RESUMEN
objective To construct the recombinant adenovirus containing p53 up-regulated modulator of apoptosis(Ad-PUMA)and investigate its growth inhibition effect on pancreatic callCer cells in vitro and in vivo.Methodls Ad-Easy system was used to construct Ad-PUMA by recombination in E.coli.The virus was Dackaged in 293 cells and subsequently identified valid.The AsPC-1 cells were infected with AdPUMA.Before and after Ad-PUMA infection,the expression of PUMA protein wag investigated by western blot,the inhibition rate of AsPC-1 cells was examined by MTY assay.The in vivo tumor suppressive effect was detected in nude mice with human AsPC-1 xenograft.PUMA protein and the apoptosis of AsPC-1 xenograft were detected by western blot and TUNEL(terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling)method.Results In vitro,the expression of PUMA protein was increased with titer of Ad-PUMA,the proliferation of AsPC.1 cells were suppressed,significantly,and the effect was in a viral dose-dependent nlanner.In vivo,the growth in nude mice of AsPC-1 infected with Ad-PUMA was significantly inhibited with an inhibition rate of 44.2%.The expression of PUMA was significantly up-regulated,and the apoptosis index wa8 significantiv increased in tumor after Ad-PUMA infection as determined by western blot and TUNEL.Conclusion The expression of PUMA call inhibit the proliferation of pancreatic cancer in vitro and in vivo,and may be used 88 a potential tool for cancer therapy
RESUMEN
Objective:To investigate whether PUMA gene transfection can increase sensitivity of pancreatic cancer cells (PC) to 5-FU-induced apoptosis. Methods: PUMA-pCEP4 containing full length PUMA cDNA or pCEP4 was transfected into human pancreatic cancer cell line AsPC-1 by lipofectamine transfection, G418 selection was used to select positive cells. AsPC-1, AsPC-1/PUMA and AsPC-1/pCEP4 cells were separately treated with serial concentrations of 5-FU(0.01-100 ?mol/L). MTT assay was used to determine the cell survival rate in each group and IC50 of 5-FU was calculated. TUNEL,FCM and DNA ladder observation were employed to study cell apoptosis. Western blotting was performed to detect the expression of PUMA protein. Results: The 5-FU IC50 values of AsPC-1, AsPC-1/PUMA and AsPC-1/pCEP4 cells were (12?1.9)?mol/L,(1.6?0.4)?mol/L and (10.4?1.6) ?mol/L, respectively, with the sensitivity of AsPC-1/PUMA cells increased by 7.5 folds. 5-FU induced cell apoptosis of AsPC-1 cells in a dose-dependent manner, with the apoptosis of AsPC-1/PUMA cells more prominent than those of AsPC-1 and AsPC-1/pCEP4 cells. Low concentration of 5-FU (0.1 ?mol/L) induced few apoptosis of AsPC-1/pCEP4 cells([1.14?0.28]%) and AsPC-1 cells ([0.9?0.23]%), and induced apoptosis in AsPC-1/PUMA cells([6.47?1.42]%). High concentration of 5-FU (1.0 ?mol/L) induced apoptosis in all groups, with that in AsPC-1/PUMA cells([34.54?9.36]%) significantly higher than those in AsPC-1/pCEP4 cells([15.8?5.15]%) and AsPC-1 cells ([12.8?3.74]%, both P