Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells.

Abstract

Over-expression of nicotinamide adenine dinucleotide phosphate oxidase (Nox) isoform enzymes was recently reported in various cancers including Burkitt’s lymphoma (BL). However, the functions of Nox isoform enzymes in BL remain poorly understood. In this study, Nox isoform expression and the effects of a Nox-specific inhibitor were evaluated in Epstein-Barr virus (EBV)-positive Raji BL cells in comparison with EBV-negative Ramos BL cells. To evaluate Nox enzyme expression in Raji and Ramos BL cells, polymerase chain reaction (PCR) and western blot analysis were performed. To verify the intracellular signaling mechanism of the Nox inhibitor-induced apoptosis of Raji cells, WST-1 assay, trypan blue exclusion method, flow cytometry, PCR, western blotting, and bromodeoxyuridine staining were conducted. Experiments using the pan-caspase inhibitor z-VAD, reactive oxygen species scavenger N-acetyl-L-cysteine (NAC), and Bim inhibitor 1 were performed. PCR and western blot results showed that Nox isoform enzymes were highly expressed in EBV-positive BL Raji cells compared with EBV-negative BL Ramos cells. The Nox2 inhibitor induced apoptosis of Raji cells in time- and dose-dependent manners. The Nox2 inhibitor also caused up-regulation of Bim and Noxa, down-regulation of Mcl-1, translocation of Bax, release of cytochrome c, and caspase cascade activation, resulting in apoptosis. Furthermore, z-VAD, NAC, and BI-1 effectively blocked the Nox2 inhibitor-induced apoptosis of Raji cells. Taken together, these results provide a novel insight into the mechanism of Nox inhibitor-induced apoptosis and evidence for Nox as a therapeutic target to treat EBV-positive malignancies.