EKB Radiosensitizes Squamous Cell Carcinoma Figure 5. IR-induced NFkB regulates radioresistance in HNSCC cells. (A) Representative autoradiogram of EMSA analysis showing complete muting of NFkB DNA binding activity in IR-induced or NFkB overexpressed cells with DIkBa. (B) Densitometric analysis of NFkB-DNA binding activity showing significant NFkB silencing with DIkBa and significant activation with p50/p65 transfection with NFkB over expression vectors, p50 and p65. (C) Histograms showing the results of MTT analysis in p50/p65 over-expressed cells treated with EKB-569 (5.0 mg). NFkB over-expression robustly induced SCC-4 cell survival. Conversely, treating NFkB over-expressed cells with EKB-569 completely (P,0.001) inhibited NFkB-induced SCC-4 cell survival. Like-wise, muting NFkB (with DIkBa) completely inhibited IR-induced cell survival. (D) Histograms showing cell viability in NFkB muted cells exposed to IR or NFkB overexpressed cells treated with EKB-569. Silencing NFkB significantly inhibited IR-induced cell viability. Like-wise, treating NFkB overexpressed cells with EKB-569 (5.0 mg) completely inhibited NFkB-induced cell viability. (E) Nuclear morphology with dual staining showing typical yet increased apoptotic characteristics in NFkB muted cells exposed to IR. NFkB overexpressed cells displayed chromatin with organized structures indicating good viability with normal nuclei. However, treatment with EKB-569 (5.0 mg) significantly inflicted chromatin with blebbing, nuclear condensation, and fragmentation in these NFkB overexpressed cells. doi:10.1371/journal.pone.0029705.g005 delineating that EKB-569 target NFkB and potentiate cell death in this setting. Discussion Primary and acquired resistance to conventional chemotherapy and radiotherapy represent the central therapeutic challenge in oncology today. Resistance may develop through varied mecha- nisms, including increased expression of cellular drug efflux pumps; mutation of the therapeutic target; increased activity of DNA repair mechanisms and altered expression of genes involved in apoptotic pathways. To overcome these resistance mechanisms, conventional cancer treatments are increasingly combined with molecularly targeted therapies. Because cytotoxic and targeted therapies have distinct biologic effects and toxicity profiles, such combinations are both rational and well tolerated. To date, the molecular pathway most frequently targeted in combination with conventional chemotherapy or radiotherapy is that of the EGFR. After activation by binding of the EGF and other natural ligands, EGFR activates prosurvival, pro-angiogenic, and anti-apoptotic pathways that may confer resistance to cytotoxic therapies. Interestingly, all these aforementioned functional pathways are known to be controlled by transcriptional master switch regulator, NFkB that also happens to be a downstream target for EGFR. In PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29705