| PurposeInorder to investigate the radiosensitization effect,the expression of PI3K/AKT pathway related protein and the difference of DNA damage and repair of XWLC-05(Yunnan-Xuanwei Lung Adenocarcinoma) Cells and A549cells when the activity of PI3K was ihibited..Material and Methods1. Use Western-Blotting to test the expression of PI3K related AKT, p-AKT308and p-AKT473proteins and DNA damage repair related DNA-PKcs, Ku70and Ku80proteins before and after the activity of PI3K inhibited by LY294002.2. Compare the radiation killing effect between XWLC-05cells and A549cells by Clonogenic assays when the PI3K/AKT pathway was blocked.3. Use commet assay to test DNA double strands break, differences of DNA repair time effect before and after the PI3K/AKT pathway blocked and explore the possibility mechanism within the process.Results1. The expression of PI3K related protein:The expression of Ser473protein and Thr308protein of XWLC-05cells were higher when compared with A549cells in control groups, indicated that the PI3K activity of XWLC-05cells was stronger than A549cells. When added with LY294002, the expression of total AKT protein haven't change in both A549cells and XWLC-05cells as control(p>0.05). With the concentration of LY294002increasing, the inhibition on expression of Ser473protein were enhanced in both cells (p<0.01). The inhibition effect was more stronger when combined with irradiation, A549cell and XWLC-05cell were best inhibited at50μM+8Gy (p<0.01).When we compared the control grorps with50μM+8Gy, it showed the difference of gray value in XWLC-05cells are greater than A549cells, indicated when combined with irradiation, the inhibition degree of LY294002to XWLC-05cells was more high than A549cells.2. Clonogenic assays showed:After added LY294002, either A549cells or XWLC-05cells showed decreasing of surviving fraction and increasing of radiosensitivity. The experiment also showed that the surviving fraction of control group of XWLC-05cells was lower than dosing group of A549cells, prompting XWLC-05cells had the highly radiosensitivity to A549cells, the two cells had difference in radiosensitivity.3. The expression of DNA repair related protein:whether added with LY294002or not, the expression of Ku70and Ku80protein hadn't change in both A549cells and XWLC-05cells(p>0.05), and the expression didn't change with concentration or dose. There were no differences of DNA-PKcs expression between A549cells and XWLC-05cells when treated with LY294002only. But when treated with both inhibitor and radiation, the expression of DNA-PKcs decreased at24h after added drug and post1h of radiation(p<0.01), the trend of XWLC-05cells is noticeable than A549cells, but had no statistical difference(p=0.07), and return to normal level after24h to irradiated. it indicated that LY294002combined with irradiation could inhibit the expression of DNA-PKcs, with time prolonging the inhibition action reduced.4. Commet assay DNA damage curve:Both A549cells and XWLC-05cells all represent that DNA damage were increased dose-dependently, and both two groups have no differences (p>0.05). Compared with control groups, DNA damage of experimental groups were enhanced when treated with LY294002; while compared with A549cells, DNA damage of XWLC-05cells is more serious. DNA repair curve:Both A549cells and XWLC-05cells all showed that DNA damage was decreased time-dependently. With the DNA repair, DNA damage of experimental groups were still stronger than control groups.Conclusions1. The activity of PI3K of XWLC-05cells is stronger than A549cells. LY294002has stronger inhibition effect to PI3K in XWLC-05cells than A549cells.2. Blocking PI3K/AKT pathway increase radiosensitivity both of XWLC-05and A549cell lines. XWLC-05cells has stronger inherent radiosensitivity than A549cells.3. Blocking PI3K/AKT pathway has weak effect on expression of Ku70and Ku80proteins. Combined with irradiation impaired the expression of DNA-PKcs at lh after irradiation, and have improved effect to XWLC-05cells. As time goes on, the impact reduced.4. Blocking PI3K/AKT pathway increaseed DNA damage of XWLC-05and A549cell lines, decrease the ability of DNA reair, and delay the time to repair. |
PDF Full Text Request