Immunopharmacology and inflammation Ethyl 3 0 ,4 0 ,5 0 -trimethoxythionocinnamate modulates NF-kB and Nrf2 transcription factors Sarvesh Kumar a,b , Brajendra K. Singh c , Ashok K. Prasad c , Virinder S. Parmar c , Shyam Biswal b , Balaram Ghosh a,n a Molecular Immunogenetics Laboratory, CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India b Department of Environmental Health Science, Johns Hopkins School of Public Health, Baltimore, MD 21205, USA c Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India article info Article history: Received 28 April 2012 Received in revised form 6 December 2012 Accepted 11 December 2012 Available online 20 December 2012 Keywords: Endothelial cell Cell adhesion molecule Anti-oxidant NF-kB Nrf2 and IkBa abstract Recently, we identified a novel cinnamate analog, ethyl 3 0 ,4 0 ,5 0 -trimethoxythionocinnamate (ETMTC) as a potent inhibitor of cell adhesion molecules (CAMs), such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. However, its mechanism of action has not been elucidated so far. Since, nuclear factor-kappa B (NF-kB) is the major transcription factor involved in the regulation of ICAM-1, VCAM-1 and E-selectin expression, we determined the status of NF-kB activation in ETMTC treated human endothelial cells. Here, we demonstrate that ETMTC inhibits TNF-a- induced nuclear translocation and activation of NF-kB by inhibiting phosphorylation and degradation of IkBa. The inhibition of IkBa phosphorylation and degradation by ETMTC was found to be due to its ability to inhibit IkB kinase activity. In addition, oxidative stress is known to regulate NF-kB activation through TNF-a signaling cascade, therefore, we examined the effect of ETMTC on TNF-a-induced reactive oxygen species generation. We observed that ETMTC significantly inhibits TNF-a-induced reactive oxygen species generation in endothelial cells. To further elucidate the anti-oxidant potential of ETMTC, we examined its effect on induction of anti-oxidant genes viz. glutamate–cysteine ligase, modifier subunit (GCLM), heme oxygenase-1 (HO1) and NAD (P)H:quinone oxidoreductase 1 (NQO1) in human bronchial epithelial cells. Interestingly, ETMTC significantly induces the anti-oxidant genes viz. GCLM, HO1 and NQO1 by activating nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Thus, ETMTC could be useful towards developing potent anti-inflammatory molecules. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Inflammation is a hallmark of many diseases like asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, rheumatoid arthritis, atherosclerosis and cancer (Baldwin, 1996; Springer, 1994; Suchard et al., 2010). It is mediated by the expres- sion of cell adhesion molecules viz. ICAM-1, VCAM-1 and E-selectin on leukocytes. Pro-inflammatory cytokines like TNF-a, IL-1b or bacterial lipopolysaccharides (LPS) activate redox sensitive tran- scription factor, NF-kB and induce the surface expression of CAMs (Ciencewicki et al., 2008; Garg and Aggarwal, 2002; Ghosh and Karin, 2002). NF-kB is sequestered in the cytoplasm with its inhibitory molecule (IkB). Rapid phosphorylation and degradation of IkBa allows NF-kB to translocate into the nucleus and regulate transcription of the targeted genes. Emerging evidence suggests that TNF-a signaling causes oxidative stress by producing reactive oxygen species which in turn activates NF-kB (Baeuerle and Baltimore, 1996; Baldwin, 1996, 2001; Rahman and MacNee, 1998). Antioxidants and free radical quenchers have also been shown to block the NF-kB activation (Verhasselt et al., 1999; Wakamatsu et al., 2005; Weber et al., 1995). Nrf2 is a basic-leucine zipper (b-ZIP) transcription factor present in the cytoplasm; upon its activation in response to inflammatory stimulus, environmental toxicant, oxidative and electrophilic stress, it detaches from its cytosolic inhibitor, Kelch-like ECH-associated protein 1 (Keap1) and translocates to nucleus and induces the expression of several anti-oxidant enzymes. (Nguyen et al., 2009; Nguyen et al., 2003; Nioi et al., 2003; Surh, 2008; Thimmulappa et al., 2002). Emerging literature suggests that there is a crosstalk between NF-kB and Nrf2. Sulforaphane, a well known Nrf2 activator inhibits NF-kB in various cell lines (Heiss and Gerhauser, 2005; Heiss et al., 2001; Surh, 2008; Xu et al., 2005). Similarly, curcumin, a well docu- mented NF-kB inhibitor also activates Nrf2 in several types of cultured cells. It possesses strong anti-inflammatory and antiox- idant activities (Balogun et al., 2003; Pugazhenthi et al., 2007). Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/ejphar European Journal of Pharmacology 0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.12.004 n Corresponding author. Tel.: þ91 11 2766 2580; fax: þ91 11 2766 7471. E-mail address: bghosh@igib.res.in (B. Ghosh). European Journal of Pharmacology 700 (2013) 32–41