inhibition. Cells incubated with PD153035 at concentrations 50, 75 and 100 nM clearly showed a significant decrease in radiation- induced NFkB DNA binding activity and mRNA expression similar to the cells incubated with EKB-569 (Figure 2 E&F). In order to determine whether EKB-569 selectively targets NFkB or the global transcription machinery in general, we analyzed the effect of EKB-569 on IR-modulated AP-1 and SP-1 transcription factors. SCC-4 cells mock-irradiated, treated with EKB-569 (0.5–5.0 mg), exposed to IR or, treated with EKB-569 (0.5–5.0 mg) and then exposed to IR were examined for AP-1 and SP-1 DNA binding activity (Figure 1 F&G). In contrast to the NF- kB pathway response, EKB-569 by itself, without radiation exposure, fails to inhibit the constitutive levels of AP-1 DNA- binding activity. On the other hand, with regard to SP-1, EKB- 569 inhibits its activity at the lower concentrations of 1 and 2 ug, but not at the higher (5 ug) concentration. More interestingly, with the addition of EKB-569 further increased the activation of AP-1 and the SP-1 induced by IR exposure. These results confirmed that the mechanism of EKB-569-mediated radiosensitization is acting specifically through NF-kB pathway. EKB-569 inhibits IR-induced transcriptional modulation of NFkB signal transduction and pathway molecules in HNSCC cells To further to substantiate our findings of IR-induced NFkB activation and EKB-569 associated selective targeting, SCC-4 cells mock-irradiated, exposed to IR or pretreated with EKB-569 (5.0 mg) and then exposed to IR were examined for transcriptional changes in 88 NFkB signal transduction and downstream target genes (Figure S1). Compared to mock-IR controls, IR exposure upregulated 74 genes, down regulated two genes, while having no effect on the expression of 12 genes. Though, originally we intended to classify the gene expression implying less stringent (overall) and stringent ($2 fold) criteria, there is only one gene, Myd88 showed less than 2 fold (1.4) while remaining 73 genes showed significant ($2 fold) upregulation compared to untreated control. Conversely, EKB-569 pre-treatment profoundly inhibited 72 of 74 IR-induced genes in this setting (Figure 3). Interestingly, expression of two genes, TLR4 and Ppm1A were significantly increased with EKB-569. A plethora of scientific literature demonstrates the functional significance of these NFkB-dependent signaling and target molecules in tumor cell radioresistance suggesting that inhibitory approaches of these molecules may benefit radiosensitization. EKB-569 regulates NFkB dependent downstream Birc 1, 2 and 5 and upregulates pro-apoptotic Bax in HNSCC cells QPCR profiling demonstrated a significant inhibition of IR- induced NFkB-dependent downstream pro-survival protein, Birc 2 and 5 upon EKB-569 treatment (Figure 3). In order to confirm the IR-induced modulations and to validate the functional significance of EKB-569-mediated regulation, we investigated whether the transcriptional machinery modulation is in fact translated to the protein level. First, immunoblotting analysis confirmed the involvement of post-translational modification of IkB in IR- induced NFkB. Further, we observed a significant setback of IR- inhibited IkBa levels upon EKB-569 treatment. This correlated well with induced NFkB activity data (Figure 1 A–D). Compared to mock-IR controls, we observed a significant induction of BIRC 2 and 5 levels (Figure 4 A&B) reflecting and correlating well with their mRNA expression levels. More importantly, treatment with EKB-569 completely (P,0.001) inhibited IR-induced BIRC2 and 5 in SCC-4 cells. Though IR did not show induced expression of EKB Radiosensitizes Squamous Cell Carcinoma BIRC 1 in this setting, we observed a conferring inhibition of this protein with EKB-569. Conversely, we observed a significant induction of pro-apoptotic Bax in cells pre-treated with EKB-569. EKB-569 confers radiosensitization in HNSCC cells To identify the efficacy of EKB-569 at the cellular or tissue level of HNSCC radiosensitization, we examined their potential in conferring functional endpoints like cell viability, survival and apoptotic death. First, trypan blue exclusion assay demonstrated that EKB-569 as a stand-alone compound induced dose- dependent inhibition of SCC-4 cell viability with a maximum (P,0.001) inhibition at 5.0 mg concentration (Figure 4C). Simi- larly, unlike the mock-irradiated control, cells exposed IR significantly (P,0.001) inhibited HNSCC cell viability (Figure 4D). More importantly, compared to IR exposed cells, EKB-569 (5.0 mg) treatment significantly (P,0.001) conferred IR- inhibited cell viability. Substantiating our cell viability data, MTT analysis revealed a dose dependent inhibition of metabolic activity with EKB-569 treatment (Figure 4E). To that end, at low concentration (0.5 mg) we did not see any significant inhibition of cell survival. However, with increase in EKB-569 concentration we observed a significant (1.0 mg, P,0.05; 2.0 mg, P,0.01 and 5.0 mg, P,0.001) inhibition of cell survival in this setting. On the other hand, compared to mock-irradsiated, cell exposed to IR showed significant (P,0.01) suppression of cell survival (Figure 4E). Addition of EKB-569 significantly conferred IR-inhibited cell survival in a dose dependent fashion. Even concentrations as low as 0.5 mg significantly conferred IR-induced cell death and we observed a complete inhibition of cell survival in IR-exposed cells with 5.0 mg demonstrating the radiosensitizing potential of EKB- 569 in HNSCC cells. Further, nuclear morphology with dual staining showed bright green chromatin with organized structures in untreated control cells indicating viable cells with normal nuclei (Figure 4F). Where as, cells treated with EKB-569 showed typical apoptotic features of bright orange chromatin with blebbing, nuclear condensation, and fragmentation. We observed a dose dependent increase in apoptosis after 0.5, 1.0, 2.0 and 5.0 mg of EKB-569. Consistent with our cell viability and survival data, we observed an induced cell death in cells exposed to IR with bright orange chromatin with blebbing, nuclear condensation, and fragmentation. More importantly, compared to IR alone, cells pre-treated with EKB-569 (5.0 mg) and exposed to IR showed extensive apoptotic characteristics and demonstrated a radiosen- sitizing potential in HNSCC cells (Figure 4F). EKB-569 targets IR-induced NFkB- regulated radiosensitization To further identify whether targeting IR-induced NFkB orchestrates EKB-569-induced radiosensitization in HNSCC cells, we adopted two approaches. First, we determined whether IR- induced NFkB regulates induced radioprotection in SCC-4 cells. To achieve this we investigated the alterations in cell viability, survival and death after muting IR-induced NFkB. Ecotopic expression of IR-induced NFkB was inhibited by transient transfection of DIkBa. Knocking-out IR-induced NFkB was confirmed with EMSA (Figure 5A&B). Compared to vector controls, knocking out IR-induced NFkB with DIkBa significantly (P,0.001) conferred IR-inhibited cell survival (Figure 5C), cell viability (Figure 5D) and enhanced IR-induced cell death (evident with bright orange chromatin with blebbing, nuclear condensa- tion, and fragmentation) dictating the role of IR-induced NFkB in radioresistance. Next, to identify that EKB-569 induced radiosen- sitization occurs at least in part by targeting IR-induced NFkB, p50/p65 over-expressed SCC-4 cells were treated with EKB-569 PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29705