塞来昔布对人鼻咽癌CNE-2细胞生长抑制及放射增敏研究

Growth inhibition and radiosensitization of Celecoxib in nasopharyngeal carcinoma cell line CNE-2

目的研究塞来昔布对人鼻咽癌细胞系CNE-2细胞生长影响及有无放射增敏作用。方法（1）细胞生长抑制研究：用MTT法检测细胞生长抑制，流式细胞术检测细胞周期分布及凋亡，透射电子显微镜观察细胞凋亡形态，sP法检测细胞COX-2表达。（2）放射增敏研究：随机设置照射对照、药物对照、单纯照射、药物＋照射组，其中成克隆实验单次照射2、4、6、8、10Gy，细胞周期分布和凋亡实验单次照射6Gy。结果塞来昔布显著抑制CNE-2细胞生长并呈浓度和时间依赖性，IC50为80μmol／L。细胞周期分布显示G0＋G1期细胞显著升高（47．03±2．76：56．17±1．95，t=4．68，P=0．010），而S、G2＋M期细胞明显下降（33．07±1．86：24．87±1．76，t=5．54，P=0．010；19．30±0．53：17．73±0．83，t=2．75，P=0．050）并呈浓度依赖性。凋亡率显著增高（1．57±0．47：10．47±0．31，t=27．39，P=0．000）并呈浓度依赖性。电镜观察到细胞皱缩、核质浓缩、核碎裂等凋亡形态学改变。SP法检测塞来昔布显著下调CNE-2中COX．2表达[17．48±0．34、12．82±0．51（t=13．20，P=0．00）]。塞来昔布的放射增敏比（D0值比为1．74：1．52）为1．15。4个组别细胞周期分布结果显示单纯照射、药物＋照射组的G2＋M期细胞明显高于照射对照、药物对照组（68．00±1．65、54．27±5．74、17．60±0．80、14．86±1．23，t=47．70，P=0．000；t=11．63，P=0．000），且单纯照射与药物＋照射组间也不同（t=3．99，P=0．020）；单纯照射、药物＋照射组的细胞凋亡率也明显高于照射对照、药物对照组（4．83±0．97、9．50±1．35、1．33±0．36、2．28±0．42，t=4．67，P=0．01；t=8．81，P=0．000），且单纯照射与药物＋照射组也不同（t=4．85，P=0．010）。结论塞来昔布能抑制人鼻咽癌CNE-2细胞生长和诱导凋亡，其机制可能涉及COX-2依赖途径。塞来昔布还能增强CNE-2细胞放射敏感性，可能机制与抑制放射后亚致死损伤修复、直接抑制细胞增殖和增强细胞对放射诱导凋亡率有关。

Objective To investigate the growth inhibition and radiosensitization of Celecoxib in human nasopharyngeal carcinoma cell line CNE-2. Methods CNE-2 growth inhibition by Celecoxib was evaluated by MTT method. Apoptosis-related changes in morphology were observed by transmission electron microscopy （TEM）. Cell cycle distribution and apoptosis rate were measured by flowcytometry （FCM）. The expression of COX-2 protein was observed by SP method after the treatment of Celecoxib. Cells were randomly planted into four groups ： irradiation control （ Ci）, drug group （ Cd）, irradiation group （ R）, and Celecoxih plus irradiation group （D ± R）. Single irradiation of 2,4,6,8, and 10 Gy were administered for colonogenic assay. Cell cycle distribution and apoptosis rate were analyzed at 6 Gy irradiation. Results The growth of CNE-2 cell was inhibited by celecoxib in a dose-and time-dependent manner, the IC50 was 80 μmol/L. After the treatment, cell ratio of G0 and G1 phases was increased （ 47.03 ± 2.76 vs 56.17 ± 1.95 , t = 4.68 , P =0.010） ,whereas the ratio of S and G2/M phases was decreased （33.07 ± 1.86 vs 24.87 ± 1.76 ,t =5.54 ,P = 0.010 ; 19.30 ± 0. 53： 17.73 ± 0.83, t = 2.75, P = 0.050） , and the apoptosis rate was increased （ 1.57 ± 0.47：10.47± 0.31, t = 27.39, P = 0. 000）in a dose-dependent manner. Apoptosis with nuclear chromatin condensation ,fragmentation and cell shrinkage was found by TEM. SP method showed that Celeib decreased COX-2 expression （ 17.48 ± 0.34 vs 12.82 ± 0.51, t = 13.20, P = 0.00）. The sensitivity ratio （ DO ） was 1.15. FCM showed that the percentage of cells in G2/M phase was significanty more in R and D ＋ R groups than in Ci and Cd groups （68.00 ± 1.65,54.27 ± 5.74,17.60 ± 0.80,14.86 ± 1.23, t = 47.70,P = 0.000 ; t = 11.63, P = 0. 000）, and also significantly different between R group and D＋ R group （ t = 3.99, P = 0. 020）. The apoptosis rate was higher in R and D ＋ R groups than Ci and Cd groups（4.83 ±0.97,9.50 ± 1.35,1.33 ± 0.86 and 2.28 ± 0.42, t = 4.67, P =0.010;t = 8.81, P = 0. 000）, D ＋ R group than R group （t =4.85, P = 0. 010）. Conclusions Celecoxib can markedly inhibit the growth and induce apoptosis in CNE-2 cells,which may depend on COX-2 pathway. Celecoxib potently enhances the radiosensitivity of CNE- 2 cells, which may due to the repair inhibit of radiation-induced DNA damage, inhibit of cell proliferation, and enhancement of cell apoptosis after irradiation.