Gefitinib Induced Apoptosis In Lung Cancer Cell Lines And The Study Of Its Interrelated Mechanism

Objective: To study aim to investigate the anti-tumor effect of small molecular tyrosine kinase inhibitor that targeted epidermal growth factor receptor gefitinib in lung cancer cell lines A549and H460 in vitro, and to explore its possible mechanisms in signal transduction pathway.Methods:1 Two cell lines A549 and H460 were selected as the study object, and MTT method was used to detect the sensitivity of the two cell lines to gefitinib. While the two cell lines, namely A549 and H460 in their well status, following studies were carried out; respectively, After the cells had adhered diverse concentrations of gefitinib were added to the medium of cells, and incubated for 72 hours. After the incubation MTT was added to each well and the absorbance was detected (at the wave-length of 490nm, and 520nm as reference). So the concentration of inhibition rate of 50 percent was calculated, namely, the cell line's IC50, and to provide reference for selecting an appropriate concentration to induce apoptosis of the two cell lines.2 The concentration of 20μM gefitinib was selected to treat A549 and H460 cells, disposed cells to gefitinib for 24 hours, the cells were dyed with DAPI, in order to observe the apoptosis induced by gefitinib under the fluorescence microscope.3 Selecting the above cells with well status, respectively seeded the cells in 25mm culture flask. After the cells totally adhereed to the well, exposed the cells to gefitinib at the following concentrations 0μM, 0.1μM, 1.0μM, 8.0μM and 2 hours later, 30ng/ml EGF was added to the medium, in order to activate the phosphorylation of EGFR and downstream signals, then incubated for 15 minutes, then cells were digested. In the cold PBS containing phosphatase inhibitor. The whole protein were distract with RIPA kit. Measure protein concentration with Bradford protein-analysis kit. Western-Blotting was carried out with the protein obtained above to determine the protein level of EGFR, p-EGFR, Akt and p-Akt. GAPDH was used as a loading control.Results:1 When A549 cells were treated with gefitinib at the concentration of 3μM, 6μM, 8μM, 10μM, 12.5μM, 25μM and 50μM, the inhibition rate of A549 were 8.26%, 40.76%, 45.87%, 45.72%, 55.69%, 86.79%, 95.67% respectively, and the rates of H460 were 0.72%, 3.44%, 17.4%, 12.52%, 22.71%, 68.91%, 94.05%. The proliferation of lung cancer cell line A549 and H460 was inhibited by gefitinib, and the effect was dose-dependent, the inhibition rate increased with the augmentation of gefitinib concentration. The IC50 of gefitinib on lung cancer cell line A549 and H460 was 10.81μM and 18.44μM respectively. It was similar to the results reported.2 Disposing the two lung cancer lines to 20μM gefitinib to induse apoptosis, and after the cells were dyed with DAPI. Grain fluorescence with dense-dyed appeared in the nucleolus, and concavo-convex's, fastened shrink or segmented nucleolus appeared in the emblematical apoptosis nucleolus. It simulated the effect of gefitinib in vitro, and observed gefitinib as a targeted therapy could induce lung cancer cells apoptosis.3 Western-blotting was used to determine EGFR and Akt phosphorylation inhibited by gefitinib: EGFR expressed in both of the two cell lines, and higher expression level in A549 cell line. EGFR phosphorylation was to some degree inhibited in the two lung cancer cell lines with 0.1μM gefitinib, and more obvious inhibition was observed with the increase of gefitinib concentration, EGFR protein phosphorylated was almost totally inhibited with mediun containing 8.0μM gefitinib in the two lung cancer cell lines. Akt phosphorylation was inhibited in both lung cancer cell lines can be observed, and the degree of inhibition was more obvious with the increase dose of gefitinib.Conclusion:1 The study selected two cell lines: A549 and H460, and MTT method was used to determine the sensitivity of the lung cancer cell line to gefitinib. The proliferation of lung cancer cell line A549 and H460 were inhibited by gefitinib, and the effect was dose-dependent. The concentration of IC50 for the effect of gefitinib on lung cancer cell line A549 and H460 was 10.81μM and 18.44μM. A concentration adjacent of gefitinib (20μM) to IC50 was selected to dispose the two lung cancer lines, and observed after dyed with DAPI, grain fluorescence with dense-dyed appeared in the nucleolus, concavo-convex's, fastened shrink or segmented nucleolus appeared in the emblematical apoptosis nucleolus. It shows the cell apoptosis. This observed the effect of gefitinib by cell experiment and apoptosis experiment in cellular level. And it showed that gefitinib is a useful drug in inducing apotosis in lung cancer cell lines.2 The experiment of EGFR and Akt phosphorylation by Western-blotting from detected Akt phosphorylation can be controlled by gefitinib, and the Akt phosphorylation is related with anti-apoptosis, so we believe the molecular mechanism on the apoptosis of lung cancer cell caused by gefitinib may be at the bottom of the Akt phosphorylation controlled. This study help us to realize the effect of gefitinib as an anti-tumor drug and the possible molecular mechanism as a targeted therapy, and provided theoretic base for the patients selection to determine whom will receive the targeted therapy and meaningful for in new targeted therapy development.