Celecoxib (Cox-2Selective Inhibitor) Induces Epithelial-to-mesenchymal Transition (EMT) In Human Lung Cancer Cell Lines Through Cox-2Independent Pathways

Nearly80%of lung cancer was non-small cell lung cancer (NSCLC) and only one fifth of the NSCLC patients have the opportunity to undergo surgical treatment. Even for the patients that the tumors can be radically resected,5-year survival rate is only around40%due to experiencing metastatic disease. Therefore, inhibition of cancer metastasis would be the key step to prolong the survival time of lung cancer patients. It is well known that Epithelial-to-mesenchymal transition (EMT) is a critical process in tumor metastasis, which is characterized by epithelial cells transition from epithelial phenotype to mesenchymal phenotype, leading to increased motility and invasion. Molecular markers for EMT include loss of epithelial cell markers, such as E-cadherin (E-cad) and up-regulation of mesenchymal molecular markers, such as vimentin.Besides, increased expression of cox-2in NSCLC cells is associated with tumor invasion and metastasis. Cycloxygenase-2protein (Cox-2) was recently found to mediate growth factors-induced EMT, such as TGF-β1. But double blind randomizedclinical phase Ⅲ trial showed that median survival time was not differentbetween the Celecoxib group and the placebo group.As the clinical outcomes of Cox-2inhibitors are still controversial, the original aim of this study was to investigatewhether Celecoxib (a cox-2selective inhibitor) would reverse EMT process triggered by TGF-β1in A549human lung cancer cells. But we found that Celecoxib induces EMT in human lung cancer A549cells throughMEK/ERK signaling pathway that partially counteracted by activation of PI3K/AKT pathway regardless of cox-2expression. Moreover, Celecoxib had no effect on the phosphorylation of Smad2and Smad3. Clinical trials of Celecoxib may be hostile due to increased risks of EMT in unselected NSCLC patients.Part I Celecoxib induces EMT process in A549cells.ObjectiveThe effect of Celecoxib on the EMT process stimulated by TGF-β1in A549cells was evaluated by western blot, immunofluorescence, and transwell assay. Methods1. A549cells were maintained in12-well plates with serum-free DMEM medium. The medium was exchanged every day, whilst various concentrations (2,4,6μ M) of Celecoxiband/or TGF-β1(1ng/ml) were added.24to72h later, the cells were harvested and the expression of E-cadand vimentinwere determined by western blot.2. E-cad and vimentin detected by immunof luorescent staining in A549cells treated with various concentrations of Celecoxib.3. Motility of A549cells after treated with Celecoxib was assessed by transwell assay.Results1. Decreased expression of E-cad and increased expression of vimentin were induced after treated A549cells with TGF-β1(1ng/ml) for72h. Cell morphology transformed from a classic cobblestone epithelial morphology to a spindle-shape, elongated shape. Furthermore, downregulation of E-cad and upregulation of vimentin induced by TGF-β1was enhanced by Celecoxib. And Celecoxib alone also promoted the expression of vimentin and decreased the expression of E-cad in a time and dose-dependent manner. Whereas, noticeable morphological changes were not found when treated A549cells with Celecoxib alone.2. Downregulation of E-cad and upregulation of vimentin were also found in A549cells after application of Celecoxib (4,8μM) by immunofluorescence.3. Significant increases in the number of migratory cells emerged by stimulated A549cells with Celecoxib (4,6μM) for18h.ConclusionsCelecoxib induces EMT process in A549cells via a TGF-β1independent pathway. PartⅡCelecoxib induces cancer cells to undergo EMT in a cox-2independent pathway.Objective To further study the role of cox-2protein in Celecoxib induced EMT. MCF-7cells (cox-2protein negative) were used as model system to assess the effect of Celecoxib in EMT process.Methods1. The expression of Cox-2protein in MCF-7and A549cells was detected by western blot.2. MCF-7cells were maintained with serum-free DMEM medium in12-well plates treated by various concentrations of Celecoxib for indicated periods.24to48h later, the cells were harvested and the expression of E-cad and vimentin were tested by western blot analysis.3. A549cells were maintained in12-well plates with serum-free DMEM medium. The medium was exchanged every day, whilst various concentrations of TGF-β1and/or Etodolacwere added.72h later, the cells were harvested and the expression of E-cad and vimentin were detected by western blot.Results1. Cox-2protein is positive expressed in A549cells, but negative in MCF-7cells.2. In MCF-7cells, Celecoxibincreased the expression of vimentin and decreased the expression of E-cad in a dose and time dependant manner.3. Etodolac induced mesenchymal-to-epithelial transition (MET) in A549cells, as it reversed TGF-β1induced downregulation of E-cad and upregulation of vimentin. Similar results were also observed when Etodolac was used alone.ConclusionsDifferent cox-2inhibitorshave diverse influence on EMT process.Celecoxib induces cancer cells to undergo EMT in a cox-2independent pathway, whereas Etodolac promotes a MET process in A549cells. It has been reported that Etodolac can not induce upregulation of E-cad in HT29, MKN45,5637and KK47cancer cell lines65’66. Therefore, clinical trials of Cox-2inhibitors in cancer patients should be cautiously carried out as the effects of Cox-2inhibitors on the EMT process depend on the characteristics of both the compounds and the cancer cells. PartⅢ Mechanism of EMT process induced by Celecoxib in A549cells.ObjectiveAim to investigate the mechanism of EMT induced by Celecoxib, phosphorylation of ERK, AKT, MEK, Smads (p-ERK, p-AKT, p-MEK, p-Smad2, p-smad3), E-cad and vimentin were examined in A549cells treated with Celecoxib and MEK/ERK, PI3K/AKT inhibitors.Methods1. P-ERK, p-AKT, p-MEK, p-Smad2and p-smad3were examined in A549cells and MCF-7cells after treated by various concentrations of Celecoxib(2-6μM) for indicated periods (5-180mins) in serum-free DMEM medium.2. A549cells were used as a model system and treated with Wortmannin (PI3K inhibitor) or AZD6244(MEK inhibitor) or MK-2206(AKT inhibitor) in combination with Celecoxib (6μM). P-MEK, p-AKT, p-ERK, E-cad, vimentin were detected after indicated time periods.Results1. P-ERK, p-AKT and p-MEK were substantially increased by Celecoxib in a dose dependant manner and lasted for up to2-3h. Phosphorylation of Smad2/3was not affected by Celecoxib with or without TGF-β1.2. PI3K inhibitor attenuated p-AKT expression but increased the activity of ERK, while MEK inhibitor reduced p-ERK expression but enhanced AKT phosphorylation. Celecoxib induced downregulation of E-cad was intensified by MK-2206. Meanwhile, AZD6244restored loss of E-cad stimulated by Celecoxib. Cox-2protein was dramatically reduced by Celecoxibplus AZD6244or MK-2206. However, decreased E-cad was found only when Celecoxib combined with MK-2206, whilst increased expression of E-cad was found when Celecoxib plus AZD6244.Conclusions1. Celecoxib induces EMT in human lung cancer A549cells through MEK/ERK signaling pathway that partially cross-inhibited by activation of PI3K/AKT pathway.2. The expression of cox-2protein did not correlate with E-cad.