Yadav et al. 10/18/11 1 3-Formylchromone Interacts with Cysteine Residue 38 in p65 and with Cysteine Residue 179 in IκBα Kinase, Leading to Downregulation of NF-κB-Regulated Gene Products and Sensitization of Tumor Cells Vivek R. Yadav 1 , Sahdeo Prasad 1$ , Subash C Gupta 1$ , Bokyung Sung 1 , Sharangdhar S. Phatak 2 , Shuxing Zhang 2 , and Bharat B. Aggarwal 1* 1 Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston 77030, Texas, USA. 2 Integrated Molecular Discovery Laboratory, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston 77030, Texas, USA $ These authors contributed equally *For correspondance: Dr. Bharat B. Aggarwal, at aggarwal@mdanderson.org Tel.: +1 713 794 1817; fax: +1 713 606 3399. ABSTRACT 3-Formylchromone (3-FC) has been associated with anti-cancer potential through a mechanism yet to be elucidated. Because of the critical role of NF-κB in tumorigenesis, we investigated the effect of this agent on the NF-κB activation pathway. Whether activated by inflammatory agents (such as TNF-α or endotoxin), or tumor promoters (such as phorbol ester and okadaic acid), 3-FC suppressed NF-κB activation. It also inhibited constitutive NF-κB expressed by most tumor cells. This activity correlated with sequential inhibition of IκBα kinase (IKK) activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and reporter gene expression. We found that 3-FC inhibited the direct binding of p65 to DNA, and this binding was reversed by a reducing agent, thus suggesting a role for cysteine residue. Furthermore, mutation of cysteine residue 38 to serine of p65 abolished this effect of chromone. This result was also confirmed by a docking study. 3-FC also inhibited IKK activation directly, and reducing agent reversed this inhibition. Further, mutation of cysteine residue 179 of IKK to alanine abolished the effect of the chromone. Suppression of NF-κB activation led to inhibition of anti-apoptotic (Bcl-2, Bcl-xL, survivin, and cIAP-1), proliferative (cyclin D1 and COX-2), invasive (MMP-9, ICAM-1), and angiogenesis (VEGF) gene products and sensitization of tumor cells to cytokines. Thus, the study shows that modification of cysteine residues of IKK and of p65 by 3-FC leads to inhibition of the NF-κB activation pathway, suppression of anti-apoptotic gene products, and potentiation of apoptosis in tumor cells. INTRODUCTION It is now generally accepted that most chronic diseases including cancer are caused by dysregulation of inflammatory pathways. Thus agents that can suppress pro-inflammatory pathways safely and effectively have potential in prevention and treatment of cancer and other diseases. While searching for such agents, we focused on chromones, which have been linked with anti-bacterial, anti-fungal, and anti-tumor activities (1). In particular, we focused on 3- formylchromone, which has been shown to exhibit anti-bacterial (2), anti-inflammatory (3), and anti- tumor activities (2,4-6). How this agent exhibits all these activities is not fully understood, but inhibition of protein tyrosine phosphatase 1B (7), chelation of bivalent cations (8), multidrug resistance (mdr) (5), p56 lck tyrosine kinase (9) and thymidine phosphorylase (10) have been implicated. Because of the critical role of NF-κB pathway in inflammation and tumorigenesis, we postulated that 3-FC mediates its effects through modulation of this pathway. Under normal conditions, NF-κB is present in the cytoplasm as an inactive heterotrimer consisting of p50, p65, and IκBα. However, when activated by various carcinogens, tumor promoters, or pro- inflammatory agents, IKK is activated, thus causing the phosphorylation, ubiquitination and degradation of IκBα by the 26S proteasome. The p65/p50 subunit is then released from the http://www.jbc.org/cgi/doi/10.1074/jbc.M111.274613 The latest version is at JBC Papers in Press. Published on November 7, 2011 as Manuscript M111.274613 Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on April 10, 2016 http://www.jbc.org/ Downloaded from