Chaachouay Hassan, Fehrenbacher Birgit, Toulany Mahmoud, Schaller Martin, Multhoff Gabriele, Rodemann H Peter
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.
Department of Dermatology, University of Tuebingen, Germany.
Radiother Oncol. 2015 Sep;116(3):409-16. doi: 10.1016/j.radonc.2015.08.012. Epub 2015 Aug 26.
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.
阻断自噬信号传导有可能改善癌症治疗效果。在本研究中，我们检测了在临床相关低氧条件（1%氧气）下自噬对人肿瘤细胞放射抗性的作用。
在常氧（21%氧气）和低氧（0.01%和1%氧气）条件下，分析非小细胞肺癌细胞系A549和H460、头颈部鳞状细胞癌FaDu、结肠癌细胞系HCT116以及小鼠胚胎成纤维细胞的克隆形成细胞存活率和低氧诱导的自噬。采用免疫荧光和电子显微镜监测自噬过程，并应用LC3、AMPK和BNIP3的蛋白质印迹分析自噬信号传导。
如增强的LC3-I向LC3-II转化所示，临床相关低氧刺激肿瘤细胞中的自噬。此外，低氧刺激的自噬通过自噬体空泡的免疫荧光染色和电子显微镜分析得到证实。将肿瘤细胞预处理至中度低氧可增加其放射抗性，在用自噬抑制剂氯喹预处理后，这种抗性显著逆转。使用针对AMPK的小干扰RNA以及AMPK缺陷细胞，结果表明1%氧气刺激的自噬不依赖于AMPK。然而，在这种条件下观察到BNIP3表达与自噬刺激之间存在相关性。
在临床相关低氧（1%氧气）条件下，自噬的刺激介导低氧肿瘤细胞对电离辐射的抗性，且这种抗性不依赖于AMPK信号传导。
