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.
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