Involvement of NOX2-derived ROS in human hepatoma HepG2 cell death induced by Entamoeba histolytica

Entamoeba histolytica is an enteric tissue-invasive protozoan parasite causing amoebic colitis and liver abscesses in humans. Amoebic contact with host cells activates intra-cellular signaling pathways that lead to host cell death via generation of caspase-3, cal -pain, Ca2+ elevation, and reactive oxygen species (ROS). We previously reported that various NADPH oxidases (NOXs) are responsible for ROS-dependent death of various host cells induced by amoeba. In the present study, we investigated the specific NOX isoform involved in ROS-dependent death of hepatocytes induced by amoebas. Co -in-cubation of hepatoma HepG2 cells with live amoebic trophozoites resulted in remark-ably increased DNA fragmentation compared to cells incubated with medium alone. HepG2 cells that adhered to amoebic trophozoites showed strong dichlorodihydrofluo-rescein diacetate (DCF-DA) fluorescence, suggesting intracellular ROS accumulation within host cells stimulated by amoebic trophozoites. Pretreatment of HepG2 cells with the general NOX inhibitor DPI or NOX2-specific inhibitor GSK 2795039 reduced Ent -amoeba-induced ROS generation. Similarly, Entamoeba-induced LDH release from HepG2 cells was effectively inhibited by pretreatment with DPI or GSK 2795039. In NOX2-silenced HepG2 cells, Entamoeba-induced LDH release was also significantly in-hibited compared with controls. Taken together, the results support an important role of NOX2-derived ROS in hepatocyte death induced by E. histolytica.