AMPK-independent autophagy promotes radioresistance of human tumor cells under clinical relevant hypoxia in vitro.

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). 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. 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. 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.