Hassan Chaachouay1, Birgit Fehrenbacher2, Mahmoud Toulany3, Martin Schaller2, Gabriele Multhoff4, H Peter Rodemann5. 1. Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Germany; Department of Radiation Oncology, Technische Universität München (TUM), and HMGU CCG - Innate Immunity in Tumor Biology, Germany; Section of Radiation Oncology, Vetsuisse Faculty, University of Zürich, Switzerland. 2. Department of Dermatology, University of Tuebingen, Germany. 3. Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Germany. 4. Department of Radiation Oncology, Technische Universität München (TUM), and HMGU CCG - Innate Immunity in Tumor Biology, Germany. 5. Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Germany. Electronic address: hans-peter.rodemann@uni-tuebingen.de.
Abstract
BACKGROUND AND PURPOSE: Blocking of the autophagy-signaling has the potential to improve cancer therapy. In the present study, the role of autophagy for radioresistance of human tumor cells was tested under clinically relevant hypoxia (1% O2). MATERIALS AND METHODS: Non-small cell lung cancer cell lines A549 and H460, head and neck squamous cell carcinoma FaDu, colon carcinoma cell line HCT116 and mouse-embryo-fibroblasts were analyzed under normoxic (21% O2) and hypoxic (0.01% and 1% O2) conditions with respect to clonogenic cell survival and hypoxia-induced autophagy. Immunofluorescence and electron microscopy were used to monitor the autophagy process and Western blotting of LC3, AMPK, and BNIP3 was applied to analyze autophagy signaling. RESULTS: Clinically relevant hypoxia stimulated autophagy in tumor cells as indicated by enhanced LC3-I to LC3-II conversion. Furthermore, hypoxia stimulated autophagy was approved by Immunofluorescence staining and electron-microscopy analysis of autophagosome vacuoles. Preconditioning of tumor cells to moderate-hypoxia increased their radioresistance that was significantly reversed following pretreatment with autophagy inhibitor, chloroquine. Using siRNA against AMPK as well as AMPK deficient cells, autophagy stimulation by 1% O2 was shown to be AMPK-independent. However, a correlation between the expression of BNIP3 and autophagy-stimulation was observed under this condition. CONCLUSION: Under clinically relevant hypoxia (1% O2) the stimulation of autophagy mediates resistance of hypoxic tumor cells to ionizing radiation, which is independent of AMPK signaling.
BACKGROUND AND PURPOSE: Blocking of the autophagy-signaling has the potential to improve cancer therapy. In the present study, the role of autophagy for radioresistance of humantumor cells was tested under clinically relevant hypoxia (1% O2). MATERIALS AND METHODS:Non-small cell lung cancer cell lines A549 and H460, head and neck squamous cell carcinoma FaDu, colon carcinoma cell line HCT116 and mouse-embryo-fibroblasts were analyzed under normoxic (21% O2) and hypoxic (0.01% and 1% O2) conditions with respect to clonogenic cell survival and hypoxia-induced autophagy. Immunofluorescence and electron microscopy were used to monitor the autophagy process and Western blotting of LC3, AMPK, and BNIP3 was applied to analyze autophagy signaling. RESULTS: Clinically relevant hypoxia stimulated autophagy in tumor cells as indicated by enhanced LC3-I to LC3-II conversion. Furthermore, hypoxia stimulated autophagy was approved by Immunofluorescence staining and electron-microscopy analysis of autophagosome vacuoles. Preconditioning of tumor cells to moderate-hypoxia increased their radioresistance that was significantly reversed following pretreatment with autophagy inhibitor, chloroquine. Using siRNA against AMPK as well as AMPK deficient cells, autophagy stimulation by 1% O2 was shown to be AMPK-independent. However, a correlation between the expression of BNIP3 and autophagy-stimulation was observed under this condition. CONCLUSION: Under clinically relevant hypoxia (1% O2) the stimulation of autophagy mediates resistance of hypoxic tumor cells to ionizing radiation, which is independent of AMPK signaling.
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