The characteristics of hypoxia-induced exosomes secreted from A549 cells and its effect on radioresistance / 中华放射医学与防护杂志

ABSTRACT

Objective To investigate the effect of exosomes secreted from human lung adenocarcinoma A549 cells under hypoxic or normoxic conditions on the radiosensitivity and invasiveness of normoxia cells.Methods A549 cells were cultured in hypoxic (1％ O2) and normoxic (21％ O2) conditions,respectively.The exosomes (N-EXO and H-EXO) secreted from normoxic or hypoxic A549 cells were collected by ultracentrifugation and its number was measured using a NanoSight detector.The appearance and size distribution of exosomes were observed by a scanning electron microscopy.The exosomal marker protein CD63 was measured by Western blot.The proliferation of cells exposed to X-rays under hypoxic or normoxic conditions were detected by CCK8 assay.The cell uptake situation of exosomes labeled with PKH67 was observed by a fluorescence microscopy.Cell migration and invasiveness were detected by a cell scratch test and transwell assay.The expression of matrix metalloproteinase 2 (MMP2) and MMP9 was detected by ELISA.Cellular radioresistance effect of exosomes was evaluated by a colony formation assay.Results The NanoSight measurement showed the number of exosomes in cell culture medium was increased after hypoxia treatment.The H-EXO and N-EXO showed typical ring cake shape.The size distribution of H-EXO was mainly between 30 nm and 200 nm,smaller than that of N-EXO (50-220 nm).Western blot assay showed that CD63 was expressed in both H-EXO and N-EXO.At 4 and 6 days after 2 Gy X-rays irradiation,cell proliferation rate of hypoxia A549 cells was significantly higher than that of normoxia cells.The green fluorescent marker of exosomes,PKH67,was distributed inside of the cell.Cell scratch test showed that the width of H-EXO group was much smaller than that of N-EXO group at 12,24 and 48 hours after exosomes treatment (t =2.96,6.76,3.35,P ＜ 0.05).Transwell assay showed that the number of transmembrane cells in the H-EXO group was more than that in the N-EXO group and the control group (t =4.84,7.88,P ＜ 0.O1).The expression levels of MMP2 (t =4.70,3.21,P＜0.05) and MMP9 (t =5.61,3.76,P＜0.05) in the supernatant of H-EXO group were significantly higher than those in the control and N-EXO groups.Cell survival assay showed that the D0 values of control,N-EXO and H-EXO group were 2.614,2.552 and 4.50 respectively,indicating that H-EXO could enhance radioresistance of A549 cells significantly.Conclusions This study finds that the number of exosomes released from A549 cells was increased under hypoxic condition but its size becomes smaller than that under normoxia.Hypoxic exosomes can promote the migration of normoxia cells andenhance cell radioresistance as well.