Research On The Effects And Mechanisms Of Indomethacin And Oxaliplatin On Human Lung Cancer Xenograft In Nude Mice

Objective:According to WHO statistics,lung cancer has the highestdisease incidence and mortality rate among malignant tumors.In China,theannual incidence of lung cancer reaches to35patients in every100,000persons.The patients of non-small cell lung cancer take the percentage of80%to90%.Patients in early stage of lung cancer are lack of obvious symptomsand clinical diagnosis can only be reached in late stage.As a result,the study ofmolecular mechanisms and inhibition drugs of lung cancer metastasis play arole in early diagnosis.The invasion and metastasis of tumors is a complex multi-gene regulationprocess,while genes interaction is becoming a hot research field.COX-2(cyclooxygenase-2) is the key enzyme for many malignant tumors,which isoverexpressed in lung cancer,especially in adenocarcinoma.COX-2canpromote tumor cells proliferation and tumor angiogenesis.Moreover,it caninhibit tumor cells apoptosis and immune function of organisms,whilestimulating invasion and metastasis of tumors.Matrix metalloproteinase-2(MMP-2),the most widely spread enzyme,candegrade extracellular matrix and basement membrane,making it possible fortumor cells invading microvascular and lymphatic vessels from primaryorgans,trespassing target organs and finally forming metastases.CD44is an extremely broadly distributed transmembrane glycoprotein onthe surface of human cells.The products of CD44have two types.They arestandard CD44(CD44s)and variant CD44(CD44v).CD44v play an importantrole in tumorigenesis and metastasis.CD44v6is a kind of splice variant ofCD44v,which can change the composition and function of the adhesionmolecules of tumor cell surface that contribute to the metastatic potential of tumor cells.Survivin,a significant member of the family of inhibitor of apoptosisproteins(IAP),is the most potent inhibitor of apoptosis factor.Survivin has thefunctions of inhibiting apoptosis and regulating cell cycle.In addition,it isclosely related to differentiation and proliferation of tumor cells,vascularization, invasion and metastasis.The trait of malignancy is unlimited proliferation.Ki67is the well-studiedprotein which can show proliferative activity.The expression of Ki67canshow malignancy proliferative rate effectively.It is closely correlated to theproliferation,invasion,metastasis and prognosis of malignancy.Indomethacin(IN) as a kind of NSAIDs has antibiotic,antipyretic andanalgesic functions,while its inhibitory effects and mechanism in lung cancerxenograft are less reported.It is still unclear whether the combination of INand COX-2inhibitor can enhance the cytotoxic effects of chemotherapy drugs.This study aims to discuss effects and mechanisms of indometacin in lungcancer xenograft,the effects of Survivin,MMP-2,CD44v6and Ki67expressions.In addition,it further studies the molecular mechanism of lungcancer invasion and metastasis.Methods:1.Xenograft animal model of human lung cancer were established byinjecting A549cells into BALB/c nude mice subcutaneously.Then the micewere randomly divided into4groups:control group,Indomethacin-treatedgroup, oxaliplatin-treated group,and Indomethacin combined withoxaliplatin-treated group.Medecines were administered respectively.Executenude mice after administering42days,cut and transplant tumortissue,immunohistochemistry detection of MMP-2,CD44v6and Survivinprotein expressions,real-time assay tumor MMP-2,CD44v6and SurvivinmRNA expressions.2.Xenograft animal model of human lung cancer were established byinjecting A549cells into BALB/c nude mice subcutaneously.Then the micewere randomly divided into4groups:control group,Indomethacin Group One(1.0mg/kg/d),Indomethacin Group Two(2.5mg/kg/d),Indomethacin GroupThree(4.0mg/kg/d).Indomethacin was administered respectively.The micewere sacrificed after administering42days,cut and transplant tumortissue,immunohistochemistry detection of Survivin and Ki67proteinexpressions,real-time assay tumor Survivin and ki67mRNA expressions.Results:1. Effects of Indomethacin combined with Oxaliplatin on tumor therapeuticeffect and synergistic in lung cancer xenograft in nude mice.1.125nude mice formed tumor.The tumor formation rate was100%.Thetumor diameter reached an average of4mm on the10th day after plantingtumor cells.1.2We performed immunohistochemistry analysis of MMP-2,CD44v6andSurvivin protein in tumor samples.Compared with the control group,statisticalanalysis of variance showed that the expression levels of MMP-2,CD44v6andSurvivin protein of Indomethacin-treated group，oxaliplatin-treated group andIndomethacin combined with Oxaliplatin-treated group were significantlyreduced (respectively,P<0.05).There was no statistically significant differencebetween Indomethacin-treated group and Oxaliplatin-treated group(P>0.05).1.3According to the real-time fluorescence quantitative analysis ofMMP-2,CD44v6and Survivin mRNA,statistical analysis of variance showedthat the expression levels of MMP-2,CD44v6and Survivin mRNA ofIndomethacin-treated group，Oxaliplatin-treated group and Indomethacincombined with oxaliplatin-treated group were significantly reduced(respectively,P<0.05). There was no statistically significant differencebetween Indomethacin-treated group and Oxaliplatin-treated group(P>0.05).2. Effects of different doses of Indomethacin on Therapeutic effect and theeffect of anti-proliferation and apoptosis in lung cancer xenograft in nudemice.2.126nude mice formed tumor.The tumor formation rate was100%.Thetumor diameter reached an average of4mm on the10th day after plantingtumor cells. 2.2We performed immunohistochemistry analysis of Survivin and Ki67protein in tumor samples.Compared with the control group,statistical analysisof variance showed that the expression levels of Survivin and Ki67protein ofdifferent doses of Indomethacin were significantly reduced(respectively,P<0.05).Compared with the Indomethacin group one,theexpression levels of Survivin and Ki67protein of Indomethacin group two andgroup three were significantly reduced(P<0.05).There was no statisticallysignificant difference between Indomethacin group two and grouptwo(P>0.05).2.3According to the real-time fluorescence quantitative analysis of Survivinand Ki67mRNA,Compared with the control group,statistical analysis ofvariance showed that the expression levels of Survivin and Ki67mRNA ofIndomethacin-treated groups were significantly reduced (respectively,P<0.05).There was no statistically significant difference between Indomethacin grouptwo and group two(P>0.05).Conclusions:1.Using Indomethacin alone or in combination with oxaliplatin is capableof significantly inhibiting the expression of MMP-2,CD44v6and Survivinprotein,as well as the expression of MMP-2mRNA,CD44v6mRNA andSurvivin mRNA.When combined with oxaliplatin,the Indomethacin can raisedthe effect of oxaliplatin against tumor.2. Different doses of Indomethacin were able to significantly inhibit thethe expression of Survivin and Ki67of lung cancer xenografts in nude mice. Ithas a dual role of anti-tumor cell proliferation and promoting tumor cellapoptosis.The expression of Survivin and Ki67has significantlycorrelation,which have synergy in occurrence and development of lungcancer.Joint detection is an important role to predict tumorprognosis.Simultaneously,inducing apoptosis,as a new way to cancertreatment,is a new direction for the treatment of lung cancer.