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

Abstract

Objective To 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. Methods A549 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. Results Flow 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. Conclusion PGE2 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. Key words: Celecoxib; Non-small cell lung cancer; Surgical stress; Anoikis