| 孔泽,汪建林,孙志强,王坚,封悦,孙菲,华秋,周梦耘,于静萍.阿帕替尼对食管鳞癌ECA-109细胞及其干性细胞辐射敏感性的影响及机制探讨[J].中华放射医学与防护杂志,2018,38(3):161-167 |
| 阿帕替尼对食管鳞癌ECA-109细胞及其干性细胞辐射敏感性的影响及机制探讨 |
| The radiosensitivity effects of apatinib on the esophageal cancer cell line ECA-109 and its stem-like cells |
| 投稿时间：2017-10-25 |
| DOI：10.3760/cma.j.issn.0254-5098.2018.03.001 |
| 中文关键词: 食管癌 阿帕替尼 血管内皮生长因子 辐射增敏 |
| 英文关键词:Esophageal cancer Apatinib Vascular endothelial growth factor Radiation sensitization |
| 基金项目:国家自然科学基金（11705095） |
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| 中文摘要: |
| 目的 探索阿帕替尼对食管鳞癌ECA-109细胞及其干性细胞辐射敏感性的影响及其分子机制。方法 采用无血清悬浮培养法富集细胞中富含肿瘤干性细胞群的球囊。将ECA-109及其干性细胞分为细胞对照组、单纯药物组、单纯照射组以及药物+照射组。CCK-8法测定细胞增殖的变化，酶联免疫吸附法（ELISA）测定血管内皮生长因子（VEGF）蛋白的浓度，流式细胞术分析细胞周期及凋亡的变化；Western blot法测定细胞中CHK2、P-STAT3蛋白的表达。结果 阿帕替尼作用于ECA-109亲本细胞及干性细胞24、48和72 h后，ECA-109干性细胞的药物半数抑制浓度（IC50）均较其亲本细胞的药物半数抑制浓度（IC50）明显升高（t=8.17、9.29、18.85，P<0.05），且其细胞抑制表达呈时间-剂量效应关系（亲本细胞：r2=0.94~0.97，P<0.05；干性细胞：r2=0.94~0.98，P<0.05）；不同浓度阿帕替尼（亲本细胞：10和20 μmol/L；干性细胞：30和40 μmol/L）联合不同剂量X射线（6和8 Gy）照射后，ECA-109亲本细胞及干性细胞的增殖抑制率均较单纯照射组明显增强（t=5.20~39.68，P<0.05）。ECA-109亲本细胞及干性细胞的VEGF蛋白水平差异具有统计学意义（t=7.45，P<0.05），且单纯药物组（20 μmol/L）及药物+照射组（20 μmol/L，6 Gy）处理后，两种细胞VEGF蛋白水平均有所下降，其中亲本细胞差异具有统计学意义（t=14.51、26.40，P<0.05）。药物+照射组ECA-109亲本细胞的G2/M期及凋亡细胞比例均较细胞对照组增高（t=8.83，11.59，P<0.05）；与细胞对照组（0 μmol/L）比较，单纯药物组的ECA-109亲本细胞CHK2、P-STAT3蛋白的表达水平显著下降（t=3.36、4.10，P<0.05）；与单纯照射组比较，药物+照射组ECA-109亲本细胞CHK2，P-STAT3蛋白的表达水平也显著下降（t=9.05、2.36，P<0.05）。结论 阿帕替尼能增强食管癌ECA-109亲本细胞及其干性细胞的放射敏感性，ECA-109干性细胞放射抵抗的原因可能与干性细胞更高表达VEGF蛋白有关。 |
| 英文摘要: |
| Objective To evaluate the radiosensitivity effects of apatinib on the esophageal cancer cell line ECA-109 and its cancer stem-like cells, and to investigate the underlying mechanism. Methods A serum-free medium (SFM) was used to culture esophageal cancer stem cell line ECA-109 and enrich the esophageal stem-like spheres. ECA-109 and its stem-like cells were divided into control group, drug treatment group, radiation group and drug plus radiation group. Cell proliferations of ECA-109 and its stem-like cells were detected with CCK-8 method. The concentration of vascular endothelial growth factor(VEGF) in the cell culture medium was determined by enzyme linked immunosorbent assay(ELISA). Cell cycle and apoptosis were detected by flow cytometry method. The expressions of CHK2 and P-STAT3 proteins were detected by Western blot assay. Results With the administration with apatinib for 24, 48 and 72 h, the half of the inhibitory concentration (IC50) of ECA-109 stem-like cells was significantly higher than that of the parent cells (t=8.17, 9.29, 18.85,P<0.05) in a time dependent manner (parental cells:r2=0.94-0.97, P<0.05; stem-like cells:r2=0.94-0.98, P<0.05). After administration with different concentrations of apatinib (parental cells:10 and 20 μmol/L; stem-like cells:30 and 40 μmol/L) combined with different dose of X-rays (6 and 8 Gy), the proliferations of ECA-109 and its stem-like cells were significantly (t=5.20-39.68,P<0.05) inhibited compared with radiation alone group. VEGF secretion from both ECA-109 cells and its stem like cells were significantly decreased in different manner (t=7.45,P<0.05). Compared with control group, the cell apoptosis rate and the percentages of cells in G2/M phase were significantly increased in drug plus radiation group (t=8.83, 11.59, P<0.05), and the expressions of CHK2 and P-STAT3 were decreased in drug group (t=3.36, 4.10,P<0.05). Compared with radiation group, the expressions of CHK2 and P-STAT3 were decreased in drug plus radiation group (t=9.05, 2.36,P<0.05). Conclusions Apatinib enhanced the radiosensitivity of ECA-109 cells and its stem-like cells, which was much more effective on ECA-109 cells and may be related to the radiation-induced inhibition of VEGF signal pathway that can further inhibit cell proliferation, promote cell apoptosis and induce cell cycle redistribution. The higher intrinsic level of VEGF protein may contribute to radioresistance of ECA-109 stem-like cells. |
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