| Objective: (1)To establish a MDR cell subline A549/CDDP of human lung adencarcinoma induced by CDDP; (2)To detect the cell growth inhibition, cell cycle arrest and apoptosis-inducing effect of docetaxel on A549 and A549/CDDP cells; (3) To explore the roles of apoptosis-related proteins including bcl-2, bax, Fas, p53 and c-myc in the anti-tumor effect of docetaxel.Methods: (1) MDR cell subline of human lung adencarcinoma, A549/CDDP, was established from A549 by exposed to a high-dose shock of CDDP first and then to increasing concentrations of CDDP until they could grow in the culture medium containing 1.0μg/ml CDDP. Sensitivity of both A549 and A549/CDDP cells to different chemotherapeutic agents was evaluated by MTT assay, cell morphology was observed by inverted microscope and transmission electron microscope (TEM) and cell cycles of them were determined by Fluorescence activated cell sorter (FACs). (2) Light microscope, TEM, colony-forming ability test and MTT assay were used to observe cell morphological changes, colony-forming ability, growth curve and inhibition rate after treatment of A549/CDDP and A549 cells with docetaxel. FACs was emplored to detected the cell cycle arrest effect of docetaxel. TEM, TUNEL and Annexin V/PI staining were used to measure apoptosis induced by docetaxel. (3) Immuocytochemistry was carried out to confirm the expression of Bcl-2, Bax, Fas and other apoptotic related protein before and after treatment of A549 and A549/CDDP cells with docetaxel.Results: (1) A549/CDDP was established successfully and the subline exhibited a MDR phenotype. The resistance of A549/CDDP cells to CDDP was 12.00-fold than that of the parental A549 cells. A549/CDDP cells showed cross-resistance to hydroxycamptothecine, vincristin and 5-fluorouracil and MDR indexes were 2.29, 8.60, 16.84, respectively. Compared with that of parental cells, size of A549/CDDP cells was a bit bigger, the ratio of nucleus to cell plasma increased, mitochondria were short and small and ageing, and vacuoles and osmiophilic lamellar body in the plasma markedly increased. Furthermore, the number of cellsin S phase of A549/CDDP increased (PO.05), while the cell proliferation rate of A549/CDDP was similar to that of A549. (2) It was shown that docetaxel could inhibit growth of human lung adenocarcinoma cell line A549 and its MDR cell subline A549/CDDP both in the time and the dose-dependent manner in the dose range of 0.1~2Hg/ml. Compared with A549 cell, A549/CDDP cell was more sensitive in 12~24 hours(P<0.01), less sensitive in 48 hours(PO.Ol), and no statistically different in 72 hours(P>0.05). (3) Docetaxel could induce the arrest of A549 and A549/CDDP cells in G2/M phase, the former was in time-dependence and the latter also showed tetraplont after 48 hours and multimicronucleation. Moreover, this G2/M phase arrest decreased after the dose increased, the number of cells in S phase increased. (4)Docetaxel induced A549 and A549/CDDP cells apoptosis, necrosis and autoschizis, the two latter were more important. Vacuoles increasing, microvilli decreasing, mitochondria swelling, a few apoptosis bodies and cytoplasm without organelles were observed by TEM. (5) Fas, c-myc and p53 proteins were expressed both in A549 and A549/CDDP cells and bcl-2 protein was expressed only in A549/CDDP cells. After treatment of docetaxel, bax upregulate in A549/CDDP cells and Fas noticeably upregulate in both cells, the expressions of bcl-2, c-myc and p53 had no visible changes.Conclusion: (1) A549/CDDP cell subline is successfully established as a reliable MDR cell subline of human lung adencarcinoma. (2) Docetaxel could markedly inhibit growth of A549 and A549/CDDP cells both in the time and the dose-dependent manner. (3) Docetaxel could induce the arrest of A549 and A549/CDDP cells in G2/M phase and mainly results in cell necrosis and autuschizis, meanwhile, apoptosis also play a role in it. (5) The upregulation of Fas might be one of the mechanisms of docetaxel. |
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