Icariin Reversed Metastatic Phenotype Of Methotrexate-resistant Lung Cancer A549 Cells

Part A: Establishment of Methotrexate-resistant A549Cell Line of Human NSCCL and Its Biological CharacteristicsObjective: To establish methotrexate(MTX)-resistant human NSCCL A549 cell line derived from the A549 cell line and observe its biological characters.Methods: A549 cells were exposed to intermittently and progressively increasing concentration of MTX. MTX-resistant human NSCCL A549 cell line was established.The morphology was observed by inverted phase contrast microscope, The sensitivity of the A549/MTX cell line to MTX was measured by cycotoxicity test. The cell growth curve was determined by MTT assay.Results: The MTX-resistant human A549 cell line was established. The A549/MTX resistance index was 6.0. Inverted phase contrast microscope showed the chang of A549/MTX. Cell growth curve demonstrated that the proliferation abilities of A549/MTX cell was decreased significantly compared with that of A549.Conclusion: We establish MTX-resistant human A549 cell line and observe its biological characteristics. It provides a new experimental model for further studying the mechanism of MTX-resistance. Part B: Icariin reversed metastatic phenotype of methotrexate-resistant lung cancer A549 cellsObjective:To investigate the effect of traditional Chinese medicine (TCM) icariin(ICA) on metastatic phenotype of methotrexate (MTX)–resistant lung cancer A549 cells and elucidate the action mechanism and therapeutic value of ICA.Methods:The half inhibition concentration (IC50 ) value of ICA in inhibiting the growth of A549/MTX cells was measured by MTT assay.The colony formation rate and the morphology of cell cluster of A549/MTX and ICA-treated A549/MTX cells were determined by douber-layer soft agar colony formation assay.The migration abilities of A549/MTX and ICA-treated A549/MTX cells were evaluated by cell scratch assay.The invasion ability of cells was tested by using Transwell chamber assay.The change of mRNA levels of nm23-h1 and c-myc genes were detected by Semi-quantitative RT-PCR.Results:MTT assay showed that the IC50 value of non-toxic ICA plus MTX was reduced compared with that induced by equal dose of MTX (35.50±1.85μmol/L vs 52.17±2.25μmol/L). The colony formation rate of ICA-treated A549/MTX cells was 0.94±0.088, less than that of A549/MTX cells (1.56±1.065,P<0.05). Cell scratching assay demonstrated that the migration capability of A549/MTX cells was stronger than that of ICA-treated A549/MTX cells at 72h (P<0.05). Transwell experiment revealed that more A549/MTX cells passed through artificial basement membrane than ICA-treated A549/MTX cells (P<0.05),indicating that the invasion capbility of ICA-treated A549/MTX cells was weaker than that of A549/MTX cells. Semi-quantitative RT-PCR showed that the expression of nm23-h1 mRNA of ICA-treated A549/MTX cells increased in compare with that of A549/MTX cells,on the contray, an decreasing tendency in the expression of c-myc mRNA was observed after treated with ICA.Conclusion: TCM ICA can reverse the metastatic phenotype of MTX–resistant A549 cells.