Effects Of Gemcitabine In Combination With Cisplatin On Lung Cancer Cell Line A549

Lung cancer, with the increasing incidence, is one of the leading cause of death among the malignant tumors worldwide. During so many therapies, chemotherapy plays an important role in the treatment of non-small cell lung cancer. Gemcitabine, a pyrimidine nucleoside antimetabolite, is one of the most promising cytotoxic agents which has shown activity in a variety of solid tumors, but appears to be most active in NSCLC. Cisplatin as a s platinum compound, is now a widely used singlely or in combination of chemotherapy for the treatment of many cancers such as lung cancer including NSCLC. Because of their complementary mechanisms of action and the nonoverlapping side effects, GEM and CDDP are ideal candidates for drug combinations. Previous studies reported a schedule-dependent interaction between gemcitabine and cisplatin. How to make full use of these two drugs, how to combine them in chemotherapy is the focus of the study. Many work have to be done about the schedule, the dose and the mechanisms of the drug combination. Our work is to investigate the effects of gemcitabine in combination with cisplatin on lung cancer cell line A549, providing useful evidence to clinical application of drug combination.Materials and Methods1. DrugsGemcitabine and cisplatin are general gifts from Hansoh company, (Jiangsu,China).2. Cell cultureHuman lung cancer cell lines A549 (adenocarcinoma) was cultured in DMEM,supplied with 10% foetal bovine serum,at 37℃in a 5% CO2 incubator. 3.3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) AssayCell proliferation was evaluated each day for four days after the MTT treatment. The absorbance, which is directly proportional to the number of living cells in the culture, was measured at 490 nm using a microplate reader (Model 490, Bio-Rad, Hercules, CA, USA). A blank with dimethyl sulfoxide (DMSO) alone was taken and subtracted from all values.4.Flow cytometry (PI)Cells from each experimental group were collected and digested with trypsin and fixed with 75% ice-cold ethanol at 4℃overnight. Cells (1×106) were centrifuged at 1500 rpm 5 minutes, and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis. The percentages of cells in the different cell cycle phases were determined using a FACS Calibur Flow Cytometer with CellQuest 3.0 software.5.Flow cytometry(Annexin V-FITC)Every group was divided into four subgroups:blank control,only Annexin V,only FITC and Annexin V+FITC.Flow cytometry performed on BD FACS CaliburTM Flow Cytometer.6.Statistical analysisThe SPSS 17.0 software was employed to analyze the data.P<0.05 was considered as statistical significance.Results1. Effects of individual drug on lung cancer cell line A549In vitro activity of GEM and CDDP was evaluated in A549 for exposure of 24h,48h,72h in various concentration. IC50 values of GEM on A549 cell line were 3.79±0.32 mg/L,1.11±0.12mg/L.0.23±0.06mg/L in 24h,48h,72h.CDDP's IC50 were 18.58±2.01mg/L,3.78±0.25 mg/L,0.76±0.10 mg/L. In comparison, both drugs work in a dose-dependent manner.2. The sequence-dependent effect of drug combination on lung cancer cell line A549According to the IC50 of individual drug, we exposed A549 in different schedules, the analysis of the cell growth inhibition by GEM in combination with CDDP was performed. When GEM is followed by CDDP, the combination Index is1, the drug interaction was significantly antagonistic; When GEM and CDDP were given at the same time, the CI is 1 which means an addictive effect.3. The sequence-dependent effect of drug combination on apoptosis of lung cancer cell line A549Apoptosis rate:Control2.82%±0.12%; G 14.54%±2.36%; C 15.06%±1.31%; GC 25.72%±3.29%; CG 10.99%±1.04%; G+C 19.24%±3.61%。In conclusion, GC(GEM is given before CDDP) got the highest apoptosis rate.4. The sequence-dependent effect of drug combination on cell cycles of lung cancer cell line A549In accordance with schedules used in the growth inhibition experiments, cells revealed a marked S phase accumulation under single drug. A G 1,G2 phase accumulation was observed when GEM is followed by CDDP, whereas a S phase accumulation was detected in the opposite schedule. A S phase accumulation also performed when the two drugs used simultaneously.ConclusionsThe effect of combination of gemcitabine and cisplatin was sequence-dependent, favoring a regime in which GEM is followed by CDDP. Our study would supply an useful reference in clinical application of drug combination.