APX-115A, a pan-NADPH Oxidase Inhibitor, Induces Caspase-dependent Cell Death by Suppressing NOX4-ROS Signaling in EBV-infected Retinal Epithelial Cells

Abstract

ABSTRACT Purpose Epstein-Barr virus is a γ-herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in malignancies such as Burkitt’s lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells. Methods Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology. Results We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor-induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK. Conclusion Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling.