Anoikis potential of Entameba histolytica secretory cysteine proteases: Evidence of contact independent host cell death

Mammalian epithelial, endothelial and various other cell types, upon their detachment from the extracellular matrix (ECM) undergo a specialized kind of apoptosis, known as anoikis. Entameba histolytica cysteine proteases have been implicated in degradation of the host ECM, which may induce anoikis in host cells. To explore this hypothesis, supernatant obtained from 2 h in-vitro cultivation of E. histolytica (SRP), was used as a source of cysteine proteases. MDA-MB-231 (human mammary epithelial adenocarcinoma) cells were treated with SRP and their detachment and apoptosis was evaluated. 25 μg/ml (with respect to protein concentration), SRP was found to be the optimal concentration to dislodge over 98% MDA-MB-231 cells from monolayer in 20 min. The detachment was followed by apoptosis of at least 41.2% cells, characterized by caspase-3 dependent inter-nucleosomal DNA fragmentation. The SRP-induced apoptosis was associated exclusively with the detached fraction. Moreover, detachment preceded apoptosis. E-64 (a cysteine protease inhibitor) abolished the SRP-induced detachment as well as inter-nucleosomal DNA fragmentation. Interestingly, SRP induced a 3.21 fold increase in the JNK activity, whilst SP600125 (a JNK inhibitor) blocked the SRP-induced inter-nucleosomal DNA fragmentation. Thus, it was concluded that spontaneously released cysteine proteases of E. histolytica can induce JNK dependent anoikis of MDA-MB-231 cells, which may be implicated in contact independent host cell death during amebiasis.