The Expression Of Folylpolyglutamate Synthetase In Methotrexate Enantiomers Resistant A549 Cell Lines And The Application In Clinical

Objective:To study the expression of folylpolyglutamate synthetase (FPGS) in methotrexate enantiomers resistant A549 cell lines (L-(+)-MTX和D-(-)–MTX)and the application of clinical specimens.Methods:A real-time fluorescence quantitative PCR method for detection of FPGS mRNA was established using SYBR Green I as fluorescence andβ-actin as reference. The method was evaluated by cycle threshold (Ct), correlation coefficient, slope, repeatability curve, melting curve and amplification efficiency curve. The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method. The expression of FPGS on protein level was determined by Western-Blot in lung cancer A549 cells, and methotrexate enantiomers resistant A549 cells.Results:The standard curves indicated the high linear relationship between cycle threshold and template concentration was observed. The correlation coefficients of FPGS andβ-actin were 0.9968 and 0.9987, and the slopes were -3.595 and -3.740, respectively. The inter-coefficient of variation was from 1.27% to 2.95%. The intra-coefficient of variation was 3.82%. The method was characterized of specific melting curve and similar amplification efficiency (slope =0.0217). The expression level of FPGS mRNA was statistical different in various MTX concentration resistant cell lines (0.80±0.09 vs 2.04±0.34,3.43±0.44 vs 8.11±0.87,2.00±0.22 vs 0.59±0.11, P<0.05)。The protein expression level of FPGS was 0.85±0.12 and 1.62±0.24 folds in L-(+)-MTX/A549 cells and D-(-)-MTX/A549 cells with the concentration of drug resistance is 15μmol/L ,compared with lung cancer A549 cells.Statistical difference was observed between two groups (P<0.05). However there had no statistical difference in MTX enantiomers resistant cell line with the concentration of drug resistance are 35μmol/L and 55μmol/L (P>0.05). After treated with methotrexate(group B), the expression level of FPGS mRNA was 0.35±0.04 folds compared with those before treatment (group A) among methotrexate resistant leukemia patients. Statistical difference was observed between group A and group B(P<0.05).Conclusion:The real-time fluorescence quantitative PCR has many advantages, such as a reproductive, specific, stable, simple and cheap method, which is suitable for the quantification of FPGS. The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow have been changed. The expression level of FPGS in drug resistant cells have been changed, especially significant difference in enantiomer-resistant cells are appeared.