塞来昔布对肺腺癌手术应激所致肺转移的影响及其作用机制

Effects of Celecoxib on lung metastasis of lung adeno-carcinoma initiated by thoracotomy stress and the underlying mechanism

目的 探讨塞来昔布（Celecoxib）对肺腺癌手术应激所致肺转移的影响及其分子作用机制.方法 利用BALB/c裸鼠,通过开胸手术同时尾静脉注射肺腺癌细胞株（A549）建立手术应激所致肺转移动物模型.将75只裸鼠随机分成3组：对照组、手术组、手术＋Celecoxib组.3组小鼠均尾静脉注射A549细胞1×106个,4周后处死所有小鼠,5％苦味酸染色比较各组小鼠肺表面结节数；苏木素-伊红（HE）染色明确各组肺结节病理性质,酶联免疫吸附试验（ELISA）试剂盒检测不同时间点小鼠血清前列腺素E2（PGE2）水平.利用A549细胞进行划痕实验、Transwell小室实验检测体外PGE2和Celecoxib对A549迁移和侵袭能力的影响,实时定量聚合酶链反应（Real-time PCR）和Western blot检测PGE2和Celecoxib对细胞基质金属蛋白酶（MMP）-2/-9、E-钙黏蛋白（E-cadherin）、糖原合成激酶-3β（GSK-3β）和细胞核β-连环蛋白（β-catenin）表达的影响.结果 HE染色确定小鼠肺表面结节系A549细胞肺转移灶.与对照组比较,手术组小鼠肺表面肺结节数明显增多[（30.2±8.8）个比（17.2±5.2）个,P＜0.05],术后第3天血清平均PGE2水平较术前明显升高达到（212.8±52.9）ng/L,而手术组给予Celecoxib则能明显抑制肺结节数增多[（9.1±4.2）个]和血清PGE2升高（102.7±11.2） ng/L,差异有统计学意义（P＜0.05）.细胞划痕和Transwell实验也显示Celecoxib能明显抑制PGE2对A549的促迁移和侵袭作用[（79.8±9.9）％比（177.8±13.2）％,P＜0.01].Western blot结果与Real-time PCR结果相似：PGE2组细胞MMP-9 mRNA和蛋白表达增加,E-cadherin表达明显下降,而经Celecoxib处理后PGE2对上述基因表达的影响被逆转.Western blot结果显示,Celecoxib能抑制PGE2刺激引起的GSK-3β活化和细胞核β-catenin表达量升高.结论 Celecoxib能明显抑制肺腺癌手术应激所致肺转移,其分子机制可能与抑制β-catenin核转位有关.

Objective To explore the effects of Celecoxib on lung metastasis of lung adeno-carcinoma initiated by thoracotomy stress and the underlying mechanism.Methods Animal models of metastasis of lung adeno-carcinoma initiated by thoracotomy stress were created by thoracotomy and injection of lung adeno-carcinoma cell line （A549） via the lateral tail vein into BALB/c nude mice.Seventy-five nude mices were randomly divided into 3 groups：control,mice receiving thoracotomy; mice receiving thoracotomy and Celecoxib.All nude mices were injected with a suspension of 1 x 106 A549 cells via the lateral tail vein and killed at 4th week.The nodules on the lung surface were stained with 5% picric acid and the number of nodules in each group was analyzed.In addition,the nodules were also cut into sections for hematoxylin and eosin （HE） staining to identify their pathological features.The serum prostaglandin E2 （PGE2） level was determined using the PGE2 enzyme linked immunosorbent assay （ELISA） kit at different time points.The effects of Celecoxib and PGE2 on cell migration and invasion were studied by wound healing and Transwell assay.Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting were applied to detect the expression of matrix metalloproteinase （MMP）-2/-9,E-cadherin,GSK-3β and β-catenin in A549 cells.Results HE staining showed that the metastatic nodules of A549 cells on the lung surface were identified.The number of metastasis in mice receiving thoracotomy was greater than that in control mice （30.2 ±8.8 vs.17.2 ±5.2,P 〈0.05） and serum PGE2 level in mice receiving thoraeotomy was increased to （212.8 ±52.9） ng/L at 31st day after surgery.However,Celecoxib inhibited the increased nodules of metastases （9.1 ± 4.2） and serum PGE2 level [（102.7 ± 11.2） ng/L] respectively （P 〈0.05）.PGE2 increased migration and invasion of A549 cells in the wound healing and Transwell assays,which could be reversed by Celecoxib [（177.8 ± 13.2） % vs.（79.8 ± 9.9） %,P 〈 0.01].Real-time PCR showed that mRNA expression of MMP-9 was increased by PGE. and reversed by Celecoxib,while E-cadherin was markedly decreased,and Celecoxib increased its expression.The results of Western blotting showed similar effects in the protein expression of MMP-9 and E-cadherin.Western blotting also showed that the effects of PGE2-induced increase in the protein level of β-catenin in the nucleus of A549 cells and activation of GSK-3β were significantly inhibited by Celecoxib.Conclusion Celecoxib markedly inhibits lung metastasis of lung adeno-carcinoma initiated by thoracotomy stress and the underlying mechanism may associate with inhibiting translocation of β-catenin into nucleus.