Effects Of Methotrexate Enantiomers On Cells Inhibittion And Its Mechanisms

8Objective:1.To investigate the effect of MTX(included (+)MTX and (-)MTX)on the proliferation of ECV304 cells and to explore its mechanisms.2. A rapid and sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) was developed and validated for the quantitative determination the concentration of methotrexate (MTX) enantiomers in the intracellular and extracellular fluid.Methods:1.ECV304, HepG2, A549 cells were cultured. The cell proliferation was determined by MTT.2. The morphological change of ECV304, HepG2, A549 cells were inspected by inverted microscope.3.Cell cycle phases of ECV304, HepG2, A549 were assayed by propidium iodide staining flow cytometry.4. Fluorescentm micrographs of A549 induced with (+)MTX, (-)MTX indicated by DAPI.5.DNA ladder were used to detect the apoptosis.6. Cell apoptosis of was determined by Annexin V-FITC (fluoresceinisothiocyanate)/PI (propidium iodide) staining.7. Liquid chromatography-tandem mass spectrometry assay (LC-MS/MS)with electrospray ionization was developed and validated for the quantitative determination the concentration of methotrexate enantiomers in the intracellular and extracellular fluid. Results:1.ECV304, HepG2 and A549 cells were treated with (+)MTX, (-)MTXat 1μmiol·L-150μmol·L-1 for 24h,48h,72h. The results showed that the proliferation of ECV304, HepG2 and A549 cells were significantly inhibited under the different conditions. The order of the inhibited efficacy was(+)MTX>(-)MTX.2. The Morphological of ECV304, HepG2 and A549 cells were found to changes by (+)MTX and(-)MTX treatment.3.After administration of 10μmol·L-1 of (+)MTX, (-)MTX for 48h, the cell cycle phases were assayed by propidium iodide staining flow cytometry. The result showed DNA replication was interfered by (+)MTX and (-)MTX treatment.4. The Morphological of A549 cells were found to changes by(+)MTX and (-)MTX treatment that included the cell shrinkage, chromatin condensation and apoptosis body.5.DNA ladder was the most recognized marker of apoptosis, and there was obvious DNA ladder in (+)MTX treated group.6. DNA ladder was the most recognized marker of apoptosis, and there was obvious DNA ladder in (+)MTX treated group.7.The different concentrations of MTX enantiomers in the intracellular and extracellular of HepG2 cells were obtained.It were shown that (+)MTX concentration much higher than (-)MTX concentration in intracellular of HepG2 cells.Conclusion:The proliferation of ECV304, HepG2 and A549 cells have the chiral selective effects by (+)MTX and(-)MTX treatment, and the inhibition on ECV304, HepG2 and A549 cells proliferation of (+)MTX was significantly stronger than (-)MTX.