Phosphorylated IkBa Predicts Poor Prognosis in Activated B-Cell Lymphoma and Its Inhibition with Thymoquinone Induces Apoptosis via ROS Release Azhar R. Hussain 1. , Shahab Uddin 1. , Maqbool Ahmed 1 , Fouad Al-Dayel 2 , Prashant P. Bavi 1 , Khawla S. Al- Kuraya 1,3 * 1 Human Cancer Genomic Research, Research Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, 2 Department of Pathology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, 3 Al-Faisal University, Riyadh, Saudi Arabia Abstract Activated B-cell lymphoma (ABC), one of the three subtypes of Diffuse Large B-cell Lymphoma (DLBCL) has the worst survival rate after upfront chemotherapy and is characterized by constitutively activated NFkB. We therefore studied the role of NFkB In a cohort of clinical DLBCL samples and ABC cell lines. In our clinical tissue microarray cohort of DLBCL samples, p-IkBa was detected in 38.3% of ABC DLBCL and was an independent prognostic marker for poor survival. In vitro, we found that Thymoquinone (TQ), a natural compound isolated from Nigella sativa caused release of ROS in ABC cells. TQ- mediated release of ROS in turn inhibited NFkB activity by dephosphorylating IkBa and decreased translocation of p65 subunit of NFkB in the nuclear compartment in ABC cell lines. This led to inhibition of cell viability and induction of mitochondrial dependent apoptosis in ABC-DLBCL cell lines. Additionally, TQ treatment also caused up-regulation of death receptor 5 (DR5), however, up-regulation of DR5 did not play a role in TQ-induced apoptosis. Finally, combination of sub- optimal doses of TQ and TRAIL induced efficient apoptosis in ABC-DLBCL cell lines. These data show that p-IkBa can be used as a prognostic marker and target for therapy in this aggressive sub-type of DLBCL and TQ may play an important role in the management of DLBCL in the future. Citation: Hussain AR, Uddin S, Ahmed M, Al-Dayel F, Bavi PP, et al. (2013) Phosphorylated IkBa Predicts Poor Prognosis in Activated B-Cell Lymphoma and Its Inhibition with Thymoquinone Induces Apoptosis via ROS Release. PLoS ONE 8(3): e60540. doi:10.1371/journal.pone.0060540 Editor: Jose Angel Martinez Climent, University of Navarra, Center for Applied Medical Research, Spain Received December 8, 2012; Accepted February 27, 2013; Published March 28, 2013 Copyright: ß 2013 Hussain et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Kkuraya@kfshrc.edu.sa . These authors contributed equally to this work. Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma accounting for 30–40% of all lymphomas. The treatment of DLBCL has been revolutionized over the last decade with the addition of Rituximab, an anti-CD20 monoclonal antibody in combination with CHOP [1], however, this disease still remains refractory to treatment in 50% of cases [2]. Gene expression studies performed have been able to identify three distinct groups of DLBCL based on their origin at different stages of differentiation[3]. Of the three groups of DLBCL, activated B cell lymphoma (ABC) tends to have a poor 5 year survival rate of 34%[4,5] as compared to germinal center B-cell (59%) and primary mediastinal B-cell lymphoma (PMBCL) (64%). The hallmark of ABC subtype of DLBCL is activation of the NFkB survival pathway that allows the malignant cell towards plasma cell differentiation [6]. Activation of NFkB pathway occurs when IkBa, an inhibitor of NFkB is degraded by either proteasomal degradation or ubiquitination allowing NFkB to enter the nucleus and exerts its transcriptional activation on growth factors such as interleukins and pro-survival and anti-apoptotic proteins such as Bcl-2, Bcl-Xl, XIAP and Survivin[7,8,9]. Thymoquinone (TQ) is a naturally occurring compound that is extracted from Nigella sativa Linn [10]. TQ has been shown to possess anti-inflammatory, anti-oxidant and anti-neoplastic activ- ity [11]. TQ has been shown to possess antitumor activities against a broad spectrum of cancer cells, including colon, ovarian, and myeloblastic leukemia[11,12,13]. The exact mode of action of TQ is not known, however, it has been shown that TQ induces its anti- tumor effect via release of reactive oxygen species (ROS)[14]. Recently, it has been shown that TQ enhances the effect of doxorubicin and oxaliplatin in various cancers[15,16]. In the present study, we first examined the expression of p-IkBa and its association with clinical and pathological parameters in a cohort of ABC-DLBCL clinical samples in a tissue microarray format. We next investigated the role of NFkB pathway on cell survival in ABC cell lines. We also demonstrated the mode of action of TQ for induction of apoptosis and found that TQ inactivated the NFkB pathway via release of ROS. Finally, in this study, we evaluate the combination therapy of TQ and TRAIL in induction of apoptosis in ABC cell lines. Materials and Methods Ethics Statement The Institutional Review Board (IRB) and Research Advisory Council (RAC) of KFSHRC approved this study: RAC # 2060008. A waiver of consent was obtained from IRB and ethics PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e60540