Important role of autophagy in endothelial cell response to ionizing radiation

• Published in: PloS one 2014-07, Vol.9 (7), p.e102408-e102408
• Main Authors: Kalamida, Dimitra, Karagounis, Ilias V, Giatromanolaki, Alexandra, Koukourakis, Michael I
• Format: Article
• Language: English
• Subjects: Allyl Compounds - administration & dosage; Autophagy; Autophagy - genetics; Autophagy - radiation effects; Biology and Life Sciences; Blockage; Cancer; Cathepsin D - biosynthesis; Cell death; Confocal microscopy; Cytoplasm - drug effects; Cytoplasm - radiation effects; Cytoplasm - ultrastructure; Democritus (460?-370?); Edema; Endothelial cells; Endothelial Cells - drug effects; Endothelial Cells - radiation effects; Endothelial Cells - ultrastructure; Endothelium; Experiments; Fluctuations; Flux; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia; I.R. radiation; Infrared radiation; Ionizing radiation; Irradiation; Lysosomal-Associated Membrane Protein 2 - biosynthesis; Lysosomes - drug effects; Lysosomes - radiation effects; Lysosomes - ultrastructure; Medicine and Health Sciences; Membrane Proteins - drug effects; Membrane Proteins - metabolism; Membrane Proteins - radiation effects; Membranes; Microscopy; Microscopy, Confocal; Oncology; Phagocytosis; Phosphatase; Post-irradiation; Proteins; Quinazolines - administration & dosage; Radiation; Radiation damage; Radiation protection; Radiation therapy; Radiation, Ionizing; Radiobiology; RNA, Small Interfering; siRNA; TOR protein; Vacuoles
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0102408

Vasculature damage is an important contributor to the side-effects of radiotherapy. The aim of this study is to provide insights into the radiobiology of the autophagic response of endothelial cells. Human umbilical vascular endothelial cells (HUVEC) were exposed to 2 Gy of ionizing radiation (IR) and studied using confocal microscopy and western blot analysis, at 4 and 8 days post-irradiation. The role of autophagy flux in HUVEC radio-sensitivity was also examined. IR-induced accumulation of LC3A(+), LC3B(+) and p62 cytoplasmic vacuoles, while in double immunostaining with lysosomal markers (LAMP2a and CathepsinD) repression of the autophagolysosomal flux was evident. Autophagy-related proteins (ATF4, HIF1α., HIF2α, Beclin1) were, however, induced excluding an eventual repressive effect of radiation on autophagy initiating protein expression. Exposure of HUVEC to SMER28, an mTOR-independent inducer of autophagy, enhanced proLC3 and LC3A, B-I protein expression and accelerated the autophagic flux. Pre-treatment of HUVEC with SMER28 protected against the blockage of autophagic flux induced by IR and conferred radio-resistance. Suppression of LC3A/LC3B proteins with siRNAs resulted in radio-sensitization. The current data provide a rationale for the development of novel radioprotection policies targeting the autophagic pathway.