Effect Of Endoplasmic Reticulum Stress On The Response Of HeLa Cells To Carbon Ion Radiation

Aims:The aims of this work were to investigate the effect of endoplasmic reticulum stress on the response of HeLa cell to carbon ion radiation and its possible mechanism.Materials and Methods:Dithiothreitol, an inducer of ER stress, was applied to induce ER stress response in HeLa cells, and MTT assay was used to analyze the influence of different concentration of dithiothreitol on HeLa cell viability. Then the cells treated with or without dithiothreitol were exposed to carbon ion beam at the Heavy Ion Research Facility in Lanzhou; cells survival situation was analyzed by the colony-forming assay; the distribution of cell-cycle phase and the apoptosis rate were obtained with flow cytometry (FCM); cell senescence was examined by SA-β-gal staining assay; cell acidic vesicular organelle (AVO) and autophagy related proteins Beclin1protein expression were observed under fluorescent microscope and assessed with FCM; Chromosome aberration were evaluated by conventional technique. In addition, we also studied the role of carbon ion radiation-induced autophagy in the survival of cells by using3-MA, an inhibitor of autophagy.Results and Conclusion:1. Our results showed that carbon ion irradiation caused HeLa cell survival decreased, and led to a significant increase of G2/M phase and apoptotic cells. We also observed an increase of AVO positive cells and the protein level of Beclin1induced by carbon ion irradiation, which suggested that autophagy was induced by carbon ion radiation. Moreover, the senescence was observed in carbon ion radiated cells.2. The acidic vesicular organelle positive cells and the expression of Beclin1were significantly reduced in3-MA treated cells; these indicated that autophagy was inhibited.3-MA treatment enhanced cell death and reduced the colony survival fraction induced by carbon ion radiation, which suggested an obvious radiosensitization effect of3-MA. Moreover,3-MA and carbon ion radiation combination treatment led to a significant increase of G2/M phase and apoptotic cells, compared with IR alone. These suggested a cytoprotective role of autophagy in the survival of cells exposed to carbon ion radiation.3. Dithiothreitol treatment enhanced cell death and reduced the colony survival fraction induced by carbon ion radiation, which suggested an obvious radiosensitization effect of dithiothreitol. Moreover, dithiothreitol and carbon ion radiation combination treatment led to a significant increase of G2/M phase and apoptotic cells, compared with carbon ion radiation alone. We also observed an increase of AVO positive cells and the protein level of Beclinl, compared with carbon ion radiation alone, which suggested an increased autophagic response in dithiothreitol and carbon ion radiation combination treated cells. More severe senescence and chromosome aberrations were also observed in combination treated cells.The results imply that continuous endoplasmic reticulum stress can change the response of HeLa cells to carbon ion irradiation, promoted cell death and inhibited proliferative activity effectively, autophagy cell death and senescence may be the pivotal mechanisms underlying the enhanced cell-killing effect induced by the combined treatment with carbon ion irradiation and ER stress inducer dithiothreitol.