Sequence-dependent Effect Of Docetaxel And Gefitinib On The Proliferation Of Lung Adenocarcinoma A549and PC-9Cells

Background and ObjectiveLung cancer is one of the leading cause of cancer-related death.in the world, with a rapid of1.5million new cases each year.-Non-small cell lung cancer (NSCLC, non-small cell lung cancer) take about80%of lung cancer, and only30%-40%of NSCLC can be resected at the time of diagnosis.A platinum-based chemotherapy-regiments were the standard treatment for advanced NSCLC. Howeverregardless of the different combination of chemotherapy drugs, the chemotherapy for NSCLC patients has reached a plateau. The median survival time was8-10months and the one year survival rate was only30%-40%.In recent years, with the rapid development of molecular biology of tumors, tumor immunology and cell biology, molecular targeted therapy came into being. Because of its less toxic reaction and targeted therapy for intracellular or surface-specific molecular specificity, molecular targeted therapy has became a hot research topic of current cancer treatment. Epidermal growth factor recptor(EGFR),a member of the ErbB family, can promote tumor cell proliferation, inhibit its apopto sis, improve its ability of movement and therapy increase tumor invasion and distant metastasis. Previous studies have shown that the EGFR is over-expressed in the lung cancer cells. Gefitinib is an oral EGFR tyrosine kinase inhibitor (Gefitinib, of ZD1839, Iressa). The FDA of United States has approved gefitinib as the second-line treatment for advanced NSCLC patients. Two randomized, phase II clinical trials IDEAL1&2studies show that gefitinib have a mild adverse reactions and well tolerated, as a second or third-line treatment in patients with advanced NSCLC who had been previously treated by chemotherapy. The subsequent phase III clinical trials NEJGSG002and WJTOG3405reported that gefitinib have a superior PFS to chemotherapy and a trendence to prolong overall survival for advanced NSCLC with EGFR mutation. Based on these studies, the FDA of United States has approved gefitinib for the first-line treatment of advanced NSCLC with EGFR mutation.Since the different mechanisms of chemotherapy drugs and EGFR-TKIs, people aimed at evaluating the synergistic cytotoxicity between gefitinib and chemotherapy. Previous studies in vitro reported that gefitinib should have enhanced toxicities in combination with conventional chemotherapeutic agents (cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, doxorubicin, etc.), which was confirmed in the animal trials,However, the combined treatment gefitinib/erlotinib and standard two-drug for patients with advanced NSCLC, such as INTACT1(gemcitabine/cisplatin with gefitinib), INTACT2(paclitaxel/carboplatin with gefitinib), TRIBUTE(paclitaxel/carboplatin with erlotinib), TALENT (gemcitabine/cisplatin with erlotinib) failed to improve time-to-disease progression, and response rate, also the efficiency and survival time was not superior to chemotherapy alone. This inconsistent results of vitro studies with clinical studies led to two point:(1) whether there are advantages patients with EGFR-TKIs and chemotherapy? TRIBUTE study examined the EGFR18-21and KRAS gene mutation of all patients, which results suggested that EGFR-TKIs combined with chemotherapy produce superior efficient in EGFR mutation patients than the wild type (53%vs.18%,p=0.012).(2) whether synergies happened in advantages crowed of EGFR-TKI combinated with chemotherapy? So the first aim of this study was to investigate the sequence-dependent effect of docetaxel and gefitinib on EGFR wild and mutation type human lung adenocarcinoma cells.The cellular mechanism of EGFR-TKIs following chemotherapy on the cell proliferation of NSCLC still remains unknown. Previous studies have shown that the concomitant treatment of EGFR-TKIs with chemotherapy unreached synergistic reasults may be cell cycle related, and the additive effects of the sequential chemotherapy followed by EGFR-TKIs may be related to EGFR phosphorylation. Recent studies reported that IGF-1dependent PI3K/AKT pathways may affect the sensitivity of NSCLC cells to gefitinib. Therefore the second purpose of this study was to investigate the effect of the sequential adminsistration of docetaxel and gefitinib on cellular signaling proteins of EGFR, ERK, AKT and IGF-1R expression, phosphorylation expression and cell cycle of NSCLC cells, to explore its cell mechanism.MethordThe EGFR and K-ras gene mutation were examined by qPCR-HRM. MTT assay was used to measure the cell proliferation. The expression and phosphorylation of EGFR, ERK, AKT and IGF-1R were determined by western blotting. Cell cycle was detected by flow cytometry. The cells were treated with docetaxel and gefitinib monotherapy and in combination.Statistical AnalysisData are presented as mean±S.E.M, and was analyzed by SPSS13.0.Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test. Results1. Cell EGFR and K-ras gene statusNo EGFR gene mutation was found in A549cells and EGFR gene exon19mutation in PC-9cells, that is consistent with previously reported in the literature.2. The sequence-dependent effect of docetaxel and gefitinib on the proliferation of human lung adenocarcinoma A549and PC-9cells.With normal cultured, A549and PC-9cells grew multiply indefinitely into logarithmicly the logarithmic growth phase, respectively two and three days. Docetaxel(10-4M-10-10M) dose-dependently inhibited both A549and PC-9cells proliferation with the IC50values of5.24×10-7mol/L and2.13×10-8mol/L, while gefitinib inhibited A549(10-2M-10-8M) and PC-9cells (10-4M-10-10M) dose-dependently with the IC50values of1.28×10-5mol/L, and4.58×10-8mol/L. Gefitinib inhibition of PC-9cell growth was significantly stronger than the A549(P <0.05) nearly1,000times.Sequential administration of gefitinib following docetaxel remarkably enhanced the inhibition of docetaxel on the proliferation of both A549(increase9.34%, P<0.05) and PC-9cells(increase8.94%, P<0.05). Synergistic effects on the cell proliferation were observed only on PC-9cell(increase7.56%, P<0.05, not on A549cells, when cells were treated with docetaxel and gefitinib with the doses of IC50concomitantly.3.Cellular signaling proteins of EGFR,ERK,AKT and IGF-1R expression, phosphorylation expression of A549and PC-9cells treated by the combination of docetaxel and gefitinib.Docetaxel and gefitinib respectively enhance and inhibition the two cell EGFR and ERK phosphorylation. The phosphorylation of EGFR and ERK induced by docetaxel on both A549and PC-9cells was significantly suppressed by subsequent exposure to gefitinib. The phosphorylation of IGF-1R on PC-9cells was significantly suppressed when cells were treatded with docetaxel and gefitinib concomitantly.4. The effect of the combination of docetaxel and gefitinib on the cell cycles of A549and PC-9cells.Cell cycle studies showed that docetaxel induced G2/M arrest for both the A549and PC-9cells, which enhanced43.28%(P<0.05) and30.13%(P<0.05) respectively. Gefitinib signifucantly induced GO/1arrest for PC-9cells (increase29.98%, P<0.05), not for A549cells. The sequential docetaxel followed by gefitinib arrested the two cells in the G2phase, compared with the control group increased about54.11%and40.77%(P<0.05) respectively. No significant effects on cell cycles were found when they were used simultaneously or gefitinib following docetaxel.Conclusion1.A549is EGFR gene wild type.while PC-9is EGFR gene mutation A549cells EGFR wild-type human lung adenocarcinoma cell line. The cell growth experiments showed that A549is resistance and PC-9is sensitive respectively to gefitinib with a difference over1000-fold, that was consistent with the results previously reported.2.Regardless of EGFR status, the synergistic effects on the proliferation of both EGFR wild-type and mutant NSCLC cells were observed when gefitinib was sequentially administrated following docetaxel, and the phosphorylation of both EGFR and ERK may contribute to this additive effect. The concomitant treatment of gefitinib with docetaxel exerted significant additive effects of cell proliferation only on PC-9cells, and the suppression of IGF-1R phosphorylation may contribute to this synergistic effects. The sequential treatment of gefitinib followed by docetaxel exert no significant additive effect on the cell proliferation and resulted in the accumulation of cells in G0/G1phase, which may decrease the effectiveness of docetaxel in subsequent cycles.