| Research background:5'-deoxy-5-fluorouridine (5'-DFUR) is an oral anti-cancer drug, widely used in clinical treatment of gastrointestinal cancer. It is a prodrug of 5-fluorouracil (5-FU), must be activated by thymidine phosphorylase (TP) in cancer tissues and converted into 5-FU. It had been reported that breast cancer and gastric cancer cells express TP , but in colon cancer it is still controversial. We have found in previous studies that in colorectal cancer tissues, TP mainly expressed in stromal cells around cancer nests, especially in macrophages. Previous study has found TP was mainly produced by macrophages in stroma and six colon cancer lines hardly expressed TP. When cancer cells do not express TP , transformation of 5'-DFUR in tissue of colorectal cancer is not clear. Previous study in vivo found that average period of nature survival was longer, average tumor weight was lighter, the average body weight decreased slowly in mouse models of colon cancer given 5'-DFUR ,compared to 5-FU,suggesting that 5'-DFUR as a highly selective anti-cancer drug was transferd to 5-FU in cancer tissue higher than normal tissue . The reason may be the TP enzyme in cancer tissue is more than normal tissues. The early study in vitro 5'-DFUR cultured 24h with blood mononuclear cells, mean value of IC50 of four cancer cells lines decreased significantly,suggesting that blood monocytes can express TP, however ,whether specific content of TP in cancer tissue is higher than in blood still need further experiment to confirm. In this study, 5-FU transfered by 5'-DFUR in cancer tissue and blood was measured by HPLC(high performance liquid chromatography) in order to deduce indirectly distribution difference of TP in tumor tissue and blood , for further study of 5'-DFUR in the transformation of colorectal carcinoma and to provide information on regulation of TP.Materials:1,Animal28 SPF-inbred BALB / c mice, 6-8 weeks, male, weight (20.00±2.34) g, purchased from Experimental Animal Center of Guangdong Province.2,The tumor cell lineBALB/c mouse colon cancer cell line (CT26), purchased from the American Type Culture Collection(ATCC).3,The experiment medicine5'-deoxy-5-fluorouridine(5'-DFUR), from the Japanese company Roche Research Center; 5-fluorouracil (5-FU) injection solution, the Jiangsu Nantong Drugs Manufacture Limited Company (the batch number: 080607); control Standard of 5-fluorouracil (5-FU) purchased from Sigma Limited Company (the batch number: 097K1352).4,Experimental apparatusShimadzu high performance liquid system ;Diamonsil C18 column (250mm×4.6mm, 5μm).Methods:1,Culture of mouse colon cancer cell line CT-261640 10% fetal bovine serum, containing penicillin 100×103 U / L and streptomycin 100 mg / L, cultured in 5% CO2 incubator at 37℃,bathed every other day and passaged by enzyme digestion every two or three days.2,Preparation of cell suspensionTook exponentially growing cells on the day of making model . Digested with 0.25% trypsin and passage into cell suspension and centrifuged at 2 000r/min for 5 minutes . Discarded supernatant and added appropriate amount of saline to adjust the cell concentration to 1×107 pcs / ml.Cell viability measured by trypan blue was above 95%. 3,Colon cancer model makingInjected 0.2ml CT26 suspension into the mouse (BALB/c) the back hypodermic. About 2 weeks later,the tumor could be visible to the naked eye on the back of mouse.4,Animal groups and drug administration28 mice were randomly divided into 4 groups:①5'-DFUR group in 15 minutes ;②5'-DFUR group in 30 minutes ;③5-FU group in 15 minutes ;④5-FU group in 30 minutes . Based on animal weight ,5-FU dosage was 0.020mg / g and solution of concentration was 1.0mg / ml while 5'-DFUR dosage was 0.038mg / g and solution of concentration is 2.0mg/ml. Each group were killed respectively to get blood and tumor tissue on scheduled time after mice were injected intraperitoneally.5,Taking samples0.5 ml blood from Orbital venous of mice was placed into 37℃water bath for 30 minutes and was Centrifuged for 5min at 3200rpm.Supernatant was stored at 4℃. 0.5g tumor tissue was weight after the filter paper cleared blood.Every 0.5g tissue and 4ml normal saline (1:8) were added to homogenizer to fix for 5 minutes, centrifuged at 3200rpm for 5 minutes. Supernatant was stored at 4℃.6,Production of blood and tumor tissue of standard curve of 5-FUDifferent distilled water containing 5-FU standard were added to seven species of 90μL serum from untreated mice into 100μL.The drug concentrationsin serum were set into 6.25,12.5,25.0, 50.0,100.0,200.0,400.0μg mL-1 ,which was used to produce serum standard curve ;Different distilled water containing 5-FU standard were added to seven species of 90μL tumor homogenate from untreated mice into 100μL.The drug concentrations of tumor homogenate were set into 1.0,2.0,4.0,16.0,32.0,64.0,μg ? mL-1,which was used to produce tumor homogenate standard curve.7,Measurement of concentration of samples 100μL serum was placed 5ml test tube and added 2mL of ethyl acetate . Mixture was centrifuged at 3200rpm for 5 min after vortex oscillation for 2 min.Supernatant was placed in another test tube . 2mL of ethyl acetate was added into the first test tube again for the second extraction, vortex oscillation,centrifugation in the same way. Merged the two supernatant and made it dry by centrifugation.Added 100μL mobile phase solution into dry test tube and mixed it. Mixed solution was placed in EP tube before 10000rpm centrifugation for 7min. 20μL of liquid supernatant was obtained to prepare for measure. Recorded the peak area of drugand calculated concentration by standard curve .the 5-FU of concentration of tumor homogenateare was measured in the same way. 8,Oservation index5-FU concentration of tumor tissues and serum in 5'-DFUR group and 5-FU group.9,Statistical analysis5-FU concentration in tumor tissue and serum of 5'-DFUR group and 5-FU group made statistics analysis separately by using statistical software. SPSS13.0.Used the Paired-samples T test.When P<0.05, that had a significant difference.Results:1,At 15,30 minutes after injecting drug 5'-DFUR, 5-FU concentrations in tumor tissue are respectively 54.64μg/g±12.80μg/g and 45.58μg/g±18.82μg/g; 5-FU concentrations in serum are respectively 8.83μg/ml±1.68μg/ml and 9.82μg/ml±2.93μg/ml;5-FU concentration in tumor tissue are respectively 6.37,4.47 times as much as that in serum in 5'-DFUR-15min group and 5'-DFUR-30min group(P<0.05).2,At 15,30 minutes after injecting drug 5-FU, 5-FU concentrations in tumor tissue are respectively 86.13μg/g±15.42μg/g and 94.68μg/g±39.89μg/g; 5-FU concentrations in serum are respectively 133.35μg/ml±20.69μg/ml and 112.70μg/ml±26.27μg/ml; 5-FU concentration in serum are respectively 1.59,1.62 times as much as that in tumor tissue in 5-FU-15min group and 5-FU-30min group(P <0.05).Conclusion:In vivo mouse model of colorectal cancer,conversion ratio of 5'-DFUR in tumor tissue is higher than that in the blood .As a result, we considere the distribution of PyNPase in tumor tissues higher than in blood . |
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