The Role of Reactive Oxygen Species In Apoptosis Induction of Mantle Cell Lymphoma (MCL); Inhibition of NADPH Oxidase 2 Induces Apoptosis In MCL

Abstract 3616

Mantle cell lymphoma (MCL) is an aggressive tumor and is characterized by deregulated growth and resistance to apoptosis. Reactive oxygen species (ROS) are important signal transduction molecules in regulation of cell growth, differentiation and apoptosis. Some of the ROS-generating NADPH oxidase (Nox) family enzymes are involved in neoplastic proliferation. We hypothesized that Nox-mediated generation of intracellular ROS conferred antiapoptotic activity and thus a growth advantage to MCL cells. (1) To investigate whether Nox gene expression might be specific, this study measured the expression levels of Nox genes 1–5 and dual oxidase 1 and 2 in two MCL cell lines (SP49 and SP53) and peripheral blood leukemic cells from a patient with MCL by quantitative, real-time RT-PCR. (2) Effects of antioxidants or small inhibitory RNA (siRNA) on viability of MCL cells were examined by Calcein or MTT assay. (3) Generation of intracellular ROS were measured by FACS analysis after cells were labeled by dichlorofluorescin (DCF). (4) Apoptosis was evaluated by Annexin V assay. (5) Signal transduction was estimated by Western immunoblots. MCL cell viability decreased in dose-dependent manner when cells were exposed to a dietary antioxidant, an inhibitor of flavoprotein-dependent oxidase, diphenylene iodonium (DPI) and vitamin E. RT–PCR analysis revealed that high level Nox2 mRNA expression was observed in all MCL cells, whereas little or no Nox1, Nox3-5 and Duox1-2 mRNAs was detected. Moreover, Nox2 expression in CD19⁺ leukemic MCL cells was significantly higher than normal CD19⁺ B cells (5.1-fold). SiRNA knockdown of Nox2 in these cell lines results in 27% reduction in intracellular ROS as measured by a fluorescent DCF assay, and 22% reduction in cell viability. SiNox2-RNAs inhibited superoxide production in MCL cells, and depletion of ROS by DPI or siNox2-RNAs induced apoptosis (28% or 11%, respectively). DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) was reduced by DPI and siNox2-RNAs. Collectively, these findings suggest that ROS generated by Nox2, at least in part, transmits cell survival signals through the AKT- ASK1 pathway in MCL cells and their depletion leads to apoptosis.