Determination Of Intracellular And Extracellular 5-Fluorouracil And Proteomics Of A549 Cells Induced By 5-Fluorouracil

Objective: Malignant tumors are most important diseases imperiling people. The mortality caused by lung cancer increased substantially in recent years. NSCLC accounts for 80-85% of all lung cancer cases. The survival rate of 80% lung cancer patients is very low. 5-Fluorouracil(5-FU) is a popular anticancer drug in clinical treatments and its resistance is becoming more serious. Therefore, it's necessary to explore the anticancer and drug resistance mechanisms. So, we carried out the following study, proliferation inhibition experiments of A549 cells by 5-FU were finished, nuclear morphology changes were observed by fluorescence microscope and apoptosis ratios were checked by FCM. Finally, apoptosis models of A549 cell were established. Intracellular and extracellular concentration of 5-FU in A549 cell were determined by HPLC in order to explore drug resistance in A549 cell prelimiarily. Next, the differential expressions of proteins in A549 cells induced by 5-FU and control group were analyzed by comparative proteomics in order to provide experimental basis to clarify its antitumor and drug resistance mechanisms and improve its chemosensitivity in clinical treatments.Methods: 1. An apoptosis model of A549 cell induced by 5-FU has been established. Growth inhibition ratios were measured by the MTT method. Percentage of apoptosis were analyzed by flow cytometer; A549 cells were treated with 5-FU of different concentration and stained with DAPI , morphology changes were observed.2. A method for the determination of intracellular and extracellular 5-FU has been established by HPLC. After the drug action, collect the intracellular and extracellular liquid respectively, and then quantitive analysis was finished by HPLC. The chromatographic conditions are as follow, chromatographic column is Hypersil ODS (4.6 mm×2.5 mm, 5μm); mobile phase is methanol-water=2:98(V/V); flow rate is 0.8ml/min; column temperature is 30℃; detected wavelength is 265 nm and the injection volume is 5μl.3. Expression changes in A549 cells induced by 5-FU were studied. Both the treated A549 cells by 150μg/ml 5-FU and the control A549 cells were cultivated for 24 h, total proteins of control cells and treated cells were extracted. Proteins were separated by 2-DE gel electrophoresis and stained by silver nitrate. Differentially expressed proteins were analyzed with PDQuest8.0 software. Finally, selected protein spots were cut out of 2-DE gels and identified under a reflector mode by MALDI-TOF-MS and PMF of proteins were acquired. Then, bio-analysis was accomplished to search for possible differentially expressed proteins with PMF by MASCOT searching engine database.Results: 1. Apoptosis occurred in all treated group cells(40,80,150μg/mL). 5-FU of different concentration inhibited the proliferation of A549 cells in a dose dependent manner from 10μg /ml to 100μg /ml and inhibition ratios were increased slowly in a dose manner from 100μg /ml to 300μg /ml for 24 h and 48 h. Inhibition ratios were increased slowly when 5-FU dose ranged from 10μg /ml to 300μg /ml for 72 h. Inhibition ratios were increased significantly from 24 h to 48 h and slowly from 48 h to 72 h at the same concentration of 5-FU. Differences were statistically significant between the treatment and control groups. IC50 of 5-FU were 153.20, 50.97, 14.66μg/ml for 24, 48, 72 h, respectively. Apoptosis percentages were in accordance with MTT results when the concentration of 5-FU ranged from 40μg/ml to 150μg/ml.2. The linear relation was very good of intracellular 5-FU over the coverage of 0.1μg/ml to 50.0μg/ml (r=0.9996). The average recovery is 102.34% and the RSD was 0.40%. The RSD of intra-day and inter-day were 1.30%, 1.04%, respectively. Meanwhile, the linear relation was quite excellent in extracellular 5-FU from 1.0μg/ml to 250.0μg/ml (r=0.9999). The average recovery is 97.65% and the RSD was 2.11%. The RSD of intra-day and inter-day were 1.32%, 1.06%, respectively. Restistances to 5-FU in A549 cells happened when concentration of 5-FU arrived at 50μg/ml and the concentration of intracellular and extracellular 5-FU were possibly influenced by drug metabolism.3,About 1100 proteins were determined by PDQuest8.0 software analysis. Further, 6 differentially expressed proteins spots between treated sample and control sample were identified by MALAI-TOF-MS analysis and MASCOT database searching. Up-regulated proteins consisted of Calreticulin, Hsp70, Prohibitin, Cytochrome C and Phosphoglycerate kinase; down-regulated proteins were Actin and Adseverin.Conclusion: 5-FU inhibited the proliferation of A549 cells and induced apoptosis. The concentration change of 5-FU in A549 cells was explored by the determination of intracellular and extracellular 5-FU by HPLC. A549 cell restistance to 5-FU possibly occurred when concentration arrived at 50μg/ml or effect time was beyond 48 h. Taken together, 6 differentially expressed proteins were possibly the potential targets that 5-FU effected A549 and Hsp70 were possibly drug resistance bio-marker of 5-FU.