inducing inhibition of apoptosis, proliferation, maturation,clonal expansion, invasion, and metastasis. NFkB is a member of the c-rel proto-oncogene family found within the promoter and enhancer region of a wide variety of genes involved in proliferation, cell cycle control [6,7], oncogenic activation [8], cell growth, differentiation and metastasis [9,10]. NFkB is retained in the cytoplasm by association with the inhibitory protein IkB. On phosphorylation, IkB is ubiquitinated and subsequently degraded by the 26S proteasome, resulting in the liberation of NFkB. NFkB can then enter into the nucleus to regulate the expression of downstream genes. Elevated NFkB activity has been linked with tumor resistance to chemotherapy and IR [11] in a number of cancer types, including head and neck cancer [12]. Conversely, inhibition of NFkB favors pro-apoptotic processes, decreases growth and clonogenic survival [13–15] and enhances chemo/radiosensitivity [16–20]. In addition to this persistant activation of growth-promoting signaling pathways, development of HNSCC also involves the accumulation of genetic and epigenetic alterations in tumor-suppressor proteins.. The activation of EGFR is a frequent event in HNSCC, and has provided the molecular basis for current efforts aimed at evaluating the clinical activity of EGFR inhibitors in HNSCC [21,22]. However, to date, the role of EGFR-dependent NFkB in the functional orchestration of HNSCC progression and metastasis is poorly realized [22,23]. Since NFkB is able to regulate more than 150 genes, and is able to functionally orchestrate many steps in carcinogenesis, tumor progression and metastasis, it is important to delineate the efficacy of potential EGFR-TK inhibitors that target the NFkB-dependent HNSCC cell survival advantage. The two most commonly employed strategies in drug development are introducing covalent (irreversible) binding of the drug target and and broadening the affected receptor tyrosine kinase targets of the drug within the cell. Currently, the second generation of EGFR TKI compounds is emerging from the drug developmental pipeline and being introduced into clinical trials. Many of these second-generation compounds form tighter covalent bonds with their target, which should theoretically increase their effectiveness by prolonging the inhibition of EGFR signaling to the entire lifespan of the drug-bound receptor molecule. In cell culture systems, such irreversibly binding TKIs can effectively kill cells that have acquired resistance to first- generation TKIs [24]. As per the other common theme of drug development, second-generation EGFR TKI have been developed that, in addition to blocking EGFR signaling, target multiple kinases in the ErbB family. The signaling network that emerges from the ErbB family of transmembrane TK receptors (of which EGFR is a member) is large, interconnected, and redundant, with many possible routes between the ligand at the cell surface and the message destination within the nucleus [25]. It is this diversity in possible signal transduction routes that allows a cell to have flexibility and, in the case of cancer cells treated with anticancer agents, facilitates resistant cell clones that bypass the inhibited receptor [26]. Blocking multiple signaling pathways with either a combination of agents or a single but multi-targeted agent has been synergistic in its effects in preclinical models [27]. Second- generation EGFR TKIs have been developed that target additional members of the ErbB family or ‘other downstream or parallel pathways such as the NFkB pathway’. EKB-569 (Pelitinib; WAY-172569), a 4-Dimethylamino-but-2-enoic acid [4-(3-chloro-4-flurophenylamino)-3-cyano-7ethoxy-quinolin-6-yl]- amide is one such second generation irreversibly-binding inhibitor of EGFR TK activity [28]. In this study, we examined the efficacy of EKB-569 in inhibiting ionizing radiation (IR)-induced NFkB activity, in modulating the transcription of 88 NFkB-dependent EKB Radiosensitizes Squamous Cell Carcinoma signal transduction molecules, in activating translation of NFkB- mediated downstream Birc1, 2 and 5 protein, in reducing cell viability, and metabolic activity and apoptosis. Further, we delineated the selective targeting of IR-induced NFkB through EKB-569 and its direct influence in HNSCC cell-fate. Materials and Methods Cell Culture Human tongue squamous cell carcinoma SCC-4 and SCC-9 cells were obtained from ATCC (Manassas, VA) and maintained as monolayer cultures in DMEM/F-12 50/50 (Mediatech Inc., Herndon, VA) growth medium supplemented with 1.5 g/L sodium bicarbonate, 2 mM L-glutamine, 15 mM HEPES, 1% NEAA, 1% MEM vitamins, 5000 I.U/ml penicillin/5000 mg/ml streptomycin, 1% sodium pyruvate, and 10% FBS (Invitrogen, Carlsbad, CA). For passage and for all experiments, the cells were detached using trypsin (0.25%)/EDTA (1%), resuspended in complete medium, counted (Countess, Invitrogen) and incubated in a 95% air/5% CO2 humidified incubator. Irradiation experiments SCC-4 and SCC-9 cells were exposed to 2Gy using Gamma Cell 40 Exactor (Nordion International Inc, Ontario, Canada) at a dose rate of 0.81Gy/min. Irradiated cells were examined for IR- induced alterations in NFkB signal transduction, selective yet, sustained NFkB activity, NFkB’s role in survival advantage and to identify the efficacy of EKB-569 on IR-induced NFkB dependent HNSCC progression. Mock irradiated cells were treated identical except that the cells were not subjected to IR. Irradiated cells were incubated at 37uC for additional 1, 3, 6, 24, 48 and 72 h. All experiments were repeated at least three times in each group. Plasmid preparation and DNA Transfection Transient transfection of NFkB p65 and p50 subunits was carried out by the lipofection method using EffecteneTM reagent (Qiagen, Inc., Valencia, CA) as described in our earlier studies [29]. NFkB inhibition was achieved using transient transfection of S32A/S36A double mutant IkBa (DIkBa, Upstate biotechnology, Lake Placid, NY) as reported in our earlier studies [29] . The mutated form of IkBa with a serine-to-alanine mutation at residues 32 and 36 does not undergo signal-induced phosphory- lation and thus remains bound to NFkB subsequently preventing nuclear translocation and DNA binding. After 18 h, transfection medium was replaced with growth medium before IR. Electrophoretic Mobility Shift Assay (EMSA) Nuclear protein extraction and electrophoretic mobility shift assay for NFkB, AP-1 and SP-1 were performed as described in our earlier studies [29]. Autoradiograms were overexposed in order to reveal the low inhibitory effects that were below the constitutive level. Densitometry analysis was performed using a BioRad Multi-Analyst software package with an integrated density program. Group-wise comparisons were made using ANOVA with Tukey’s post-hoc correction. A P value of ,0.05 is considered statistically significant. For the competition assay, the nuclear extract was pre-incubated with unlabeled homologous NFkB oligonucleotide followed by addition of [c-32P]-ATP labeled NFkB probe. Supershift analysis was performed as described earlier [29]. Immunoblotting Total protein extraction and immunoblotting were performed as described in our earlier studies [29]. Rabbit polyclonal anti- IkBa, Birc1, 2, 5 or Bax antibody (Santa Cruz) were used to detect PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29705