The Role Of S-adenosylmethionine In Proliferation, Invasion And Metastasis In Non-small Cell Lung Cancer

?Background? Lung cancer ranks first in all cancer morbidity and mortality.Non small cell lung cancer(NSCLC)represented 85% pathological type in all lung cancer.Most late NSCLC patients lost surgery chance because of Lymphovascular invasion and metastasis.The genes targeted drugs epidermal growth factor receptor Tyrosine Kinase Inhibitors(EGFR-TKIs)have effects on partial late NSCLC patients,but the drugs resistance brings difficulty in treatment,wherever mesenchymal–epidermal transition(MET)and Wnt/?-catenin pathway activation and high expression contribute to EGFR-TKIs resistant and both of them positively regulated each other.S-Adenosylmethionine(SAM)known as dietary supplements and methylation reagent plays an important role in many kinds of cancers,which can inhibit ?-catenin pathway in liver and colon cancer.In liver cancer,it is confirmed to inhibit proliferation by suppress hepatocyte growth factor(HGF)pathway,and HGF is the ligand of MET.However,its role in NSCLC is unclear,since the epigenetic alterations,including DNA methylation and histone modifications are important in lung cancer development.Considering the effect of SAM on MET,?-catenin pathway as well as on tumor proliferation,the potential effect and mechanism of SAM in NSCLC need to be further examined and application of SAM in advanced NSCLC patients can provide more strategies for EGFR-TKI resistance.?AIMs? To investigate the effect of SAM and its metabolite MTA on tumor proliferation,invasion and metastasis in both NSCLC cell line and xenograft nude mice.To examine its effect on MET,EGFR,?-catenin and its targeted genes expression and underlying molecular mechanism.To provide the theoretical basis for future molecular targeted therapy.?Motheds?1.To investigate the therapeutic potential of SAM and MTA in NSCLC,we performed soft agar colony formation assay,invasion assay,MTT proliferation,wound-healing assay in vitro and xenograph nude mice experiment in vivo using A549 or H292 cell.2.After treatment with SAM and MTA,We used real-time PCR,Western Blot and Immunofluorescence to examine the EGFR,and EGFR active gene Interleukin 8(IL-8),MET,?-catenin and ?-catenin targeted genes-c-myc,CCND1 mRNA and protein expression in A549 and H292 cell line.3.We used real-time PCR,Western Blot and Immunofluorescence to examine upper group genes mRNA and protein expression by overexpression SAM synthesis Enzymes encoded gene-Methionine Adenosyltransferase 1A(MAT1A)with MAT1 A overexpression plasmids and catalytic mutant plasmids to verify potential epigenetics role of SAM in NSCLC.4.To investigate the transcriptional role of SAM and MTA,the targeted genes promoter activity were examin ed by using dual Luciferase Reporter Assay System.5.Through overexpression MAT1 A and knock down MAT1 A with plasmid and siRNA respectively in epithelial cell Hep3 B,we built SAM abundance and SAM deficient status to examine the tumor metastasis related genes expression with gene metastasis Array.?Results?1.SAM and its metabolite MTA can inhibit the colony formation,proliferation,invasion and metastasis in A549 and H292 cell line.SAM gavage can suppress tumor growth in xenograft nude mice.2.Both SAM and MTA can inhibit the MET ? EGFR,CCND1 ? c-MYCmRNA and protein level as well as the ?-catenin protein level which display the dose responsible relationship.3.Overexpressing MAT1 A can also suppress the MET?EGFR,CCND1?c- MYC expression by raising endogenous SAM level.but catalytic mutant destroyed the effect.4.Overexpressing MAT2 A and MAT2 B can enhance the MET ? EGFR,CCND1?c-MYC mRNA expression,but silence suppress the expression.5.Both SAM and MTA can inhibit AP-1 promoter activity.Also,mutant AP-1 site in IL-8 promoter reduces the activity,which indicates SAM and MTA may inhibit the targeted genes expression through AP-1 pathway.6.In Hep3 B cell,raising endogenous SAM by overexpressing MAT1 A can downregulate 65/76 genes expression related to tumor metastasis including HGF and MET.?Conclusion?1.SAM and its metabolite MTA can inhibit tumor progress in NSCLC.2.SAM and MTA can suppress MET,EGFR,EGFR activator gene IL-8,?- catenin and ?-catenin targeted gene CCND1,c-MYC's expression which may relate to tumor development in NSCLC.3.The effects of SAM and MTA are suggested to be through or partially through epigenetic way and AP-1 binding site can be the potential target to regulate the genes expression.4.MAT1 A is a tumor suppress gene,adversely,MAT2 A and MAT2 B are tumor enhancer genes.