Research On The Effects And Reasons Of Indomethacin Combined With Oxaliplatin On Transplantation Tumor Model For Human Lung Cancer In Nude Mice

Objective: About120million patients worldwide are diagnosed withlung cancer each year, and the incidence and mortality of lung cancer showedan increasing trend, the prognosis is poor, the incidence of lymph nodemetastasis earlier, more common clinical. Because early symptoms of lungcancer are secret, metastasis has occurred in the majority of patients have beendiagnosed. Invasion and metastasis is one of the important reasons that affectthe prognosis of patients with lung cancer, and it is a significant method toexplore the lymph node metastasis related cytokines and to explore theinhibitors of lymphatic metastasis for curb ing lung cancer progression andimproving the prognosis of patients.With developing constantly of molecular biology, the study that on themechanism of invasion and metastasis of lung cancer and its specific factorexpression is unceasingly thoroughing as well.EGFR (epidermal grwth factor receptor) epidermal growth factorreceptor is an important member of the ErbB receptor family, which wasoverexpressed in a variety of tumor tissue, especially upregulated in the oftumor with lymph node metastasis. Zhu Congzhong[1]using tissue chiptechnology and immunohistochemical method detected EGFR andcyclooxygenase-2(COX-2) expression in the lung tissue was positivelycorrelated, both the two factors are closely related with lymph node metastasisof lung cancer, activation and regulation of COX2and EGFR are related withsignaling pathways contain mitogen-activated protein kinase(mitogen-activated protein kinase, MAPK) and phosphatidylinositol3-kinase(phosphatidylinositol3-kinase, PI3K)，there is a direct or indirect mutualinteractivation among them. C-erbB-2(HER-2, Neu) as an important member of the epidermal growthfactor family, is a product of the Proto-Oncogene encoding. HER-2is mostlythrough the activation of the oncogene amplification and overexpression，furthermore，ultimately regulate cell proliferation, differentiation, migration,adhesion, survival, apoptosis, accelerate cell proliferation，accelerated cellcycle to enhance aggravation by certain signal transduction pathways，meanwhile it will inhibition of apoptosis in malignant cells and becomeresistant to most chemotherapy drugs.Chemokine receptor4(CXCR4) belong to the CXC receptor subfamily,which is the exclusive receptor of chemokines CXCL12scilicetstroma-derived factor-1(SDF-1). With the Chemokine specificreceptor-mediated effecting, they constitute the CXC-L12/CXCR4biologicalaxis to form an coupling of molecules。It is closely related to informationtransfer between cells, cell migration. Recent studies have found that theSDF-1/CXCR4axis plays an important role in tumor cell migration, invasionand metastasis[2-3]β-catenin (β-catenin) is a key molecules of cadherin-catenin complex(E-cadherin/catenin) and Wnt signaling pathway. The abnormal accumulationof β-catenin, causing cell proliferation or tumorigenesis,and closely associatedwith lymph node metastasis of lung cancer.Indomethacin(Indomethacin, In), a non-steroidal anti-inflammatory drugs(NSAIDs), is few reported with regard to the inhibitory effect and mechanismof lung cancer xenografts in the literature. But in recent years, the study that Incombined with COX-2inhibitors whether or not can enhance the cytotoxiceffects of chemotherapeutic drugs and anti-the lymphatic generated effectgradually increases. Oxaliplatin (oxaliplatin) is a third generation platinumbroad-spectrum anticancer drugs, and alkylated by generating conjugate rolein the DNA, forming intra-chain and inter-chain crosslinking, therebyinhibiting DNA synthesis and replication. The purpose of this experiment is tostudy the role of indomethacin combine oxaliplatin to resist human lungcancer xenografts in nude mice and the effect to expression of EGFR, beta-catenin, C-erbB-2, CXCR4, in order to investigate the mechanism ofinvasion and metastasis of lung cancer, for the treatment of lung cancer toseek a new target.Method:25nude mice,4-5-week-old, BALB/c, male, weighing20～24grams,fed with sterile feed and sterile purified water in a rearing environment withambient temperature, suitable humidity and no specific pathogens. With F12kmedium containing10%newborn bovine serum,100U/ml penicillin and100U/ml streptomycin, human lung cancer A549cells are cultivated in anincubator of37℃with5%C02and cells are digested with0.25%trypsin andare transfer of culture when cells grow adherently to70%～80%confluence.PBS washed and digested the A549cells in logarithmic growth phase with0.25%trypsin and to prepare a single cell suspension，then collected it in acentrifuge tube, centrifuge1000rpm equilibrium centrifugation5minutes,discard the supernatant. Dilute them with serum-free F12k dilution medium toa cell density of1×10~7/ml after counted with CBC board. The human lungcancer A549cells suspension0.2ml was injected subcutaneously in the leftRegio axillaries of nude mice to establish transplantation tumor model for lungcancer in nude mice.25nude mice formed tumor. Measuring the longdiameter(a) and short diameter(b) of tumor volume with sliding caliper, toestimate tumor approach volume according to formula V=ab2/2。After thetumor diameter reached an average of4mm, removed the nude mouse whichhad the smallest tumor volume and then randomly divided them into fourgroups,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, C-erbB-2, CXCR4, β-catenin protein expressions,RT-PCR assay Tumor EGFR, C-erbB-2, CXCR4, β-catenin mRNAexpressions. Results:All the nude mice having an inoculation of lung carcinoma A549cellsdeveloped tumors. We performed immunohistochemistry analysis of EGFR,C-erbB-2, CXCR4, β-catenin protein in tumor samples. Statistical analysisshowed that the expression levels of EGFR, C-erbB-2, CXCR4, β-cateninprotein of Indomethacin-treated group and Indomethacin combined withoxaliplatin-treated group，Compared with control group，were significantlyreduced (respectively, P <0.05)， and the expression levels ofCompared-treated group were lower than Indomethacin-treatedgroup(respectively, P <0.05). Compared with control group，There was nodifference in the expression levels EGFR, C-erbB-2, CXCR4, β-cateninprotein of oxaliplatin-treated group(P>0.05).According to the real-time fluorescence quantitative analysis of EGFR,C-erbB-2, CXCR4, β-catenin mRNA, statistical analysis showed that theexpression levels of EGFR, C-erbB-2, CXCR4, β-catenin mRNA ofIndomethacin-treated group and the Indomethacin combined withoxaliplatin-treated group were significantly reduced than the controlgroup(respectively, P<0.05), meanwhile the expression levels ofCombination-treated group were lower than Indomethacin-treatedgroup(respectively, P <0.05); compared with the control group, the expressionlevels of EGFR, C-erbB-2, CXCR4, β-catenin mRNA of oxaliplatin-treatedgroup were no significant difference (P>0.05).Conclusion:1Indomethacin were able to significantly inhibit the the expression ofEGFR, C-erbB-2, CXCR4, β-catenin protein and EGFR, C-erbB-2, CXCR4,β-catenin mRNA of lung cancer xenografts tumor in nude mice. It hinted thatIndomethacin could inhibit the expression of lung cancer correlation factorsEGFR, C-erbB-2, CXCR4,β-catenin，accordingly，to inhibit the growth andinvasion of transplantation tumor for human lung cancer in nude mice.2Compared with single-agent, Indomethacin combination withoxaliplatin-treated group were able to more significantly inhibit the theexpression of EGFR, C-erbB-2, CXCR4, β-catenin of lung cancer xenografts tumor in nude mice. Indicating Indomethacin may have againsted theresistance caused by oxaliplatin, so the two drugs in combination has asynergistic effect to improve the efficacy of anti-tumor and anti-lymph nodemetastasis.