塞来昔布对手术应激产生的非小细胞肺癌抗失巢凋亡的影响

Effect of celecoxib on anoikis resistance in non - small cell lung cancer induced by surgical stress

目的探讨塞来昔布（celecoxib）对肺腺癌手术应激所致非小细胞肺癌细胞A549的抗失巢凋亡的影响及其分子作用机制。 方法采用多聚2-羟乙基甲基丙烯酸酯（poly-HEMA）覆盖细胞培养板培养A549细胞，分别设立对照组、前列腺素E2（PGE2）处理组、PGE2联合塞来昔布处理组，处理48 h后应用流式细胞术检测各组中细胞发生失巢凋亡情况。采用Western blot方法检测各组细胞的丝裂原细胞外激酶（MEK）/细胞外信号调节激酶1/2（ERK1/2）通路和磷酸肌醇3激酶（PI3K）/蛋白激酶B（Akt）通路激活以及下游凋亡相关蛋白Mcl-1和Bim的表达。应用BALB/c小鼠开胸手术模拟手术应激，进行尾静脉注射A549细胞建立肺转移动物模型。将小鼠分成对照组、手术组和手术＋塞来昔布组（完全随机化分组），4周后处死小鼠并观察肺转移，同时采用免疫组织化学方法检测转移产生的肺结节中Mcl-1和Bim的表达。 结果流式细胞术检测结果表明poly-HEMA培养的A549细胞凋亡率为（41.0±2.4）%，而PGE2处理组的凋亡率为（22.8±3.2）%，与对照组比较差异有统计学意义（P＝0.012），PGE2联合塞来昔布处理组的凋亡率为（36.6±2.4）%与PGE2组比较，差异有统计学意义（P＝0.034）。Western blot结果显示，PGE2能激活MEK/ERK1/2通路，上调ERK1/2的磷酸化水平，并降低Bim的表达，而塞来昔布能明显抑制PGE2的作用。另外PGE2对PI3K/Akt通路无明显作用，但塞来昔布可以抑制该通路，并下调Akt的磷酸化水平，抑制Mcl-1的表达。体内实验中，手术组的肺结节数明显多于对照组，而塞来昔布组肺结节数与手术组比较明显减少，免疫组织化学检测肺结节中的Mcl-1和Bim蛋白的表达水平，结果与体外实验一致。 结论PGE2是手术应激中产生的主要细胞因子，PGE2具有明显的体外抗失巢凋亡作用，且塞来昔布在体内外皆能通过抑制MEK/ERK1/2通路和PI3K/Akt通路来升高促凋亡蛋白Bim的表达和降低抗凋亡蛋白Mcl-1的表达，从而达到促进失巢凋亡，减少肺癌细胞转移的作用。

ObjectiveTo investigate the effect and its molecular mechanism of celecoxib on anoikis resistance in non-small cell lung cancer cell line A549 induced by lung cancer surgical stress. MethodsA549 cells were cultured using cell culture plate covering poly-（2-hydroxyethyl methacrylate） HEMA. The control group, prostaglandin E2 （PGE2） treatment group, and PGE2 combined celecoxib treatment group were established. Anoikis of cells was assessed by flow cytometry after treatment for 48 h. Western blotting was used to detect the activation of mitogen extracellular kinase （MEK）/extracellular signal-regulated kinase 1/2 （ERK1/2） pathway and phosphatidylinositol 3 kinase （PI3K）/protein kinase B （Akt） pathway and the expression of apoptosis related proteins, Mcl-1 and Bim, in each group. BALB/c mice were used to simulate the surgical stress, and A549 cells were injected into the tail vein to establish the animal model of lung metastasis. Mice were divided into control group, surgery group and surgery plus celecoxib group （completely randomized grouping）, the mice were killed after 4 weeks and lung metastasis was observed, and the expression of Mcl-1 and Bim in pulmonary nodules was detected by immunohistochemical method. ResultsFlow cytometry results showed that apoptosis rate of A549 cells cultured in poly-HEMA was （41.0±2.4）%, that in PGE2 treated group was （22.8±3.2）% （as compared with the control group, P=0.012）, and that in the PGE2 combined with celecoxib treatment group was （36.6±2.4）% （as compared with the PGE2 group, P=0.034）. The results of Western blotting showed that PGE2 could activate the MEK/ERK1/2 pathway, up-regulate the phosphorylation level of ERK1/2, and down-regulate the expression of Bim, while celecoxib could significantly inhibit the effects of PGE2. In addition, PGE2 had no obvious effect on PI3K/Akt pathway, but celecoxib could inhibit the pathway, and down-regulate the phosphorylation level of Akt and inhibit the expression of Mcl-1. Immunohistochemically, the expression level of Mcl-1 and Bim protein in the pulmonary nodules was consistent with that of experiments in vitro. ConclusionPGE2 is the main cytokine production in the surgical stress. This study shows that PGE2 has obvious effect in vitro on anoikis resistance, and celecoxib inhibits the MEK/ERK1/2 pathway and the PI3K/Akt pathway to increase the expression of pro-apoptotic protein Bim and decrease the expression of anti-apoptotic protein Mcl-1 in vitro and in vivo, which can promote anoikis to reduce lung cancer cells metastasis.