Inhibitory Effects And Underlying Mechanism Of CCR5 Antagonist On Non-small Cell Lung Cancer A549 Cells

As the leading cause of morbidity and cancer-related death worldwide,lung cancer has a serious threat to human health.Non-small cell lung cancer(NSCLC)is the most common subtype,accounting for nearly 80% of lung cancer cases.Because of the insidious onset and rapid development of lung cancer,more than 75% patients are diagnosed at advanced stages,and are inoperable.The advanced and metastatic NSCLC patients have a poor prognosis due to the lack of curable drugs.So,a novel and more effective strategy for targeting cancer therapy should be developed.CCR5,one of the coreceptors for human immunodeficiency virus type-1(HIV-1),play a key role in the process of HIV infection.As a target for therapeutic intervention,CCR5 are widely applied in the design and development of AIDS drugs.With the rapid development of tumor molecular biology,tumor immunology and cell biology,considerable progress has been made in understanding the role of CCR5 in tumorigenesis and development.CCR5 overexpression is shown in NSCLC patients with predicted poor prognosis.It plays a crucial role in NSCLC processes,which has an indirect effect on cancer progression by controlling the antitumor immune response.The expression of CCR5 could promote tumor growth and contribute to tumor metastasis,in different types of malignant tumors.Our previous work demonstrated that the CCR5 antagonist Maraviroc(MVC)could inhibit the invasion and migration of the A549 cells,and inhibit the cell growth at high concentrations.So,CCR5 targeted therapy was considered as a novel strategy for the treatment of NSCLC.In this study,we combined computational simulation and experimental assays to discover the potential candidates for targeted therapy in NSCLC and to explore the potential mechanisms underlying the antitumor effect.The main works and conclusions are listed as follows.(1)A hierarchical virtual screening was employed to find novel CCR5 antagonists with antitumor activity.In this study,the crystal structure of the CCR5 was recovered from the RCSB Protein Data Bank.A series of molecular docking were utilized as a query to identify novel chemical entities from structural databases.To pursuit the promising hits,the docking score of MVC was fixed as the threshold value for the screening.A total of 15 hits were obtained after employing the docking score-filter.Refinement by drug-likeness analysis,molecular dynamics simulations and binding free energy analysis,the potential inhibitors(nifeviroc)was identified.(2)To investigate the effect of CCR5 antagonists on inhibiting A549 cells growth and its mechanism.CCK8,Edu cell imaging assay,clone formation assay and flow cytometry were used to detect the effect of CCR5 antagonists on A549 cell growth,cell cycle and proliferation.The results showed that nifeviroc had a superior growth-inhibitory activity on A549 cell than MVC.Nifeviroc inhibited cell growth,probably through inducing G0/G1 phase cell cycle arrest.Western blotting results suggested that the G1/G0 phase arrest by nifeviroc was due to the expression of p21 was up-regulated,while the expression of cyclin D1 and CDK4 proteins was markedly reduced.(3)To explore the effects of CCR5 antagonists on apoptosis in A549 cells.Nifeviroc-induced apoptosis in A549 cells was confirmed using Hochest 33258 staining and Annexin V-FITC/PI double staining assays.Compared with the MVC,nifeviroc exhibited more powerful potency.Using the specific fluorescent dyes DCFH-DA and JC-1 to determine the ROS production and mitochondrial membrane potential,the result showed nifeviroc trigger apoptosis in A549 cells,accompanied by an increase in the intracellular ROS level and disrupting mitochondrial membrane potential.(4)To explore molecular mechanisms underlying the anticancer effect of CCR5 antagonists on A549 cells.(1)RT2 Profiler? PCR array was carried to explore gene-expression profile changes of A549 cells treated with nifeviroc.There were 84 differentially expressed genes that had been screened,including 17 up-regulated genes and 33 down-regulated genes.Among them,the apoptosis-related differentially expressed genes were categorized into the extrinsic and intrinsic apoptosis pathways.(2)Activation of caspase-9,caspase-8,caspase-7 and caspase-3 in nifeviroc-induced apoptosis were detected by western blot and caspase activity assay.Bcl-2 and its family proteins play pivotal roles in the regulation of the cytochrome c release and mitochondria functions.The mitochondrial apoptotic pathway relevant assays results showed nifeviroc increased the pro-apoptotic protein Bax expression and decreased anti-apoptotic protein Bcl-2 expression.It led to the permeabilization of the outer mitochondrial membrane(OMM),disrupted mitochondrial membrane potential and promoted the release of cytochrome c.Cytochrome c promoted cleavage of caspase-9.Activated caspase-9 in turn cleave caspase-3,caspase-7 and PARP.Additionally,the expression of survivin in nifeviroc-treated cells was significantly down-regulated.Taken together,nifeviroc induced apoptosis in A549 cells via activation of caspase,modulation of the protein levels of Bax,Bcl-2 and survivin.(5)To investigate the effect of CCR5 antagonists on adhesion,invasion and migration in A549 cellsThe effects of nifeviroc on the adhesion,invasion and migration of A549 cells were evaluated using the cell matrix adhesion assay,wound-healing assay,transwell chamber assay.The cell matrix adhesion assay showed that nifeviroc significantly inhibited the adhesion of A549 cells to fibronectin.It also markedly suppressed the invasion and migration of A549 cells in vitro.(6)To explore the mechanisms of CCR5 antagonists inhibited migration and invasion in A549 cells.The results of human extracellular matrix and adhesion molecules assay showed that nifeviroc suppressed the abilities of migration and invasion in A549 cells possibly through downregulating the expression levels of matrix metalloproteinase(MMP-1,MMP-2,MMP-9),while enhance the expression of tissue inhibitor of matrix metalloproteinases(TIMP-1,TIMP-2).(7)Molecular modeling study on the interaction between CCR5 and antagonists.Based on the analysis mentioned above,MD simulation and binding free energy analysis were used to explore the binding modes of antagonists and CCR5.Results showed that nifeviroc retained the important common binding mode known for MVC.They occupied an almost similar space in the binding site at the bottom of a pocket.The aromatic nucleus reached deep into the pocket to form hydrophobic interactions with residues Tyr37,Trp86,Thr195,Glu283,Tyr251 and Thr259,respectively.As the allosteric modulators of CCR5,MVC and nifeviroc stabilized CCR5 in an inactive conformation.The analyses of root-mean-square fluctuation(RMSF)showed that CCR5-nifeviroc and CCR5-MVC shared similar distributions and trends for dynamic features.The N-,C-terminin and intracellular and extracellular loops of CCR5 had higher conformational flexibility,while alpha helixes and beta strands were more rigid.At the transmembrane regions,nifeviroc exhibited stronger rigidity than MVC.This indicated that the combination of nifeviroc and CCR5 increased the structural stability of the binding pocket and the transmembrane domain,which was helpful to maintain the binding state.Binding free energies calculations by MM-GBSA method indicated that the binding affinity of nifeviroc was higher than that of the MVC.It could be inferred that nifeviroc bound more tightly to CCR5 than MVC.During the binding process,van der Waals interactions provided the substantial driving force.The most favorable contributions were from Trp86 and Glu283.In summary,a successful Virtual Screen-based identification of CCR5 antagonists with antitumor activity was established which leads to discovery of nifeviroc.This study showed nifeviroc plays a role in induction of the apoptosis and inhibition of the proliferation,invasion,metastasis of non-small cell lung cancer A549 cells.It has a potential development prospect as a new drug for lung cancer treatment.The theoretical results of this study offer important clues for the deep mechanism researches.