there is a distinct calpain-dependent but caspase-independent pathway for apoptosis-like death in oxidatively stressed E. histolytica trophozoites.