Study On Expression And Relationship Of Phosphl-Ated Tyrosine Kinase Receptors In Methotrexate Enantiomers Drug-Resistant Cell Lines

Objective The aim of this study is to establish methotrexate(MTX) enantiomersresistant human NSCCL A549cell line and observe their biological characters,compare with expression profile of phosphorylated RTKs in resistant cell and parentalcell by liquichip technology.To find cancer phosphoprotein biomarkers associatedwith mechanism of MTX resistance and understand the relationship between theresistance to MTX and Phosphorylated tyrosine kinase receptor family,and to provideevidence for clinical selected targeted chemotherapy drugs and therapy program.Methods:①A549cells were exposed to intermittently and progressively increasingconcentration of MTX enantiomers. Both MTX enantiomers resistant human NSCCLA549cell lines were established;②The morphology was observed by inverted phasecontrast microscope;③T he cell growth curve was determined by MTT assay;④MTTreduction assay detected drug fast index of L-(+)-MTX/A549and D-(-)-MTX/A549cells;⑤Plate scrape experiment determined migration ability of L-(+)-MTX/A549,D-(-)-MTX/A549cells;⑥Double soft-agar clone formation experiment detectedclony efficiency and colony size of L-(+)-MTX/A549, D-(-)-MTX/A549cells;⑦Theexpression of7phosphorylated RTKs in three cells was detected by the LuminexxMAP systerm simultaneously and analyzed the correlation among them.Result:①The MTX enantiomers resistant human A549cell lines were established.The L-(+)-MTX/A549resistance index was6.10and D-(-)-MTX/A549resistanceindex was20.64;②Inverted phase contrast microscope showed the chang ofL-(+)-MTX/A549and D-(-)-MTX/A549;③Cell growth curve demonstrated that theproliferation abilities of L-(+)-MTX/A549was decreased significantly,The rate ofcell proliferation of D-(-)-MTX/A549was similar to that of A549;④After72hours, the number of L-(+)-MTX/A549that entered scratch zone was fewer thanD-(-)-MTX/A549;⑤The number and rate of colony formation in D-(-)-MTX/A549,L-(+)-MTX/A549and parent cells was not significant(P>0.05), but the size ofD-(-)-MTX/A549was larger than L-(+)-MTX/A549cells;⑥The MFI of p-EGFR,p-HER-2, p-IGF-1R, p-HGFR, p-PDGFRβ, p-Tie-2, p-VEGFR-2were26.91±1.81、16.56±1.15、6.69±0.57、25.94±2.02、11.66±1.14、68.75±3.76、16.94±1.84inD-(-)-MTX/A549cells; these were54.13±2.75、16.94±1.44、6.56±0.54、26.06±2.52、12.06±1.61、70.88±3.78、16.75±1.48in L-(+)-MTX/A549cells; thesewere54.38±2.88、33.75±2.49、6.47±0.59、28.25±2.64、11.56±1.55、72.44±4.03、17.00±1.67in parent cells. Significant correlation was noted between p-EGFR andp-Her-2, p-Her-2was closely correlated with p-VEGFR and p-HGFR. Otherphosphorylated RTKs was no significant correlation among the three groups cells.Conclusion:In tumor cells movement ability and Proliferation capacity,D-(-)-MTX-induced NSCLC A549cells has greater capacity than L-(+)-MTX,thusexplain that MTX enantiomer has difference in tumor Proliferation and metastasisafter resistant;There is differential expression of p-EGFR in methotrexateenantiomer-resistant A549cell lines.