The Reseach Of Low Concentration Of Etoposide Inducing The Senescence Of A549 Cells By Mdm2-Rb Signal Pathway

Abstract:Objective:To observe low concentration of etoposide induce the senescence of cultured human lung adenocarcinoma A549 cells treated with different concentration of etoposide in this study.To research the role of Mdm2-Rb signal pathway in cell senescence and mechanism of low-dose metronomic chemotherapy by detecting the expression of phosphorylated Rb protein,Mdm2 protein and cell cycle distribution.Method:A549 cells were devided into four groups:control group (cells+culture medium), experimental group 1 (cells+medium containing 1μM etoposide), experimental group 2(cells+medium containing 5μM etoposide), experimental group 3(cells+medium containing 25μM etoposide). The cells were cultured for 48 hours for tetrazolium (MTT) colorimetric testing of A549 cells proliferation; cytochemical methods testing of senescence associated-β-galactosidase activity; flow cytometry testing of cell cycle distribution; immunocytoc-hemical methods testing of the expression of phosphorylated Rb protein and Mdm2 protein. Results:①Control group cells grew well, showed rosette-like arrangement of spindle or polygonal cells.The nucleus were in the center of the cells containing abundant cytoplasm. The growth inhibiting of experimental group 1 cells was slightly.The cell morphology had no significant change compared with control group cells. The experimental group 2 cells were more scattered, arranged in small piles,cells shrinkage,round,orbicular-ovate or polygonal, cytoplasm reduction.Some cells had pseudopodium. The growth inhibiting of experimental group 3 cells were obvious.Some cells were round, floated on the fluid. The morphology change of the living cells were not obvious compared with control group cells.②Cell proliferation inhibition rate increased gradually with the increasing concentration and lasting action time in the A549 cells treated with different concentration of etoposide, the difference among the four groups being significant (P<0.05).③Senescent cells percentage increased significantly among the experimental groups compared with control group cells(P<0.05).④The percentage of G1-phase cells of experimental group 2 increased significantly while experimental group 3 decreased significantly (compared with control group,P<0.01). The percentage of S-phase cells of experimental group 2 decreased significantly while experimental group 3 increased significantly(compared with control group,P<0.01). The percentage of G2-phase cells among the four groups was no significant difference (P>0.05).⑤The expression of phosphorylated Rb protein and Mdm2 protein in experimental group 2 decreased significantly In contrast to the others (P<0.01).The comparison in experimental group 1, experimental group 3 and control group was no obvious difference (P>0.05).Conclusion:①In vitro low concentration of etoposide can increase the activity of senescence associated-β-galactosidase in human lung adenocarcinoma cell line A549 cells, inducing cell senescence.②Low concentration of etoposide can decrease the expression of phosphorylated Rb protein and Mdm2 protein, leading to G1 arrest.③Low concentration of etoposide may leading to G1 arrest by Mdm2-Rb signal pathway, inducing cell senescence.