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A2682 - Strategies to Overcome Endotoxin Induced Radiotherapy Resistance in Non-Small Cell Lung Cancer Cells In Vitro
Author Block: M. Gökyildirim1, F. Subtil2, U. Grandel1, F. Grimminger1, R. Engenhart-Cabillic2, W. Seeger3, U. Sibelius1, K. Hattar1; 1Justus-Liebig-University Giessen, Internal Medicine IV/V Giessen, Universities of Giessen and Marburg Lung Center (UGMLC), The German Center for Lung Research (DZL), Giessen, Germany, 2Philipps-University Marburg, Universities of Gießen and Marburg Lung Center (UGMLC), The German Center for Lung Research (DZL), Marburg, Germany, 3Justus-Liebig-University Giessen, Internal Medicine II Giessen, Universities of Giessen and Marburg Lung Center (UGMLC), The German Center for Lung Research (DZL), Giessen, Germany.
RATIONALE: Lung cancer (LC) is the leading cause of cancer death. Radiotherapy is a component of tumor therapy alone or in combination with surgery, chemotherapy or targeted therapy, like anti-epidermal growth factor receptor (EGFR)-strategies. Pulmonary infections are common complications in patients with LC and worsen the prognosis. These patients often show an acquired resistance to radiotherapy. Gram-negative bacteria are common pathogens in patients with LC. Their virulence is caused by cell wall components, especially by lipopolysaccharides (LPS). LPS is known to activate multiple pathways in pulmonary epithelial cells. But it is not clear how LPS could induce radiotherapy resistance in non-small cell lung cancer (NSCLC) cells. METHODS: NSCLC cell lines with different EGFR-status (A549/wildtype, H1975/mutated and H520/deficient) were pre-incubated with LPS and irradiated. Colony formation assays were performed to quantify the survival. The plating efficiency and the survival rate were calculated. Additionally, protein analyses were performed. Up-regulated target proteins were inhibited in LPS-treated cells before irradiation. Also, DNA-double strain breaks and interleukin (IL)-6 expression were quantified. RESULTS: In all cell lines, ionizing radiation induced a dose-dependent reduction in clonogenic survival. However, in LPS pre-treated cells the effect of ionizing radiation was severely attenuated and the sensitivity to radiotherapy was remarkably decreased in the human cell lines H1975 and A549, while in H520 the radio-sensitivity was unchanged. This effect was most pronounced when 10 µg/ml LPS were used. In H1975 and A549 cells the survival fraction increased significantly in the presence of LPS. The proteome arrays show an upregulated phosphorylation of the cAMP response element-binding protein (CREB) and EGFR after the LPS treatment and radiation. Pharmacological intervention with an irreversible EGFR tyrosine kinase inhibitor restored radio-sensitivity in LPS pre-treated cells. The inhibition of CREB phosphorylation by targeting the CREB-binding protein (CBP) was equally effective. This outcome could be reproduced when DNA-double strain breaks were quantified as a surrogate for irradiation-dependent cellular damage. Surprisingly, the CBP inhibition leads to an increase in IL-6 expression. CONCLUSION: LPS induces radiotherapy resistance by EGFR- and CREB-dependent mechanisms. This means that bacterial infections could directly affect response to radiotherapy in NSCLC. This was shown by LPS treatment of H1975 and A549 cells which induces a radiotherapy resistance. The inhibition of possible target proteins like CREB has an influence on DNA-double strain breaks and IL-6 expression. Finally, these findings could have implications for overcoming radiotherapy resistance in NSCLC.