| Epidermal growth factor receptor tyrosine kinase inhibitor gefitinib is a prominent representative of the cancer targeted drugs. Overseas studies have shown that gefitinib can make tumors shrink in non-small cell lung cancer patients who has a EGFR mutation-positive. The "2011non-small cell lung cancer clinical practice guidelines (China Version)" determined gefitinib as the first-line therapy in the EGFR mutation-positive patients. However, data from clinical teatment and experiment shows that the acquired resistance to gefitinib limited its further application. In this study, we establish gefitinib-resistant PC9cell line to detect the effect of gefitinib in the phosphorylation of proteins that is in the EGFR signaling pathway, detect the expression of PI3K downstream transcription factor FoxO3a in the resistant cell lines, detect the correlation between the phosphorylated Fox03a and IC50, to investigate the relationship between the phosphorylated FoxO3a and resistance.ObjectivesCulture human lung adenocarcinoma cell line PC9in vitro, and establish gefitinib-resistant PC9cell line. Detect the effect of gefitinib in the phosphorylation of proteins that is in the EGFR signaling pathway, detect the expression of PI3K downstream transcription factor FoxO3a in the resistant cell lines, detect the correlation between the phosphorylated FoxO3a and IC50, to investigate the relationship between the phosphorylated FoxO3a and resistance.Methods1. Cell culture PC9cell and PC9/GR cell are culture at ambient conditions of37℃±0.3℃, relative humidity≥95%, and CO2concentration5%±0.1%in vitro.2. The establishment of a PC9/gefitinib resistance cell line The PC9/GR cells developed an acquired resistance to gefitinib following a stepwise increasing dose of gefitinib combined with intermittent high-dose challenge. The PC9/GR can stably grow in the0.5μmol/L gefitinib..3. Effect of Gefitinib on cellular growth inhibition during different resistance period. 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/L respectively.4. The effective of Gefitinib on the EGFR signaling pathways protein level in PC9cell line The PC9Cell line is treated with different concentration(0.5,1,2,4μmol/L) of gefitinib, wash the culture after1hour and then extract the protein. Detect the change of EGFR signaling pathways protein level by Western Blot. The PC9Cell line is treated with1μmol/L Gefitinib after different treatment time (0,1.5,3,6,12h), wash the culture and then extract the protein. Detect the change of EGFR signaling pathways protein level by Western Blot.5. The effective of Gefitinib on the EGFR signaling pathways protein level in different period of PC9/GR The concentration of gefitinib1μmol/L was induced to the different period of PC9/GR after treatment with1hour, wash the culture and then extract the protein. Detect the change of EGFR signaling pathways protein level by Western Blot.Results1. Effect of Gefitinib on morphological alterations of PC9cell line Inverted microscope was employed to observe the morphological alterations of PC9and PC9/GR cells. The cellular morphology of PC9is oval and the cellular morphology ofPC9/GR is fusiform.2. The effective of Gcfitinib on the growth inhibition The cellular sensitivity of Gefitinib was determined by MTT assay. The IC50value of PC9is0.05±0.011μmol/L. The IC50value of PC9/GR1is0.11±0.015μmol/L. The IC50value of PC9/GR2is0.23±0.061μmol/L. The IC50value of PC9/GR3is0.45±0.047μmol/L. The IC50value of PC9/GR4is0.83±0.103μmol/L. The IC50value of PC9/GR5is1.7±0.153μmol/L.3. The effective of Gefitinib on the EGFR signaling pathways protein level in PC9cell line As the Western Blot shows, the expression of protein level of phosphorylated Akt and phosphorylated Erk are gradually reduced with increasing concentrations of gefitinib in a dose-dependent. The expression of protein level of phosphorylated Akt and phosphorylated Erk are gradually reduced with increasing treatment time of gefitinib in a time-dependent.4. The effective of Gefitinib on the EGFR signaling pathways protein level in different period of PC9/GR As the Western Blot shows, the expression of protein level of phosphorylated EGFR, phosphorylated FoxO3a, phosphorylated Akt and phosphorylated Erk have a significantly difference. All the protein level are increasing with the increasing induced time.Conclusion1. Establish a PC9/gefitinib resistance cell line successfully.2. Gefitinib can inhibit the phosphorylation level of Akt and Erk significantly in PC9cell line.3. The effective of gefitinib on the EGFR signaling pathway is gradually weakened after the increasing induced time. The resistance progress and phosphorylation of FoxO3a closely related... |
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