Elucidation of intracellular signaling pathways required for cell death in Caco2 cells induced by Entamoeba histolytica

Abstract

Entamoeba histolytica, which causes amoebic colitis and occasional liver abscesses in humans, can induce host cell death through apoptosis and necrosis. Recently, we have demonstrated that E. histolytica can induce cell death in neutrophils via diphenyleneiodonium (DPI)-sensitive NADPH oxidase (NOX)-derived reactive oxygen species (ROS). Although there are enzyme systems similar to the phagocyte NADPH oxidase system in many non-phagocytic cell types, the signaling role of NOX-derived ROS in cell death of human colon epithelial cells induced by E. histolytica remains obscure. Also, it has been well known that elevation of intracellular Ca2+ and activation of calpain play important role in Entamoeba-induced cell death. However, detailed signaling mechanism of colon cell death induced by E. histolytica remains to be identified. In this study, we investigated a signaling role of reactive oxygen species (ROS) and calpain in the Entamoeba-triggered cell death model. Incubation of colon epithelial Caco2 tumor cell lines with amoebic trophozoites resulted in intracellular ROS generation, calpain activation, and cell death in a caspase-independent manner. Pretreatment with DPI, an inhibitor of NOX, strongly decreased E. histolytica-induced cell death in Caco2 cells. The knock-down of NOX1 by siRNA significantly inhibited E. histolytica-induced cell death and ROS response in Caco2 cells. We also observed degradation of survival factors such as NFB and STAT3, 5 in Caco2 cells following exposure of the amoeba. Incubation with E. histolytica markedly caused protein dephosphorylation and proteolysis of SHP-1, 2 phosphatases in Caco2 cells. The Entamoeba-induced degradation of survival factors, protein dephosphorylation, and proteolysis of SHP-1, 2 phosphatases were markedly inhibited by calpain inhibitor calpeptin. In addition, knockdown of m-calpain by siRNA blocked E. histolytica-induced cell death, degradation of survival factors, protein dephosphorylation, and proteolysis of SHP-1, 2 phosphatases in Caco2 cells. These results suggest that intracellular ROS generation and/or calpain-mediated degradation of survival factors, protein dephosphorylation, and proteolysis of SHP-1, 2 phosphatases are closely linked to cell death of colon epithelial cells induced by amebic adhesion during the early phase of intestinal amebiasis.