High-dose Gefitinib Pulsatile Overcome The Human Lung Adenocarcinoma PC9/GR Cells In Vitro

Objective: Currently, lung cancer has been the first rank of malignant tumor, with non-small cell lung cancer(NSCLC) being the predominant form of the disease, accounting for 80-85%. EGFR-TKIs are classic drugs for non-small cell lung cancer patients with EGFR mutation. However, clinical efficacy is limited by acquired resistance. Recent years, results of several related clinical experiments showed that the re-initation of EGFR-TKIs can postpon the disease progression, delay the disease progression and prolong the survival time. The aim of present study was to investigate the antitumor effect of the high-dose pulsatile dose of gefitinib to EGFR-TKI resistant cell lines PC9/GR, evaluate the efficacy and provide theoretical basis for clinical treatment.Methods: Gefitinib, as an single agent, was treated on non-small-cell lung cancer EGFR-TKI resistant cell lines PC9/GR. The anti-proliferative effects were detected by MTT assay and calculation the half maximal inhibitory concentration(IC50)by OD value. According to IC50 value, we divided the experiment into the control group, the routine gefitinib administration group with the IC50 value, gefitinib pulsatile administration group with the 2IC50 value and gefitinib high dose pulsatile administration group with the 4IC50 value. The morphological changes of apoptotic cells were observed by fluorescence microscope after related drug treatment; The cell apoptosis and the cell cycle phase distribution were analyzed by flow cytometry. The expression of EGFR/p-EGFR 、 ERK /p-ERK 、 AKT/p-AKT were measured using western blotting.Results: We examined the anti-proliferative effects of gefitinib on PC9/GR using MTT assay. The PC9/GR cells were exposed to various concentrations of gefitinib for 72 h and calculated the IC50 value was(5.44±0.91) μmol/L. The growth-inhibitory curves revealed gefitinib dose-dependent growth inhibition characteristic. According to IC50 value, we divided the experiment into the control group, the routine gefitinib administration group with the IC50 value, gefitinib pulsatile administration group with the 2IC50 value and gefitinib high dose pulsatile administration group with the 4IC50 value. So, in PC9/GR cell line, the treatment group was control, 5 μmol/L, 10 μmol/L, 20 μmol/L. By Annexin V-FITC staining the cells after treatment with the different dose of gefitinb, we found the advanced apoptotic cells obviously increased and can observe the cells were irregular in shape, such as condensation and fragmentation of nuclei in high dose pulsatile administration group. Meanwhile, the results of apoptosis assay show that 20 μmol/L treatment group had significantly high apoptosis rates than other groups. The statistical analysis showed that the difference to gefitinib were significant(p<0.05). The population of cells arrest in the G0/G1 phase increased significantly in 20 μmol/L treatment group and a corresponding increase of arrest at S-phase. According to the western blot, we found that the high dose pulsatile administration can effectively inhibit the expression of the EGFR/PI3K/AKT and EGFR/MEK/ERK pathway by decreased the expressions of p-EGFR, p-AKT, p-ERK.Conclusion: In conclusion, the present study demonstrated that high dose gefitinib pulsatile can overcome the TKI acquired resistance by blocking EGFR signaling pathway. We speculate that high dose gefitinib pulsatile may increase the ATP-binding affinity of TKI, making further efforts to inhibit the phosphorylation of EGFR and induce cells apoptosis. This study suggests that high dose pulsatile gefitinib may be good to NSCLC patients which with acquired resistance to EGFR-TKI.