The Impact Of MTOR Inhibitor RAD001 Combined Gefitinib On Human Non-small-cell Lung Cancer Cell Lines Which Are Egfr-wild And Resistant To Gefitinib

Objective: The impact of RAD001 combined Gefitinib on the cell proliferation and apoptosis of the human non-small cell lung cancer NCI-H460 and NCI-H661, which are Primary Resistant to Gefitinib.Methods: Our objects of study are human non-small cell lung cancer NCI-H460, and NCI-H661 cell line .They are resistant to gefitinib.The EGFR of the two cell lines are wild.The PIK3CA and K-RAS genes in H460 are mutation, but in H661 are wild.Experiment is divided into control group, Gefitinib group, RAD001 group, Gefitinib and RAD001 combination group. Drug after 72 hours using MTT assay the inhibitory rate of two cell lines in each group, and calculate the combination index (CI); using flow cytometry assay the apoptosis rate of two cell lines in each group. Drug after 24 hours using western blot detect the protein expression of the mTOR downstream p70S6K and p-p70S6K in each group of the two cell lines.Results: 1.MTT Results: In lung cancer NCI-H460 cells, the IC50 of Gefitinib is 19.46±0.0.17μM, the IC50 of RAD001 is 86.80±1.64nM, the IC50 of the two-drug combination is 0.51±0.32μM/nM, which is significantly lower than the single Gefitinib or RAD001 drug. The combination index (CI) of the two drugs in NCI-H460 is 0.0326. In lung cancer NCI-H661 cells, the IC50 for the single agent Gefitinib is 34.96±0.28μM, the IC50 of the single agent RAD001 is 23.72±1.14, the IC50 of the two-drug combination is 10.14±2.17μM/nM, which is significantly lower than the single drug Gefitinib or RAD001.The combination index (CI) of the two drugs in NCI-H661 is 0.8419. The combination indexes of Gefitinib and RAD001 in the two cell lines are less than 1, which indicate they are synergistic effect.2. Flow cytometry Results: In lung cancer NCI-H460 cells, the apoptosis rate in the control group is 2.11%, Gefitinib group is 11.44%, RAD001 group is 5.27%, and the two combination therapy group is 13.20%. In lung cancer NCI-H460 cells, the apoptosis rate of the control group is 3.59%, Gefitinib group is 8.62%, RAD001 group is 7.40%, and the two combination therapy group is 14.34%. The apoptosis rates of the two combination therapy groups are higher than the single drug groups. 3. Western Blot test results: The p-p70S6K protein level in the control group of NCI-H460 cells is higher than the NCI-H661 cells. In two cell lines, the p-p70S6K protein levels in RAD001 group and the two drug combination group are lower than those in the control group.Conclusion: 1. RAD001 can reverse the primary gefitinib resistance of the EGFR-wild human non-small cell lung cancer cell lines.2. The reversal effect of RAD001 in the EGFR-wild human non-small cell lung cancer cell lines maybe related to the K-RAS and PIK3CA mutations.3. PIK3CA mutation and K-RAS mutation activate PI3K/AKT/mTOR pathway.