Mechanisms By Which Celecoxib Inhibits Vasculogenic Mimicry In Lung Cancer

Background and Objectives:Lung cancer is the leading cause of cancer-related death worldwide,and non-small cell lung cancer(NSCLC)is the most common of lung cancer cases.More than 40% of NSCLC patients are initially diagnosed at the advanced stage and lose the chance of surgery.Despite progress in radiochemotherapy,targeted therapy and immunotherapy,cancer relapse and metastasis restrict the effect of these treatments.Efforts to better understand the mechanisms underlying cancer relapse and metastasis,and to develop potential strategies to overcome these problems,are urgently needed.Neovascularization is a key factor that contributes to the growth,invasion and metastasis of tumors,and vasculogenic mimicry(VM)is an important form of neovascularization found in highly invasive tumors,including lung cancer.In VM,tumor cells mimic endothelial cells to form vessel-like structures transporting blood elements.Recently,VM has been reported to correlate with the poor prognosis of lung cancer patients and to possibly contribute to the resistance to anti-angiogenic agents.Despite mounting studies focus on VM,the mechanisms underlying VM formation remain unclear.Celecoxib(CXB)(Celebrex?,Pfizer)is a member of non-steroidal anti-inflammatory drugs(NSAIDs)that selectively inhibit cyclooxygenase-2(COX-2).This agent plays the roles of suppressing inflammation,tumor growth and angiogenesis and sensitizing radiochemotherapy.We explored the effects of CXB on VM in lung cancer and found that VM was inhibited by this agent and the inhibition was not directly related to COX-2 levels,indicating that CXB may affect VM in a COX-2-independent manner.This finding is consistent with recent reports,i.e.CXB has COX-2-independent properties.The current study speculated that CXB may repress VM in lung cancer by targeting the molecules other than COX-2.Methods:1)In vitro examination of CXB's effects on VM in lung cancer cellsThree distinct lung cancer cell lines(A549,H1650 and H460)were cultured.Tube formation by tumor cells was observed in 3D Matrigel,and PAS staining confirmed that the tubes had VM features.After adminitering ionizing radiation(5 Gy),we tested the tube-forming abilities of tumors cells in 3D Matrigel.CXB at certain concentrations was then added into the culture medium,and 24 h later,the Matrigel tube formation assay was repeated to determine the effects of CXB on VM in lung cancer cells.2)Bioinformatic prediction of new targets of CXBA total of 84 genes related to vascularization were chosen to constitute the database of potential targets.The 3D structure of each protein expressed by these genes was searched in Uni Prot and PDB databases,and the structure of CXB downloaded from Pub Chem database.The MGLTools 1.5.6 software was used to optimize the structures of the ligand and receptor into pdbqt format.After setting the docking box,we completed the molecular docking with Autodock_vina 1.1.2 in Cento S6.5 Linux system.The docking sites and residues were identified with Py MOL and PLIP.Comparing the binding sites of CXB and reported inhibitors,we revealed that CXB could bind to the functional pocket of proteins APN,AKT1,ITGAV and NOS3.3)Preliminary validation of the newly predicted targetsThe four predicted targets(APN,AKT1,ITGAV and NOS3)were confirmed by biological assays.Due to the difficulty in purchasing antibodies against NOS3,we tested proteins APN,AKT1 and ITGAV,and used COX-2 as a control.A)We mixed CXB and the four proteins(APN,AKT1,ITGAV and COX-2)respectively,and tested the fluorescence quenching effects of CXB on each protein with Spectra Max M2 at three different temperatures(30°C,34°C and 39°C).Direct binding was assessed based on the fluorescence intensity of each CXB-protein system.B)In the three cell lines(A549,H1650 and H460),the differencial expressions of APN,AKT1,ITAV and COX-2 were detected.Under the irradiation at a dose of 5Gy,the differencial expressions of the four genes were detected.In the presence of CXB,the gene expressions were also determined.C)Female nude mice(aged 5 weeks)were injected with tumor cells to establish xenograft models and randomly divided into 4 groups(CXB,RT,RT and CXB,and control groups).Radiation was given at a dose of 15Gy(320KV),and CXB was administered by gavage feeding.The tumor tissues were dissected and immunofluourescently stained with the double stain containing m CD31 and h E-cad antibodies and with periodic acid-Schiff(PAS)to observe VM.Results:1)CXB inhibited VM in lung cancer.All the three cell lines(A549,H1650 and H460)were able to form vessel-like structures in 3D Matrigel and were positive for PAS staining,indicating the formation of VM.After 5Gy irradiation,the cells exhibited significantly enhanced VM-forming ability(P<0.01).After administration of CXB,the VM completely disappeared,and the inhibition of VM by CXB seemed not associated with COX-2 levels.2)Four proteins were predicted targets of CXB.Among the 84 proteins related to vascularization,55 had identified 3D structures.Molecular docking revealed that the binding free energy was smaller than-8.0 kcal/mol for 14 proteins,smaller than-9.0 kcal/mol for 6 proteins,and smaller than-10.0 kcal/mol for 1 protein.The last 7 proteins were selected for further validation.Comparative docking showed that CXB could bind to the functional sites of 5 proteins(APN,NOS3,ITAV,AKT1 and COX-2).3)CXB directly interacted with three of the five proteins.Fluourescence spectrum analysis demonstrated that increasing CXB decreased the fluourescence intensity of each of the 3 CXB-protein systems(AKT1,ITAV and COX-2),and increasing temperature enhanced the fluourescence intensity of each system(AKT1,ITAV and COX-2),suggesting that CXB may directly bind to the three proteins.In lung cancer cells,increasing CXB decreased the expressions of the four genes(APN,AKT1,IGTAV and COX-2).In different cell lines,irradiation induced elevated expressions of three genes(APN,AKT1 and IGTAV),and CXB significantly suppressed the expressions of these three genes(APN,AKT1 and IGTAV).In xenograft models,the tissue was double positive for m CD31 and h E-cad by immunofluourescence staining,indicating the formation of VM.Irradiation could enhance the VM-forming ability of cancer tissues while CXB could inhibit VM formation.Conclusions:1)Lung cancer cells are able to form VM,and this property can be enhanced by irradiation while inhibited by CXB,and the inhibitory effects of CXB seem not related to COX-2 levels.2)CXB can directly interact with three proteins(APN,AKT1 and ITAV)apart from COX-2 to different extent,which is a COX-2-independent “off-target” effect of CXB.3)In different lung cancer cell lines,irradiation can induce the expressions of APN,AKT1 and ITAV to different extent,and CXB is able to inhibit VM and enhance the radiation sensitivity of lung cancer cells..4)Prediction of “off-target” receptor proteins for CXB by bioinformatic methods and validation of the protein targets by in vitro and in vivo experiments have theoretical significance and practical implications as an example of “conventional drug in new use”.