| Objective: To evaluate Celecoxib's radiosensitizing effect and to analyze the effect of autophagy and autophagic cell death on Celecoxib radiosensitizing human glioma SHG-44 cell lines.Methods: The human glioma cell SHG-44 was managed in vitro. Group of drug (Group D) was divided according to the different concentration of Celecoxib (0μM, 30μM, 50μM and 100μM);and the group irradiation (Group R) divided according to the different dose of 6-MV X-ray(0Gy, 1Gy, 2Gy, 4Gy, 6Gy and 8Gy), the other group was formed by Celecoxib combined with irradiation (Group D+R). MTT assay was performed to determine the effects of Celecoxib on SHG-44 cell growth; Celecoxib's radiosensitization was measured by clongenic assay; the cell cycle redistribution and apoptosis was analyzed by Flow-cytometric analysis. Meanwhile, acridine orange (AO), FITC-LC3-Ⅱand transmission electron microscope were used to detect autophagy.Results:1. The outcome of MTT assay suggested that Celecoxib may produce a concentration- dependent and time-dependent inhibition of SHG-44 cell proliferation(P<0.05). Celecoxib showed radiosensitizing effect to a certain extent in SHG-44 cells by colony forming assays, the sensitizating enhancement ratio SERD0=1.20.2. Flow-cytometric analysis showed that Celecoxib 30μM induced G2/M arrest and enhanced prolongation of G2/M arrest that induced by irradiation 8Gy, (C,D,R and D+R G2/M: 9.87±1.34%, 20.14±2.52%, 29.15±1.99%, 34.26±2.20%, respectively; P<0.05),however, apoptotic cell death was not apparent (R: 8.2±0.93%, D+R: 9.7±1.24%, P=0.158). Furthermore prolongation of G2/M arrest was demonstrated dose-dependent manner (2, 6 and 8Gy G2/M: 14.06±1.37%, 18.72±3.77%, 29.15±1.99%, respectively; P<0.05).3. To investigate autophagy, first we performed TEM, SHG-44 cells treated with irradiation and / or Celecoxib showed prominent formation of autophagic vesicles with lamellar structures or residual digested material, without the nucleus condensation or segmentation. Next, cells incubated by acridine orange (AO) after irradiation 8Gy and/or Celecoxib 30μM, which emitted red fluorescein in acidic vesicular organelles (C, D, R and D+R: 1.08±0.21, 1.94±0.06, 2.71±0.14, 6.19±0.56; P < 0.05), and FITC-MAP1- LC3-Ⅱantibody was used to label LC3-Ⅱ, which was bounded on the membrane of autophagosomes. The level of autophagy of the group combination cells showed more than the R group or the Celecoxib group(C, D, R and D+R: 9.34±1.05%, 18.46±2.67%, 24.81±2.04%, 43.51±3.26%, P<0.05).4. As the dose of radiation increased, G2/M phase was prolonged, while the level of autophagy increased in a corresponding. Statistical software for correlation analysis showed that there is a linear correlation between the prolongation of G2/M arrest and the level of cellular autophagy (correlation coefficient r=0.97,P<0.05). 5. As the level of autophagy increased, the clongenic survival decreased exponentially. Statistical analysis showed that there is a negative correlation between the logarithm of cell survival fraction and the level of cellular autophagy (correlation index R=0.98,P<0.05).Conclusions:1. Celecoxib enhances the radiosensitivity of glioma cells SHG-44 in vitro.2. Celecoxib enhances SHG-44 cells G2/M phase arrest by radiation-induced and promoted cell autophagy. To induce autophagic cell death may be one of the mechanisms of Celecoxib radiosensiting glioma cells SHG-44.3. Induction of autophagy and autophagic cell death may be in association with the cells G2/M phase arrest. |
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