| 12-O-tetradecanoylphorbol-13-acetate (TPA) is a compound of phorbol esters. TPA could induce tumor cells differentiation, including human lung cancer cells. Howerer, whether TPA has other effects, such as reducing invasion ability on human lung cancer cells are not clear.ObjectiveTo observe the effects of adhesion, migration, invasion ability and the sensitivity to cisplatin of human lung adenocarcinoma A549 cell line after treated by TPA.Methods1 cell culture All cells were maintained in RPMI-1640 medium, supplemented with 10% heat-inactivated fetal calf serum. Cells were cultured at 37±0.3℃in atmosphere of 95% air and 5% CO2.A549/TPA cells:A TPA-resistant subline, named A549/TPA cells, was established by maintaining A549 cells in the presence of 5ng/ml TPA over 9 months. The surviving cells were maintained in the presence of TPA at concentrations of 5ng/ml and passaged every 3 days.A549/R-CDDP cells:A cisplatin-resistant subline, named A549/R-CDDP cells, was established by maintaining A549 cells in the presence of cisplatin over 10 months. The resulting cells were 10-fold resistant to cisplatin than A549 cells. The surviving cells were maintained in the presence of cisplatin at concentration of 2μg/ml and passaged every 3 days.2 Experimental Methods Cell proliferation and morphological changes were observed after TPA treatment by MTT assay, cell number count and colony formation assay. The cell cycle was determined by flowcytometry and immunocytochemistry. The cell adhesion, invasion, and migration ability were tested on Matrigel matrix membrance and by Transwell assay. The changes of CXCR4 expression were measured by flow cytometric analysis.Results1 Growth inhibition Treatment group of 1ng/ml,5ng/ml TPA compared with control the inhibition rate was not statistically significant for A549 cells, but forlOng/ml of TPA, the inhibition rate was statistically significant. A large number of suspension cells were produced from the third day when A549 cells were treated with 5ng/ml TPA, while the control group had no such phenomenon. The cell number of A549/TPA treated by TPA was statistically significant with control from the fourth day, the logarithmic growth phase was lost, and the cell density decreased. Results of colony forming assay showed that survival fraction was statistically significant when A549, A549/TPA cells were treated by TPA. Survival fractions of A549 cells on treatment of lng/ml,5ng/ml TPA were 44.2%,72.1%, and for A549/TPA cells,52.3%.2 Morphological change Treatment of A549 cells by lng/ml,5ng/ml TPA showed 8.41%,26.38% vacuolized cells in colony formation assay. A549/TPA cells group had the similar phenomenon.3 Cell cycle analysis TPA reduced Go phase cells and increased G1 phase cells of A549. For A549/TPA cells it was much more obvious.4 Sensitivity of A549 cells to cisplatin TPA enhanced sensitivity to cisplatin on A549 cells and A549/R-CDDP cells. IC50 were 0.46μg/ml,0.16μg/ml when A549 cells were treated by cisplatin combined with TPA respectively. For A549/R-CDDP cells 6.98μg/ml,3.02μg/ml respectively. 5 Invasion ability test of A549 cells5.1 Adhesion Adhesion cell number on the matrigel matrix surface after TPA treatment decreased 73.91% of the control.5.2 Migration The control group formed a network structure of "lattice" morphology of cells on the matrigel matrix surface and the "pipelines" by cells linking. Cells treated by TPA scattered individual and did not form a typical structure as control.5.3 Invasion The cell number across the matrigel matrix membrance treated by TPA was only 47.73% of the control.5.4 CXCR4 expression After 24h of A549 cells treated by TPA, CXCR4 expression rate and fluorescent intensity compared with the control were not changed significantly. When A549 cells were treated by TPA for 5 days these were statistically significant about suspension cells and adherent cells.Conclusions1 TPA inhibits the proliferation of A549 cells and increases the sensitivity of A549 and A549/R-CDDP cells to cisplatin.2 TPA reduces the adhesion, migration, invasion ability and the expression of CXCR4 of A549 cells. |
