EKB Radiosensitizes Squamous Cell Carcinoma Next, to demonstrate that the inhibition of NFkB signaling pathway is not a EKB-569 compound-specific effect and that the proposed combination (IR and EGFR inhibition) can be carried out on to the clinic with any other EGFR compound, we incubated the SCC-4 cells with other commonly used irreversible EGFR blockers, afatinib and neratinib (HKI-272). Afatinib and neratinib dose-dependently inhibit NF-kB DNA-binding activity (Figure 2 A, B, C & D). The inhibition of NFkB was found to be persistent up to at least 72 h (Figure 2H). To further validate whether EKB-569 directly inhibits NFkB activity, we examined the inhibitory effect on the activity of unpstream kinases. Data presented in Figure 2G shows EKB-569 and other related EGFR inhibitors, afatinib (300 nM) and neratinib (200 nM) significantly block the expression of IR-induced upstream IkB kinase beta (IKK-b). Additionally, we confirmed that EKB-569-mediated inhibition of NF-kB is EGFR-dependent. EGFR-knockdown experiments with a widely used specific EGFR inhibitor, PD153035 were performed to confirm the EGFR-mediated NFkB Figure 2. Effect of EGFR inhibitors on NFkB DNA binding activity, EGFR mRNA and, EGFR and IKKb protein levels. (A) Representative autoradiogram showing the NFkB-DNA binding activity in the nuclear extracts of human SCC-4 cells exposed to IR (2Gy) or treated with 50, 100 or 200 nM HKI-272 (neratinib) prior to IR exposure. Neratinib treatment significantly inhibited IR-induced NFkB DNA binding activity (Left panel). Representative autoradiogram showing the NFkB-DNA binding activity in human SCC-4 cells exposed to 50, 100 or 200 nM neratinib (Right panel). Compared to the mock-IR cells, neratinib induced a dose-dependent suppression of NFkB activity in these cells. (B) Representative autoradiogram showing the NFkB-DNA binding activity in human SCC-4 cells exposed to IR with or without Neratinib (200 nM) and harvested after 1, 3, 6, 24, 48 and 72 h. Neratinib persistently inhibited IR-induced NFkB-DNA binding activity at all time points investigated. (C) Representative autoradiogram showing the NFkB-DNA binding activity in human SCC-4 cells exposed to 100, 200 or 300 nM afatinib. Compared to the mock-IR cells, afatinib induced a dose-dependent suppression of NFkB activity (Left panel). Representative autoradiogram showing the NFkB-DNA binding activity in SCC-4 cells exposed to IR or treated with 100, 200 or 300 nM afatinib and exposed to IR. Afatinib treatment significantly inhibited IR-induced NFkB DNA binding activity (Right panel). (D) Representative autoradiogram showing the NFkB-DNA binding activity in human SCC-4 cells exposed to IR with or without afatinib (300 nM) and harvested after 1, 3, 6, 24, 48 and 72 h. Afatinib treatment persistently inhibited IR-induced NFkB-DNA binding activity at all time points investigated. (E) Representative autoradiogram showing the NFkB-DNA binding activity in human SCC-4 cells exposed to IR or treated with 50, 75 or 100 nM PD 153035 hydrochloride (a potent EGFR-TK inhibitor) and exposed to IR. PD153035 treatment induced a significant dose-dependent inhibition of IR-induced NFkB DNA binding activity. (F) Real-time QPCR analysis showing EGFR mRNA levels in SCC-4 cells mock- irradiated, exposed to 2Gy and in cells treated either with EKB-569 (5.0 mg) or PD153035 (50 nM) and exposed to IR. (G) Immunoblot showing complete suppression of radiation induced EGFR and IKKb levels in SCC-4 cells pretreated with EKB-569 (5.0 mg), afatinib (300 nM), neratinib (200 nM) or PD153035 (75 nM). (H) QPCR analysis showing complete and sustained (up to 72 h) suppression of radiation induced EGFR transcriptional levels in SCC-4 cells treated with either afatinib (300 nM) or neratinib (200 nM). doi:10.1371/journal.pone.0029705.g002 PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29705