Hydrogen peroxide induces apoptosis-like death in Entamoeba histolytica trophozoites

Abstract

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 H 2 O 2 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 H 2 O 2 -exposed trophozoites with preservation of membrane integrity. Biochemical alteration in ion fluxes is also a key feature in PCD, and H 2 O 2 -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 2 O 2 -treated trophozoites. Pretreatment with the cysteine protease inhibitor E-64d, the extracellular and intracellular Ca 2+ chelators EGTA and BAPTA/AM, and the Ca 2+ influx inhibitor verapamil prior to H 2 O 2 exposure abolished DNA fragmentation. The oxidatively stressed trophozoites also showed an increased calpain activity, indicating involvement of Ca 2+ -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 caspaseindependent 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.

The metabolites of glutamine prevent hydroxyl radical-induced apoptosis through inhibiting mitochondria and calcium ion involved pathways in fish erythrocytes

“…An hypothetical model for the pathway of apoptosis could be that • OH stimulates Ca 2 þ influx that triggers calpain activities, which in turn cleaves Bid or Bax that induces the release of cytochrome c with subsequent caspase-9 and caspase-3 activation that results in DNA fragmentation in fish erythrocytes. Nevertheless, Nandi et al revealed that H 2 O 2 induces a distinct calpain-dependent DNA fragmentation without the involvement of caspases in E. histolytica trophozoites [99]. Furthermore, our results showed that zVAD-fmk completely prevented the caspase-9 and caspase-3 activation, but did not fully inhibited DNA fragmentation in the • OH-induced carp erythrocytes.…”

Ca2+ and caspases are involved in hydroxyl radical-induced apoptosis in erythrocytes of Jian carp (Cyprinus carpio var. Jian)

“…This is in good line with the report that oxidative stress leads to the influx of Ca 2? that activates calpain with subsequent DNA fragmentation in E. histolytica trophozoites (Nandi et al 2010). Nevertheless, no reports have published the caspase-3 independent but calpain-dependent DNA fragmentation in fish cells.…”

The first suicides: a legacy inherited by parasitic protozoans from prokaryote ancestors

“…This recent finding adds weight to the speculation of Debrabant and colleagues [83] that caspase-dependent and caspase-independent pathways may exist in different Leishmania spp. In addition, it has been proposed that a clan CA cysteine protease forms part of the apoptosis molecular pathway in P. falciparum [80], and calpain activity has been linked to apoptosis in E. histolytica [84]. There is no reason to exclude the possibility that cascades involving more than one clan of cysteine proteases may operate in the execution of apoptosis in some parasitic protozoans…”

“…Despite their lack of mitochondria, the parasitic protozoans Giardia intestinalis, Entamoeba histolytica , Tritrichomonas foetus and T. vaginalis can be induced to exhibit some apoptosis features, such as chromatin condensation, translocation of phosphatidylserine to the outer leaflet of the plasma membrane, DNA fragmentation, and the formation of apoptotic bodies which, in some cases, are inhibited by pancaspase inhibitors [60,84,102]. This also brings into question theories that associate the origin of apoptosis in eukaryotes with the progenitors of mitochondria.…”