The novel NF-jB inhibitor DHMEQ synergizes with celecoxib to exert antitumor effects on human liver cancer cells by a ROS-dependent mechanism Nadia Lampiasi a,⇑ , Antonina Azzolina a , Kazuo Umezawa b , Giuseppe Montalto c , James A. McCubrey d , Melchiorre Cervello a a Institute of Biomedicine and Molecular Immunology ‘‘Alberto Monroy’’, National Research Council (C.N.R.), Via Ugo La Malfa 153, 90146 Palermo, Italy b Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi, Kohoku-ku, Yokohama, Kanagawa, Japan c Department of Internal Medicine and Specialties, University of Palermo, Via del Vespro 143, 90127 Palermo, Italy d Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, 600 Moye Blvd, Greenville, NC 27858, USA article info Article history: Received 30 August 2011 Received in revised form 7 February 2012 Accepted 7 February 2012 Keywords: DHMEQ Celecoxib NF-jB CD95/CD95L Liver cancer cells abstract In a previous work of ours dehydroxymethyl-epoxyquinomicin (DHMEQ), an inhibitor of NF-jB, was shown to induce apoptosis through Reactive Oxygen Species (ROS) production in hepatoma cells. The present study demonstrated that DHMEQ cooperates with Celecoxib (CLX) to decrease NF-jB DNA bind- ing and to inhibit cell growth and proliferation more effectively than treatment with these single agents alone in the hepatoma cell lines HA22T/VGH and Huh-6. ROS production induced by the DHMEQ–CLX combination in turn generated the expression of genes involved in endoplasmic reticulum (ER) stress and silencing TRB3 mRNA significantly decreased DHMEQ–CLX-induced cell growth inhibition. Moreover, the DHMEQ–CLX combination was associated with induction of PARP cleavage and down-regulation of the anti-apoptotic proteins Bcl-2, Mcl-1 and survivin, as well as activated Akt. CD95 and CD95 ligand expression increased synergistically in the combination treatment, which was reversed in the presence of NAC. Knockdown of CD95 mRNA expression significantly decreased DHMEQ–CLX-induced cell growth inhibition in both cell lines. These data suggest that the DHMEQ–CLX combination kills hepatoma cells via ROS production, ER stress response and the activation of intrinsic and extrinsic apoptotic pathways. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Several strategies have been suggested for the treatment of HCC patients but, unfortunately, it still has a high lethality. Therefore, novel approaches are required to contrast this tumor. The nuclear transcription factor-jB (NF-jB) has been impli- cated in carcinogenesis because it plays a critical role in cell sur- vival, inflammation and cell growth. Recent studies indicate that NF-jB is essential for promoting inflammation-associated cancers and it is therefore a potential target for cancer prevention [1]. Sev- eral reports have indicated that NF-jB is constitutively activated in a variety of cancer cells, including hepatocellular carcinoma [2]. Dehydroxymethyl-epoxyquinomicin (DHMEQ) is a novel NF-jB inhibitor which induces apoptosis and cell-cycle arrest in several cancer cell types [3–5]. We previously demonstrated that DHMEQ promotes ROS generation in human liver cancer cells and that oxi- dative stress induces ER stress response, DNA damage and release of cytochrome c with activation of the caspase cascade [6]. There are two isoforms of cyclooxygenases (COXs), COX-1 and COX-2, the latter being induced by a variety of stimuli. COX-2 is markedly elevated in many types of tumor, including HCC, as selec- tive COX-2 inhibitors (COXIBs) show anti-proliferative and pro- apoptotic effects in human liver cancer cells [7,8] suggesting that COXIBs might be effective in HCC treatment. Accumulating evi- dence suggests that COXIBs inhibit cell proliferation through a COX-2-independent mechanism [9–11]. The molecular mechanism underlying CLX-mediated apoptosis seems to be associated with the induction of ER stress response through calcium [12] and with the down-regulation of the anti-apoptotic protein survivin [13]. CLX induces the expression of functional death receptors, such as CD95, and a rapid down-regulation of myeloid cell leukemia-1 (Mcl-1) protein, suggesting the activation of intrinsic and extrinsic apoptosis pathways in HCC [14]. CLX has been reported to act syn- ergistically with different drugs in promoting the apoptosis of hu- man liver tumor cells [15]. There are two different apoptotic pathways: death receptor and mitochondrial [16,17]. Ligation of cell surface death receptors, including CD95 with its specific ligand, triggers a death receptor apoptotic pathway. Fas-associated Death Domain (FADD) is first recruited to the death receptor, followed by the association with pro-caspase 8, which is activated by cleav- age. Activation of the mitochondrial apoptotic pathway depends on 0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2012.02.008 ⇑ Corresponding author. Tel.: +39 091 6809513; fax: +39 091 6809548. E-mail address: lampiasi@ibim.cnr.it (N. Lampiasi). Cancer Letters 322 (2012) 35–44 Contents lists available at SciVerse ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet