The Study Of Reversal Effects And Mechanism Of DC-CIK Cells Combined With Gefitinib Against Lung Adenocarcinoma Cell Line H1975

Objective:To observe the effect of DC-CIK cells combined with gefitinib on the cell proliferation,as well as the expression of EGFR/AKT/m TOR signaling pathway in human lung adenocarcinoma H1975 cell with EGFR-790 M mutation.To evaluate the influence and potential molecular mechanism of DC-CIK cells combined with gefitinib on lung adenocarcinoma H1975 cell,accessing the potential of DC-CIK cells to reverse the resistance of lung adenocarcinoma H1975 cells to gefitinib,and to provide experimental basis and theoretical evdience for the treatment of the EGFR-TKI acquired drug resistance.Methods: Mononuclear were obtained from the peripheral blood of healthy volunteer,and were induced to mature DC and CIK cells in vitro.The phenotypic changes of DC and CIK cells were analyzed by flow cytometry.MTT assay was used to detect the effect of different concentrations of gefitinib and different effector-target ratios of DC-CIK cells on the proliferation of lung adenocarcinoma H1975 cells with EGFR-T790 M mutation.The expressions of p-EGFR,p-AKT,p-m TOR and ?-actin in the control group,gefitinib group,DC-CIK cells group and combination group were detected by Western-Blot.Result:1.At the 9th day of DC cells,the expression rates of CD40+,CD80+,CD86+ and HLA-DR+ were significantly higher than those at the 0th day:(72.97±4.98)% vs.(14.13±4.17)%,(70.23±3.95)% vs.(0.43±0.06)%,(83.23±3.00)% vs.(14.43±0.64)%,(90.87±0.42)% vs.(29.40±0.62)%,indicating that the induced DC cells have been matured.2.The expression rates of CD3+,CD8+,CD3+CD8+ and CD3+CD56+ increased gradually with the prolongation of culture time in CIK cells which induced in vitro;And at the 14 th day,the proportions of CD3+CD8+ cells and CD3+CD56+ cells [(81.50±0.7)%,(22.50±1.64)% ] was significantly higher in CIK cells which co-cultured with DC cells than those in CIK cells cultured alone [(72.90±1.56)%,(16.63±0.83)](P<0.05).3.The cytotoxicity of DC-CIK cells on lung adenocarcinoma H1975 cells enhanced with the increase of the effort-target ratio.When the effort-target ratios were 2.5:1,5:1,10:1,20:1 and 40:1,the inhibition rates were (27.78±1.36)%,(36.92±2.34)%,(44.65±2.61)%,(58.28±1.21)and(78.63±1.60)% respectively(P<0.05).4.The inhibitory effects of each concentration of DC-CIK cells combined with gefitinib on lung adenocarcinoma H1975 cells were higher than any other single groups(P<0.05),and all of the combined effect values(Q value)were between 0.85 and 1.15.5.These was no significant difference in the phosphorylation levels of EGFR/AKT/m TOR signal pathway between the gefitinib group and control group(P>0.05).When compared with control group,the expression levels of p-EGFR,p-AKT,and p-m TOR in DC-CIK cells group and DC-CIK combined with gefitinib group were significantly lower(P<0.05);and the levels in DC-CIK cells combined with gefitinib group were futher lower than those in DC-CIK cell group(P<0.05).Conclusion: 1.The cytotoxicity of DC-CIK cells to lung adenocarcinoma H1975 cells enhanced with the increase of the effort-target ratio,and its combined effect with gefitinib showed additive effect.2.In addition to killing the tumor cells directly,the molecular mechanism of DC-CIK cells on lung adenocarcinoma H1975 cells may also be related to the down-regulation of p-EGFR,p-AKT,and p-m TOR in lung adenocarcinoma H1975 cells,and the reversal mechanism of gefitinib resistance may be of down-regulation the EGFR/AKT/m TOR signaling pathway,thereby restoring the sensitivity of lung adenocarcinoma H1975 cells to gefitinib.