奈达铂对不同EBV状态鼻咽癌细胞放射增敏的实验研究

目的 观察奈达铂对人鼻咽癌细胞系CNE2（EBV-）和C666（EBV＋）的抑制作用及放射增敏作用,并探讨其作用机制.方法 不同浓度奈达铂（0～100μg/ml）作用于人鼻咽癌CNE2（EBV-）和C666（EBV＋）细胞,MTS法检测奈达铂的细胞增殖抑制作用,并计算IC50值;以IC5和IC10奈达铂浓度作为增敏剂量,MTS法和克隆形成法检测细胞放射敏感性的变化,流式细胞术检测细胞凋亡百分率及细胞周期的改变.结果 MTS实验奈达铂对CNE2和C666细胞IC50值分别为34.32μg/ml和63.69μg/ml（24h）;增敏实验分为空白对照组、NDP1组（CNE2 0.34μg/ml,C666 0.69μg/ml）和NDP2组（CNE2 0.64μg/ml,C666 1.27μg/ml）,4Gy照射后三组细胞存活率,CNE2细胞为（90.0±0.3）%、（80.2±0.5）%、（53.3±0.7）%（24h）,（76.7±0.1）%、（60.0±0.6）%、（40.0±0.2）%（48h）,（66.7±0.7）%、（40.2±0.4）%、（30.0±0.6）%（72h）;C666细胞为（92.3±0.2）%、（61.5±0.7）%、（50.3±0.3）%（24h）,（77.7±0.2）%、（44.6±0.9）%、（36.2±0.4）%（48h）,（53.8±0.2）%、（34.6±0.3）%、（23.1±0.4）%（72h）;以克隆形成结果拟合存活曲线,CNE2细胞SF2为0.46、0.26、0.14,C666细胞SF2为0.43、0.31、0.20;CNE2和C666细胞SER（SF2）为1.77和1.39（NDP1）,3.29和2.15（NDP2）;照射后三组细胞凋亡率,CNE2为（4.55±0.12）%、（9.02±0.75）%和（14.55±0.45）%,C666细胞为（4.02±0.33）%、（7.11±0.32）%和（10.36±0.58）%;三组细胞G2/M期比例,CNE2为（3.49±0.25）%、（17.55±0.55）%和（32.09±0.48）%,C666为（3.65±0.18）%、（13.75±0.51）%和（30.11±0.77）%;两种细胞系组间比较差异均具统计学意义（P＜0.05）.结论 奈达铂对不同EBV状态的人鼻咽癌CNE2和C666细胞具有放射增敏作用,增敏作用CNE2细胞（EBV-）优于C666细胞（EBV＋）,其增敏机制与增加细胞凋亡、调节G2/M期比例有关.

Experimentally study the radiosensitizing effects of nedaplatin on NPC cells at different EBV status

Objective We examined the anticancer activity and radiosensitizing effects of nedaplatin （NDP） on human nasopharyngeal carcinoma cells （CNE2 and C666） at different Epstein-Barr virus status, and explore the mechanism. Methods After incubating with different concentration of NDP （ 0-100μg /ml）, cytotoxic effects of NDP and inhibition concentration （IC50） were investigated by MTS assay. The cell viability and survival from ionizing radiation （IR） were evaluated using MTS and clonogenic survival assay, respectively. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Results MTS results showed that the IC50 value of CNE2 cell was 34.32μg/ml for and 63. 69μg/ml for C666 cell after incubating with concentration for 24 hours. The radiosensitization experiment including three groups ： control group, NDP1 group （ CNE2 0.34ug/ml, C666 0. 69μg/ml）and NDP2 group （CNE2 0.64μg/ml, C666 1.27μg/ml）. After irradiated with X-ray at the dose of 4Gy, cell survival rates of each group for CNE2 cell were （90.0±0.3 ） % , （ 80.2 ±0.5 ） % , （ 53.3 ± 0.7）%（24h）;（76.7 ±0. 1）%,（60.0 ±0.6）%, （40.0±0.2）% （48h）, （66.7 ±0.7）%, （40.2 ±0.4）%, （30.0±0.6）% （72h）; for C666 cell, the rates were （92.3±0.2）%, （61.5 ±0.7）%, （50.3±0.3）%（24h）,（77.7±0.2）%, （44.6±0.9）%, （36.2±0.4）%（48h） and （53.8±0.2）%, （34.6±0.3 ） %, （23.1 ± 0.4） % （72h） ; The cell survival curve in accordance with one-hit multi-target model by the colony formation assay were fit to conclude the radiation biological parameters, the survival fraction in 2Gy（SF2） for CNE2 cell were 0. 46 ,0. 26 ,0.14 and 0. 43 ,0. 31,0. 20 for C666 cell; the sensitizing enhance rate （SERSF2） were 1.77（CNE2） and 1.39 （C666） in NDP1 group, 3.29 （CNE2） and 2.15 （C666） in NDP2 group; The percentage of cell apoptosis in three group were （4.55 ±0.12）%, （9.02 ±0.75）%, （14.55±0.45）% for CNE2 cell, （4.02±0.33 ） %, （7.11± 0.32） %, （ 10.36 ± 0.58 ） % for C666 cell, respectively; Cells within G2/M phase were （3.49 ±0.25）%, （17.55 ±0.55）%, （32.09 ±0.48）% for CNE2 cell, （3.65 ±0.18 ） %, （ 13.75 ± 0.51 ） %, （ 30. 11± 0.77 ） % for C666 cell. Differences among the three groups were statistically significant （P 〈 0. 05 ）. Conclusions NDP proved to have radiosensitizing effects on CNE2 （ EBV - ） and C666 （ EBV ＋ ） cells, and present more effective on CNE2 （ EBV - ） cells, the radiosensitizing effects may act through enhance apoptosis and adjust G/M phase distribution of tumor ceils.