MTX-resistant A549 Cells Enantiomer Of VEGF, MMP-2 Expression And Invasion And Differences

Objective: To study methotrexate(MTX) enantiomers resistant human non-small cell lung cancer A549 cells, vascular endothelial growth factor(VEGF), matrix metalloproteinase-2(MMP-2) gene and protein expression and its relationship with cell Research invasion ability differences.Methods:(1) Increasing concentrations combined with low-dose sustained-induced method to establish different enantiomeric MTX resistant cell lines, named as: L-(+)-MTX / A549, D-(-)- MTX / A549, MTT assay and detection of drug resistance index MTX enantiomers of A549 cell growth inhibition, inverted phase contrast microscope morphological changes;(2) Transwell invasion assay L-(+)- MTX / A549, D-(-)-MTX A549 / differential and parental cell invasion ability;(3) Real-time PCR detection of L-(+)- MTX / A549, D-(-)- MTX / A549 and parental A549 cells VEGF, MMP-2m RNA expression;(4) Western blot detection of L-(+)- MTX / A549, D-(-)- MTX / A549 and A549 parental cells, the expression of VEGF and MMP-2 protein.Results:(1) An inverted phase contrast microscope, L-(+)- MTX / A549, D-(-)-MTX / A549 and A549 parental cells showed fusiform adherent growth. L-(+)- MTX / A549 resistance index of 5.8 for moderate resistance; D-(-)- MTX / A549 resistance refers to 15.7, highly resistant. Two kinds of MTX enantiomers inhibit the growth of A549 cells in a dose and time dependent, L-(+)- MTX inhibition of A549 cells was significantly stronger than the D-(-)- MTX.(2) Transwell chamber to detect D-(-)-MTX / A549 cells in cell number(242 ± 32) through artificial basement membrane Matrigel was significantly higher than that of L-(+)-MTX / A549(104 ± 15) and the parental cells cell(127 ± 15), the difference was statistically significant(P <0.05). L-(+)-MTX / A549 cells through the basement membrane Matrigel artificial number(104 ± 15) cells is slightly less than the parental cells(127 ± 15), but the difference was not statistically significant(P> 0.05).(3) D-(-)-MTX / A549 cells and the ratio of VEGF and MMP-2m RNA internal control are less than L-(+)-MTX / A549 cells and the parental cells and the ratio of VEGF and MMP-2m RNA internal reference, the difference was statistically significant(P <0. 05), L-(+)-MTX / A549 and the parental cell group VEGF and MMP-2m RNA ratio and the ratio between internal reference no statistically significant(P> 0. 05).D-(-)- MTX / A549 cells, VEGF, MMP-2 expression levels greater than L-(+)-MTX / A549 cells and parental cells.(4) By Western blot analysis D-(-)- MTX / A549 cells express VEGF and MMP-2 protein was significantly higher than that of L-(+)- MTX / A549 and the parental cells, the difference was statistically significant(P <0.05). L-(+)- MTX / A549 cells express VEGF and MMP-2 were higher than the parental cells slightly decreased, but there was no significant difference(P> 0. 05).Conclusions:(1) The establishment of different enantiomers of MTX-resistant cell lines, respectively, L-(+)- MTX / A549 and D-(-)- MTX / A549, exist between the two chiral resistant cell lines the difference is the key to this study, research work on the invasive ability of enantiomers resistant human non-small cell lung cancer A549 cells laid a solid foundation for further study of methotrexate(MTX);(2) MTX enantiomers resistant cells, VEGF and MMP-2 expression levels associated with tumor aggressiveness, D-(-)- M TX / A549 cells showed high expression of VEGF and MMP-2m RNA and protein, and L-(+)- MTX / A549 and there are differences between the parental cells. Enantiomers after MTX resistance changes lead to tumor invasiveness, relatively non-pharmacological effects of D-(-)- MTX can enhance the ability of drug-resistant cell invasion and metastasis.(3) VEGF and MMP-2 difference in MTX-resistant cells enantiomers expression and the ability of resistant cell invasiveness association for further clinical study of VEGF and MMP-2 in the MTX-resistant cells were transferred enantiomer Invasion provide the scientific basis for the mechanism of action, clinical provide the basis for a more rational use of MTX enantiomer drugs.