Research On The Effects Of Indomethacin Combined With Oxaliplatin Affect Lymph Node Metastasis On Transplantation Tumor Model For Human Lung Cancer In Nude Mice

Objective: Lung cancer is the leading cause of cancer death in the worldnow. In recent years the incidence rate and the mortality rate of lung cancer inour country are considerably on the rise,which has become a serious threat tohuman life and health.Since early lung cancer showed only a cough, chest pain,chest tightness, not enough attention has been paid by patient, so distantmetastasis has occurredat the moment of clinical diagnosis., losing the chanceof operation. In our country, non small cell lung cancer accounts for80%,including adenocarcinoma, squamous cell carcinoma and large cell carcinoma.As advanced lung cancer treatment methods, radiotherapy and chemotherapyhave a certain effect, but the prognosis of the patients is not ideal, the five yearsurvival rate is still low, the average survival time is only10-12months.Invasion and metastasis is one of the most important reasons that affect theprognosis of patients with lung cancer, So the related molecular mechanismsof metastasis and targeting therapy of lung cancer is so important for advancedlung cancer patients.Epidermal growth factor receptor (EGFR) is akind of170KD tyrosinekinase receptor, a member of the HER/ErbB family. EGFR is expressed in avariety of solid tumor tissue.EGFR has correlated with cell proliferation,angiogenesis, tumor invasion, metastasis and the inhibition of apoptosis[1-3].E-cadherin (E-cad)， a calcium-dependent intercellular adhesionmolecule，plays an important role in the maintenance of cell morphology, cellmovement, and cell adhesion function.Intercellular adhesion molecule-1(ICAM-1) is a transmembraneglycoprotein belonging to the immunoglobulin superfamily, is the ligands oflymphocyte function-associated antigen-1(LFA-1). ICAM-1participates in the progressiong of cancer and inflammation when binds its ligand.CD44v6is a transmembrane glycoprotein belonging to the cell adhesionmolecules family. Mainly involved in mediating the interactions betweencell-cell and cell and the extracellular matrix and tumor invasion andmetastasis.Indomethacin(Indomethacin, In), a nonspecific cyclooxygenase-2(Cyclooxygenase-2, COX-2) inhibitor, is a kind of non-steroidalanti-inflammatory drugs, mainly used for fever and relieve inflammatorypain.Studies have shown that it has anticancer effect[4]. Oxaliplatin (oxaliplatin)is a third generation platinum broad-spectrum anticancer drugs, and alkylatedby generating conjugate role in the DNA, thereby inhibiting DNA synthesisand replication.The purpose of this experiment is to study the role of the non-specificCOX-2inhibitor indomethacin to resist human lung cancer xenografts in nudemice and the effect to expression of EGFR, CD44v6, ICAM-1, E-cad, in orderto investigate the mechanism of invasion and metastasis of lung cancer, andthe synergetic effect with oxaliplatin, to provide a theoretical basis for the lungcancer treatmentMethod:25nude mice,4-5-week-old, BALB/c, male, weighing20~24grams,raising in the IVC environment. With F12k medium containing12%newbornbovine serum,100U/ml penicillin and100U/ml streptomycin, human lungcancer A549cells are cultivated in an incubator of37℃with5%C02and cellsare digested with0.25%trypsin and are transfer of culture when cells growadherently to70%~80%confluence.Made the A549cells in logarithmicgrowth phase with into a single cell suspension，then injected subcutaneouslyin the left Regio axillaries of nude mice to establish transplantation tumormodel for lung cancer in nude mice. According to formula V=xy2/2(x the longdiameter,y the short diameter), to estimate tumor approach volume。After thetumor diameter reached an average of4mm, removed the nude mouse whichhad the smallest tumor volume and then randomly divided them into four groups,6nude mice each group: control group, Indomethacin-treated group,oxaliplatin-treated group, and Indomethacin combined with oxaliplatin-treatedgroup. Medecines were administered respectively. Execute nude mice afteradministering42days, cut and transplant tumor tissue, immunohistochemistrydetection of EGFR, E-cad, ICAM-1,CD44v6protein expressions, RT-PCRassay Tumor EGFR, E-cad, ICAM-1,CD44v6mRNA expressions.Results:24nude mice having an inoculation of lung carcinoma A549cellsdeveloped tumors，the tumor formation rate was96%. the tumor formate in the5~14days after planting the tumor cells.14days after tumor cell implantation,tumordiameter was4mm.We performed immunohistochemistry analysis of EGFR, E-cad,ICAM-1，CD44v6protein in tumor samples. Statistical analysis showed thatthe expression levels of EGFR, CD44v6, ICAM-1protein ofIndomethacin-treated group， oxaliplatin-treated group and Indomethacincombined with oxaliplatin-treated group，Compared with control group，weresignificantly reduced,and the expression level of E-cad wasincreased(respectively, P <0.05)，and the expression levels of EGFR, CD44v6,ICAM-1proteinof Combination-treated group were lower thanIndomethacin-treated group and oxaliplatin-treated group,and the expressionlevel of E-cad was increased (respectively, P <0.05). The expression levels ofIndomethacin-treated group and oxaliplatin-treated group have no difference(P>0.05).According to the real-time fluorescence quantitative analysis of EGFR,E-cad, ICAM-1, CD44v6mRNA, statistical analysis showed that theexpression levels of EGFR, CD44v6, ICAM-1mRNA ofIndomethacin-treated group， oxaliplatin-treated group and Indomethacincombined with oxaliplatin-treated group were significantly reduced than thecontrol group and the expression level of E-cad was increased(respectively,P<0.05), meanwhile the expression levels of EGFR, CD44v6, ICAM-1mRNA of Combination-treated group were lower than Indomethacin-treated group and oxaliplatin-treated group,the expression level of E-cad wasincreased(respectively, P <0.05); the expression levels of EGFR, E-cad,CD44v6, ICAM-1mRNA of Indomethacin-treated group andoxaliplatin-treated group were no significant difference (P>0.05).Conclusion:Indomethacin alone or in combination with oxaliplatin both could be ableto inhibit the espression of EGFR, CD44v6, ICAM-1, promote the expressionof E-cad of lung cancer xenografts tumor in nude mice. Compared withsingle-agent, Indomethacin combination with oxaliplatin-treated group wereable to more significantly inhibit the the expression of EGFR, CD44v6,ICAM-1, promote the expression of E-cad. Indomethacin could effect theexpression of lung cancer metastasiscorrelation factors,EGFR, E-cad, CD44v6,ICAM-1，accordingly，to inhibit the invasion of transplantation tumor forhuman lung cancer in nude mice.The combination of Indomethacin andoxaliplatin have synergistic anti-tumor effect，so indomethacin could enhancethe anti-cancer effect of oxaliplatin.