Effect Of Resistance To Gefitinib On Phosphorylation Of FoxO3a In PC9Cells

Gefitinib is the representative of the epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) tyrosine kinase inhibitor (Tyrosine Kinase Inhibitor, TKI). It is attracted widespread attention as tumor-targeting drugs in non-small cell lung cancer clinical practice. It has shown that gefitinib reduced the tumor burden in non-small cell lung cancer patients with EGFR mutation-positive.2011"NCCN Clinical Practice guideline-Non-small cell lung Cancer" clearly recommend gefitinib as first-line chemotherapy for patients with EGFR mutation. However, the acquaired resistance to gefitinib is difficult to avoid in clinical therapy. It is a serious problem and restricts the application of the EGFR TKI. FoxO (Forkhead box O)3a transcription factor is a key growth factor by regulating its downstream target genes in survival, proliferation, differentiation and apoptosis in tumor occurrence and paid more and more attention in tumor therapy.Objectives To systematic study the alteration of FoxO3a phosphorylation in PC9cells and PC9/GR cells and analysis the difference of nuclear translocation charactors of FoxO3a between PC9cells and PC9/GR cells.Methods1. Cell culturePC9cell and PC9/GR cell are culture at ambient conditions of37℃±0.3℃, relative humidity≥95%, and CO2concentration5%±0.1%in vitro. 2. Sensitivity of PC9cells and PC9/GR cells to gefitninib. The cellular sensitivity of Gefitinib was determined by MTT assay (72-hour exposure). The concentration of Gefitinib are The concentrations of gefitinib are0.5,1,5,10,50,100,500,1000,5000,1OOOOnmol/Lrespectively.3. Effect of serum starvation or sitimulation of EGF on signaling pathway in PC9cells and PC9/GR cells. PC9cells and PC9/GR cells were treated with serum starvation for time course and EGF in variety of concentrations (0,12.5,25,50ng/ml), and for different time (0,5,10,20min), for evaluat of signaling proteins by using Western blot.4. The effective of inhibitor of signal kinases on the EGFR signaling pathway in PC9cell and PC9/GR cells. Serum-starved PC9cells and PC9/GR cells were pretreated with LY294002or PD98059for30min, then treated with50ng/ml EGF for20min. Cells lysed in RIPA buffer and western blotting with anti-phospho-Akt, anti-phospho-FoxO3a, anti-phospho-ERK and anti-GAPDH antibodies.5. Characterization of intracellular translocation of FoxO3a induced by EGF in PC9cells and PC9/GR cells. We transfected PC9cells and PC9/GR cells with FoxO3a-GFP plasmid, observed the subcellular location of GFP, recorded the translocation condition in serum-starvation, EGF treatment and pretreatment with Gefitinib, as well as identified the relationship between phosphorylation and translocation.Results1. Biological characteristics of PC9cells and PC9/GR cells It is not found significant morphological difference between PC9cells and PC9/GR cells. There is nor T790M mutation in EGFR20exon in PC9cells and PC9/GR cells. The IC50value of PC9cells and PC9/GR cells was: IC50(PC9)=13.5nmol/1, IC50(PC9/GR)=1968nmol/12. Reactivity of signaling pathway to serum starvation and EGF in PC9cells and PC9/GR cells.Serum starvation reduces the phosphorylation of Akt, ERK and FoxO3a in time-dependent manner, and to baseline in60min. EGF induces the phosphorylation of Akt, ERK and FoxO3a in time-and dose-dependent manner, and to peak in20min with50ng/ml. The phosphorylation of FoxO3a induced by EGF could be blocked by gefitinib and PI3K/Akt inhibitor LY294002but ERK inhibitor PD98059.3. Characterization of intracellular translocation of Fox03a induced by EGF in PC9cells and PC9/GR cells.Serum starvation induced FoxO3a-GFP located in nuclus in PC9cells and PC9/GR cells in time-dependent manner.EGF induced FoxO3a-GFP cytoplasmic location in PC9cells and PC9^GR cells in time-dependent manner.EGF induced translocation of FoxO3a-GFP could be blocked by Gefitinib. Distribution of FoxO3a-GFP in cytoplasm could be return by EGF treatment for60min in PC9/GR cells but not in PC9cells.Conclusion1. There is nor alteration in morphological character and mutation on EGFR20exon T790M site between PC9cells and PC9/GR cells.2. The IC50to Gefitinib in higher in PC9/GR cells than that in PC9cells.3. Serum starvation reduces the phosphorylation of Akt, ERK and FoxO3a in time-dependent manner, and makes FoxO3a-GFP located in nuclus faster in PC9cells than in PC9/GR cells.4. EGF induces the phosphorylation of Akt, ERK and FoxO3a in time-and dose dependent manner, and makes FoxO3a-GFP cytoplasmic location faster in PC9/GR than in PC9cells.5. FoxO3a phosphorylation and FoxO3a-GFP nuclear exclusion could be blocked by LY294002and Gefitinib.