ekb-569 and Carcinoma--Squamous-Cell

Our earlier studies indicated that ionizing radiation (IR) induces NF-κB-dependent clonal expansion of therapy resistant tumor cells. Herein, we investigated whether mitigation of NF-κB-dependent telomerase activation by EGFR tyrosine kinase inhibitor can enhance IR-induced celling killing. SCC-4 and SCC-9 cells exposed to IR with or without Pelitinib were examined for NF-κB and hTERT transcription using luciferase reporter assays. NF-κB-dependent hTERT transcription was confirmed by either muting NF-κB or by using hTERT constructs lacking NF-κB binding sites. hTERT, mRNA, telomerase activity and cell survival of tumor cells were analyzed using QPCR, TRAP and clonogenic assay, respectively. Pelitinib inhibited IR-induced NF-κB, telomerase activity and hTERT transactivation. Ionizing radiation-induced telomerase activity is regulated at the transcriptional level by triggering TERT promoter activation. Functional NF-κB mediates telomerase activity by binding to the κB binding region in the promoter region of TERT. Elimination of the NF-κB recognition site on telomerase or muting NF-κB compromises IR-induced telomerase promoter activation. We found that Pelitinib inhibited IR-induced TERT transcription, transactivation and telomerase activation in IR-exposed and NF-κB-overexpressed cells. Furthermore, Pelitinib potentiates IR-induced cell killing. Our results strongly suggest that IR-induced NF-κB-mediated cell survival is supported by telomerase activation. We propose that if this pathway can be inhibited with Pelitinib treatment, one could further enhance therapeutic outcome in squamous cell carcinoma.

Topics: Aminoquinolines; Aniline Compounds; Carcinoma, Squamous Cell; Enzyme Activation; ErbB Receptors; Humans; NF-kappa B; Polymerase Chain Reaction; Protein Kinase Inhibitors; Telomerase

EKB-569 (Pelitinib), an irreversible EGFR tyrosine kinase inhibitor has shown potential therapeutic efficiency in solid tumors. However, cell-killing potential in combination with radiotherapy and its underlying molecular orchestration remain to be explored. The objective of this study was to determine the effect of EKB-569 on ionizing radiation (IR)-associated NFκB-dependent cell death. SCC-4 and SCC-9 cells exposed to IR (2Gy) with and without EKB-569 treatment were analyzed for transactivation of 88 NFκB pathway molecules, NFκB DNA-binding activity, translation of the NFκB downstream mediators, Birc1, 2 and 5, cell viability, metabolic activity and apoptosis. Selective targeting of IR-induced NFκB by EKB-569 and its influence on cell-fate were assessed by overexpressing (p50/p65) and silencing (ΔIκBα) NFκB. QPCR profiling after IR exposure revealed a significant induction of 74 NFκB signal transduction molecules. Of those, 72 were suppressed with EKB-569. EMSA revealed a dose dependent inhibition of NFκB by EKB-569. More importantly, EKB-569 inhibited IR-induced NFκB in a dose-dependent manner, and this inhibition was sustained up to at least 72 h. Immunoblotting revealed a significant suppression of IR-induced Birc1, 2 and 5 by EKB-569. We observed a dose-dependent inhibition of cell viability, metabolic activity and apoptosis with EKB-569. EKB-569 significantly enhanced IR-induced cell death and apoptosis. Blocking NFκB improved IR-induced cell death. Conversely, NFκB overexpression negates EKB-569 -induced cell-killing. Together, these pre-clinical data suggest that EKB-569 is a radiosensitizer of squamous cell carcinoma and may mechanistically involve selective targeting of IR-induced NFκB-dependent survival signaling. Further pre-clinical in-vivo studies are warranted.

Topics: Aminoquinolines; Aniline Compounds; Carcinoma, Squamous Cell; Cell Death; Cell Line, Tumor; DNA, Neoplasm; ErbB Receptors; Gamma Rays; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Linear Energy Transfer; Neoplasm Proteins; NF-kappa B; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-rel; Radiation-Sensitizing Agents; Signal Transduction; Transcription, Genetic; Up-Regulation

Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells, via transactivation of the EGF receptor (EGFR). H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. The aim of this study was to develop an 'In-Cell Western' (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling, to enable the role of the EGFR in H. pylori-induced phosphorylation of ERK in epithelial cells to be ascertained. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. H. pylori strains both with, and without a cag PAI, and Helicobacter felis, significantly increased pERK levels in A431 cells. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. A significantly lower reduction was observed with cag+ strains in A431 but not AGS cells. The cag PAI was not necessary for EGFR signal transactivation. These data suggest that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.

Topics: Aminoquinolines; Aniline Compounds; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Epithelial Cells; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Helicobacter Infections; Helicobacter pylori; Humans; MAP Kinase Signaling System; Organic Chemicals; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; Stomach Neoplasms; Transcriptional Activation