Abstract:

Objective To observe the inhibition effect of cisplatin (CP) combined with rapamycin (RAPA) or 3-methyladenine (3-MA) on lung adenocarcinoma A549 cells, so as to provide evidence for improving the chemotherapeutic effectiveness of CP. Methods From March 2013 to June 2014, we accessed human lung adenocarcinoma A549 cells from the scientific research center of the Fourth Hospital of Hebei Medical University. Cells’ proliferation inhibition rate and the 50% inhibitory concentration (IC50) of CP, RAPA and 3-MA were obtained by conducting tetramethylazozolium 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT) experiment and those IC50s were used in the inhibitory experiment. Four groups were studied: control group (without drug intervention), CP group (15 μmol/L of CP), CP+RAPA group (10nmol/L of RAPA followed by 15 μmol/L of CP), CP+3-MA group (add 3 μmol/L of 3-MA for 1 hour and then add 15 μmol/L of CP). The A549 cells in logarithmic growth phase were seeded into 6-well plates with a concentration of 1.0×10⁶/ml. When the cells grew to cover about 70%-80% of the bottom area of the well, diluted drugs were added and cells then were cultured for 24 h, 48 h, 72 h. The growth and migration of A549 cells were detected by cell scratch test, and the expression of mTOR, LC3-Ⅱ and Bax mRNA and proteins were detected by RT-PCR and Western blot test. Data were processed and analyzed using SPSS17.0 statistical software. Results The IC50 results of MTT experiment were 15 μmol/L for CP, 10 nmol/L for RAPA, and 3 μmol/L for 3-MA. Scratch experiments showed that the weakest migration ability was in CP+RAPA group at 24 h and CP+3-MA group at 48 h and 72 h. RT-PCR showed that the expressions of LC3-Ⅱ mRNA in CP+RAPA group (2^-△△Ct: 24 h 1.866±0.069, 48 h 1.803±0.083, 72 h 1.836±0.056) were significantly increased (24 h F=149.780, 48 h F=111.599, 72 h F=167.855, P<0.01) compared with CP group (2^-△△Ct: 24 h 1.489±0.031, 48 h 1.325±0.007,72 h 1.428±0.080), while the expressions of Bax mRNA in CP+3-MA group (2^-△△Ct: 48 h 1.864±0.104, 72 h 1.935±0.068) were increased significantly (48 h F=52.853, 72 h F=202.118, P<0.01) compared with CP group (2^-△△Ct: 48 h 1.346±0.080, 72 h 1.462±0.029). Western blot test showed that the expressions of LC3-Ⅱ protein in CP+RAPA group (2^-△△Ct: 48 h 0.556±0.010, 72 h 0.571±0.009) were significantly higher than that of CP group (2^-△△Ct: 48 h 0.426±0.0107, 72 h 0.492±0.009) (48 h F=372.056, 72 h F=930.500, P<0.01), and the expressions of Bax protein in CP+3-MA group (2^-△△Ct: 48 h 0.897±0.022, 72 h 0.916±0.005) were significantly higher than that of CP group (2^-△△Ct: 48 h 0.463±0.011, 72 h 0.581±0.007), (48 h F=1100.412, 72 h F=5715.778, P<0.01). Conclusion CP combined with RAPA or 3-MA can inhibit the growth of A549 cells better than CP alone. CP combined with 3-MA has the strongest effect when used for a long time.

Key words: Lung neoplasms, Cisplatin, Sirolimus, Tumor cells, cultured, Depression, chemical, Drug screening assays, antitumor