Effect Of Lumiracoxib On Bcl-2 Modulating Apoptosis Of Human Non-small Cell Lung Cancer Cells

Non-small cell lung cancer(NSCLC) is one of the leading cause of cancer morbidity and death and the poorest of prognosis cancer throughout the world.The 5-year survival rate remains 4～14%despite recent important therapeutic advances.The main reasons are relapse in region and metastasis in distant place.The basical and clinical studys have been found that NSCLC is one of the cancer that can transfer early.More than fifty percent patients diagnosed are advanced NSCLC and the overall prognosis remains poor. Although the rapid development of chemotherapy,therapeutic effect seem to have reached a plateau.Thus it is imperative to develop more effective in NSCLC agents. Recently it has been found that cyclooxygenase-2(COX-2) is overexpressed in NSCLC and seems to associate with carcinogenesis,tumor growth and prognosis.So the selective inhibitor of COX-2 is one class of agents that was used for the prevention and treatment of NSCLC.Lumiracoxib(LUM) is a highly selective cyclooxygenase-2(COX-2) inhibitor with anti-inflammatory,analgesic and antipyretic activities comparable with class -specific,but with much improved gastrointestinal safety.No study has revealed that LUM has anti-proliferation and induce apoptosis activity on human NSCLC cell lines in vitro.Although some antineoplastic agents including Docetaxol(DOC) have been demonstrated significant clinical activity against NSCLC,the use was limited by the side effects.So it is very important to investigate whether COX-2 inhibitors are beneficial for the treatment of NSCLC if given in combination with the effectively antineoplastic agents, such as DOC.In this study,we have firstly investigated the growth-inhibitory effect of LUM alone or in combination with DOC in human NSCLC cell lines A549 and NCI-H460 and the possible mechanism,especially about whether the apoptosis pathway of Bcl-2 induced by Lumiracoxib is through ERK signal transduction.This may be helpful for elucidating the relationship between the expression of COX-2 and the development of NSCLC in order to provide the theory foundation.1.Effect of LUM on the growth of A549 and NCI-H460 cellsWe analyzed the effects of LUM on the proliferation ofA549 and NCI-H460 cell lines. A dramatic dose-dependent augmentation of CPIR was seen in cells incubated with LUM at concentrations of 15～240μmol·L^-1 for 48 hours.The higher the concentration of LUM was,the stronger the cytotoxic effect reached,which suggested an obvious dose-dependent manner of LUM.The r value of dose-effect curves was: r=0.957(P＜0.01) for A549 and:r=0.912(P＜0.01) for NCI-H460.And the IC₅₀ value of LUM was 2597.17μmol·L^-1 and 832.72μmol·L^-1(P＜0.01) for A549 and NCI-H460 cells,respectively.The inhibitory effects were augmented with the prolongation of culture time,suggesting a time-dependent manner of LUM on above cell lines.2.The synergistical inhibition of LUM combined with DOC in A549 and NCI-H460 cellsTo investigate the synergistically inhibitory effects of LUM and DOC on the proliferation of A549 and NCI-H460 cell lines,five doses of LUM(15～240μmol·L^-1) were used to combine with DOC(0.2～2μmol·L^-1).CDI was used to analyze the synergistically inhibitory effect of drug combination.LUM combining with different concentrations of DOC showed different extent synergistically inhibitory effect on the proliferation of the two cell lines in appropriate concentrations.LUM and DOC yielded synergistic interactions across a wide concentration range(CDI＜I).3.Possible mechanisms of LUM in A549 and NCI-H460 cells3.1 Morphological evidence of apoptosis induced by LUM or combined with DOC in A549 and NCI-H460 cellsA549 and NCI-H460 cells treated with LUM at concentration of 120μmol·L^-1 for 48 hours.Morphological evidence of apoptosis was demonstrated by using acridine orange(AO) fluorescence staining.It shows that AO permeated through cell membrane and made the nuclei appear green.LUM caused the typically apoptotic changes,such as chromatin condensation,membrane blebing,deformed and fragmented nuclei and reduction in cell volume,and so on.The number of dead cells was increased when the cells were treated with the combination of LUM(120μmol·L^-1) and DOC(1μmol·L^-1).3.2 Effect of LUM alone or combined with DOC on cell cycle in A549 and NCI-H460 cellsA Flow Cytometry(FCM) assay was performed to analyze apoptotic rate of A549 and NCI-H460 cells treated with LUM or DOC or a combination of the two agents for 24 hours.The higher the concentration of LUM was,the higher the apoptotic rate reached,which suggests an obvious dose-dependent manner of LUM.The apoptotic rate was augmented with the prolongation of culture time,suggesting a time-dependent manner of LUM on above cell lines.The apoptotic rate of LUM combinated with DOC was higher than that drugs was used alone.This demonstrates that LUM in combination with DOC had synergistic apoptotic-induction effect on the two cell lines. 3.3 Cell cycle perturbation caused by LUM in A549 and NCI-H460 cellsListmode software was used to analyze the kinetic changes of cell cycle distribution. For A549 and NCI-H460 cells exposed to various concentrations of LUM,the S-phase fraction decreased while G₀/G₁ fraction increased in a dose-dependent manner.And the percentages of cells in G₀/G₁ phase increased after 12,24,36,48 hours,respectively, when compared with untreated control cells.It indicates that LUM might arrest the cell cycle at the G₀/G₁ phase.3.4 Effect of LUM on the level of PGE₂ and cAMP in A549 and NCI-H460 cellsThe levels of PGE₂ and cAMP were determined by radioimmunoassay(RIA). Compared with the control,the PGE₂ production was reduced and the level of cAMP was increased after the treatment with 15,30,60,120,240μmol·L^-1 of LUM, respectively.It indicates that LUM may inhibit the COX-2 activities by decreasing its secretion which depends on up-regulating the level of cAMP.3.5 Effects of LUM on cyclooxygenase-2 gene expression in A549 and NCI-H460 cellsWe performed the Western blot analysis to examine whether COX-2 protein is expressed in A549 and NCI-H460 cells.COX-2 protein was highly expressed in A549 and NCI-H460 cells.After treatment with 15～240μmol·L^-1 LUM for 24 h,LUM significantly decreased the expression level of COX-2 protein in A549 cells,but not in NCI-H460 cells.It indicates that COX-2 pathway is not required for the antitumor of LUM.3.6 Effects of LUM on the level of ERK2,phosphorylation ERK,and Bcl-2 protein in A549 and NCI-H460 cellsCells growing to confluence were treated with IL-1β(1 ng/ml) prior to stimulation with LUM(0,15,30,60μmol·L^-1) for 24 hours.Western blot analysis was used to examine the level of ERK2,p-ERK and Bcl-2 protein in human NSCLC cell lines.We found that LUM both inhibited the up-regulated ERK2 and p-ERK induced by IL-1βand down-regulated Bcl-2 protein expression in A549 and NCI-H460 cells.This results show that LUM-induced Bcl-2 gene expression may be via inhibiting the ERK2 and p-ERK in A549 and NCI-H460 cells.3.7 Effect of LUM on the expression of Bcl-2 and Bax in A549 and NCI-H460 cellsWestern blot analysis was used to examine the expression of Bcl-2 and Bax in A549 and NCI-H460 cells.It was found that LUM decreased the expression of Bcl-2 protein, However,the expressions of Bax protein increased in A549 dose-dependently and kept stable on NCI-H460 cells.The ratio of Bcl-2/Bax decreased correspondingly.4.ConclusionThe results in the present study demonstrated that LUM had antiproliferative effect in A549 and NCI-H460 cells,with an obvious dose-and time-dependent manner.LUM,in combination with anticancer drugs DOC,had a significantly synergistically growth-inhibitory effect on the two cell lines,which lead to the apoptosis and cell cycle arrest.The antitumor effect of LUM might be related to significantly inhibiting the COX-2 activities dependently on the up-regulated level of cAMP.LUM might induce Bcl-2 gene expression via inhibiting the ERK2 and p-ERK in human NSCLC cell lines.