Effect Of Aspirin Combined With5-fluorouracil On Hepg2Cells

ObjectiveHepatocellular carcinoma （HCC） is the incidence of the fifth in the world. It relatesmortality ranked third in the world of malignant tumor, second only to gastric cancer andesophageal cancer. The malignant degree is high, and the natural survival time is short.Because its pathogenesis is concealed, patients in clinical diagnosis is always in middle-latestage, only10%-20%patients can be operation excision. The results showed that after5years of hepatocellular carcinoma recurrence rate is as high as60-70%. It is visible that theeffect of chemotherapy doesn’t reach our expectations, which has become further improvecurative effect of hepatocellular carcinoma dilemma.5-fluorouracil （5-FU） has been widely used in chemotherapy of hepatocellularcarcinoma before operation and after operation. It transforms into vivo5-fluorouracildeoxyribonucleoside （5F-dUMP）, and inhibites thymidylate synthase, preventsdeoxyuridine monophosphate （dUMP） methyl into deoxythymidylic acid （dTMP）, thusaffects synthesis of DNA. But the long-term use of large doses of toxicity to bone marrowand digestive tract is larger, the effective dose and the toxic dose of medication is similar,which limits its application.Aspirin （acetyl salicylic acid, ASA）, as an analgesic, anti-inflammatory and salicylicacid inhibited platelet aggregation drugs, clinical application has110years. It has beenclinically for the treatment of acute and chronic rheumatism disease. Study in recent yearsshow that aspirin also has anti tumor effect. Research finds that aspirin can inhibit the invitro culture of primary hepatocellular carcinoma （HCC） cells in nude mice. HCCorthotopic transplantation tumor growth also has good inhibition. Aspirin on tumor growthinhibition of cyclooxygenase-2（COX-2）, and does not cause gastrointestinal bleeding andulcers in nude mice. The experiment in vitro, inhibition to aspirin combined with5-FU on human hepatocellular carcinoma cells, in order to enhance the efficacy of5-FU, reduce thedosages and time, thereby reducing the toxicity of fluorouracil. Then provide theoreticalsupport for the inhibitory effect of the clinical application of combined aspirin and5-FU inhepatocellular carcinoma.Aim to investigate the effect of aspirin combined with fluorouracil in the inhibition ofcell proliferation, whether there is a synergistic apoptosis. To investigate the mechanism ofaspirin combined with5-fluorouracil induced apoptosis of hepatoma cells.Research methods:Cells cultured in vitro,（1） group: divided into four groups, respectively: blankcontrol group,5mg/ml fluorouracil group, aspirin group and （90, respectively in the180,360mg/L） three concentration group,5mg/ml fluorouracil+aspirin （90,180,360mg/L）was the combination group.（2） indexes and observation methods: the human hepatocellular carcinoma HepG2cells in vitro by fluorouracil, aspirin alone（90,180,360mg/L）or combined treatment, cellproliferation was detected by CCK-8assay, continue to culture1H at a wavelength of450nm, the determination of the absorbance value of Kong Guang ELISA analyzer （A）.Records of the results of human hepatoma HepG2cells, inhibition rate calculation formula:the inhibition rate of=[1-in experimental group A/A values of the control group] x100%.（3）HepG2cell apoptosis detection ofAnnexin V PE/PI fluorescent double labelingmethod.From the bottom of the culture flask to hepatocellular carcinoma HepG2cells bytrypsin digestion. The configuration,1200rotating centrifugal5min;Each cell in each hole lx105cells were inoculated on culture plates, each group had3holes （aspirin group each concentration3holes）; after inoculation with24h cell densityreached80%, the replacement of the culture medium were added; each hole （90,180、360mg/L） aspirin or10mg/ml5-fluorine uracil or two drug United, after treating cellscultured for48hours induced cell apoptosis; spare not containing EDTA by trypsindigestion culture plate of cells; PBS cells were washed two times （2000rpm, centrifugal5min） collected from3×105cells; learn from the5OOul Buffer （1×Binding bindingbuffer） was added to the cell suspension in addition;5U1Annexin V-FITC and lOul PI;room temperature, light,10min reaction;（4） Western Blot to detect the apoptosis related proteins COX-2, Bcl-2expression and Caspase-9protein.On the basis of protein concentration, protein samples were taken with30ug, thedilution Volume2times and5x buffer in4:1volume mixing ratio, after100℃, lOminwater treatment, cooling, ice sample; first with the70V voltage pre electrophoresis（30min）, and then in the stacking gel voltage70V, the separation gel voltage of100Vunder the conditions of electrophoresis, until the bromophenol blue indicator migration intothe gel bottom can stop stop electrophoresis; polyacrylamide gel electrophoresis, slowlyremove the separation gel, switch to electric conversion buffer processing30min; thecorresponding antibody and skimmed milk powder5%TBS/T solution with1:1000dilution,and PVDF film the night table, at4℃incubation conditions, after washing with TBS/T;using1:5000diluted labeled two antibody; after developing ECL chemiluminescencedetection, Quantity one （Bio-Rad） semi quantitative software（5）statistical analysis: the data with the mean standard deviation（Rtes）said that,between the two groups using t test, compared among groups using single factor analysis ofvariance （one-way ANOVA） with P<0.05as the difference has statistical significance.Results:1. Aspirin alone can inhibit proliferation of hepatocellular carcinoma cells, and in theexperimental range was positively correlated with the dosage.2. It is more effective that aspirin combined with5-FU on the proliferation of hepatomacells apoptosis induced by inhibition of HepG2than the drug alone.3. Aspirin alone or combined with fluorouracil by down regulating the expression ofapoptosis related proteins Bcl-2, increase the expression of Caspase-9protein and apoptosis.The aspirin down-regulation expression and down regulate the expression of COX-2Bcl-2.ConclusionAspirin can inhibit the proliferation of HepG2cells, induced apoptosis of hepatoma cell,combined with fluorouracil synergistic effect. The mechanism may be that aspirin decreasesthe expression of COX-2, thereby reducing the expression of Bcl-2, increase the expressionof Caspase-9to cut down the apoptosis of HepG2cells.