Effect Of Gefitinib In Combination With Oxaliplatin On Lung Cancer Cell Line A549

Lung cancer is the leading cause of cancer death with highest incidence in our country. Comprehensive therapy has been more and more important for lung cancer. The drugs of molecular target therapy, such as gefitinib, have been widely used in clinic. Gefitinib is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), which has been approved for the treatment of advanced non-small cell lung cancer (NSCLC) by FDA since 2003. Gefitinib monotherapy has demonstrated clinically meaningful antitumor activity, but the effective power was only 10%-20%. So how to make full use of gefitinib, how to combine gefitinib with chemotherapy, is the emphasis of the next study. Oxaliplatin (L-OHP) is the third-passage platinum-derived antitumor drug that has been widely used in NSCLC patients. In the present study, series of basic experiments had been carried out to evaluate the antitumor effect of gefitinib combined with L-OHP on the lung cancer cell line A549, providing preclinical evidence for optimizing the schedule of gefitinib combined with chemotherapy in the treatment of lung cancer.The work described here include three parts that will be detailed below:(1) to investigate the effects of gefitinib in combination with oxaliplatin on lung cancer cells in different sequences administration in vitro;(2) to evaluate the effects of gefitinib in combination with oxaliplatin in different sequences administration in vivo;(3) to analyze the underlying mechanisms responsible for different antitumor effect.Firstly, determination of IC50 was performed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. In vitro ,we exposed cells to oxaliplatin and/or gefitinib. IC50 value of gefitinib in 24h, 48h, 72h were 92.3±3.10μmol/L, 32.5±1.05μmol/L, 5.21±0.80μmol/L. IC50 values of oxaliplatin on A549 cells were 1.21±0.03μmol/L, 0.64±0.05μmol/L and 0.23±0.02μmol/L. The analysis of the cell growth inhibition by gefitinib in combination with oxaliplatin in three different schedules was performed. when oxaliplatin was given before or simultaneously with gefitinib, CI<1 or =1, the activitives of the drugs were synergistic or additive. While, oxaliplatin given after gefitinib, CI>1, the activitives of the drugs were antagonistic. Our results provided evidences of strong synergy between the drugs when gefitinib was given following oxaliplatin.Secondly, we established the transplanted tumor model of nude mice with the aim of evaluation the antitumor effect of different combination of gefitinib and oxaliplatin to confirm above observation in vitro. BALB/c nude mice beared 2×107 A549 cells per mouse were randomized into six groups:(1)control;(2)oxliplatin;(3)gefitinib;(4)oxliplatin followed by gefitinib;(5)simultaneous gefitinib and chemotherapy followed by gefitinib;(6)oxliplatin followed by gefitinib. After continous observation of 35 days, the weight change of nude mice and the tumor growth were evaluated. As a result, compared with other groups, the weight increase in the group of chemotherapy followed by gefitinib have no statistical difference P(>0.05), and the tumor grew the most slowly, the inhibition rate was 58.9%(P<0.05). The data above suggested that gefitinib and oxaliplatin did not add to each other's toxicity. And the group of oxliplatin followed by gefitinib had the strongest antitumor power on transplanted tumor among six groups.Lastly, to investigate the mechanism of schedule-dependent synergy in this study, we carried out a biological analysis of cell-cycle modulation and apoptosis induction by the drugs. Gefitinib mediated a prolongation of cell cycle progression in G0/G1 phase(40.3%) on lung cancer cells. Oxaliplatin induces S arrest(70%). Chemotherapy induce DNA damage results in cell cycle arrest, primarily at the G2/M and G0/G1 checkpoints, providing the opportunity for DNA damage repair prior to mitosis. Subsequent gefitinib exposure increased the arrest at G2/M phase(37.9%, P<0.05), so prevented the repair of chemotherapy-induced DNA damage, cells could not repair normally. As a result, sequential chemotherapy followed by gefitinib exposure could induced more G2/M phase arrest. Similiarly, in our study, an enhancement of apoptosis(22.3%, P<0.05) was observed when gefitinib followed oxaliplatin.Our observations indicate that inhibition of cell cycle progression and induction of cell apoptosis contribute to the antitumor effects of oxaliplatin followed by gefitinib on human lung cancer cells. The effect of gefitinib combined with oxaliplatin is sechedule-dependent. Oxaliplatin followed by gefitinib exhibited a maximum effect, the mechanism was possibly related to inhibition of damage restore and maintaining of oxaliplatin-induced apoptosis. Our present study would provide useful reference for the clinical application of gefitinib.