PUMA基因转染胰腺癌AsPC-1细胞增强对5-FU致凋亡的敏感性

PUMA gene transfection increases sensitivity of pancreatic cancer cell line AsPC-1 to 5-FU-induced apoptosis

目的：探讨PUMA基因转染是否增强胰腺癌AsPC-1细胞对5-FU致凋亡的敏感性。方法：采用脂质体转染法将PUMA-pCEP4和空载体pCEP4质粒转染入胰腺癌AsPC-1细胞中,G418筛选阳性细胞。将系列浓度（0.01～100μmol/L）的5-FU分别作用于AsPC-1、AsPC-1/PUMA和AsPC-1/pCEP4细胞72h,MTT法测定各组细胞的存活率并计算IC50,FCM、断裂DNA琼脂糖凝胶电泳和TUNEL法检测细胞凋亡情况,Western blotting检测各组细胞PUMA蛋白表达的变化。结果：AsPC-1、AsPC-1/PUMA和AsPC-1/pCEP4细胞的5-FUIC50分别为（12±1.9）、（1.6±0.4）和（10.4±1.6）μmol/L,AsPC-1/PU-MA细胞对5-FU作用的敏感性增加了7.5倍。5-FU以剂量依赖方式诱导AsPC-1细胞凋亡,但对AsPC-1/PUMA细胞所诱导的凋亡比AsPC-1和AsPC-1/pCEP4更明显。低浓度5-FU（0.1μmol/L）轻微引起AsPC-1/pCEP4[（1.14±0.28）%]和AsPC-1细胞凋亡[（0.9±0.23）%],但能诱导AsPC-1/PUMA细胞明显凋亡[（6.47±1.42）%];高浓度5-FU（1μmol/L）诱导各组细胞凋亡,但AsPC-1/PUMA细胞凋亡率[（34.54±9.36）%]明显高于AsPC-1[（12.8±3.74）%]和AsPC-1/pCEP4细胞[（15.8±5.15）%],差异均有统计学意义（P〈0.01）;FCM、断裂DNA琼脂糖凝胶电泳和TUNEL方法检测显示相同的结果。PUMA蛋白在AsPC-1/PUMA细胞中的表达明显高于AsPC-1和AsPC-1/pCEP4细胞。结论：PUMA基因转染明显地增强了胰腺癌AsPC-1细胞对5-FU致凋亡作用的敏感性。

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 μmol/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）μmol/L,（1.6±0.4）μmol/L and （10.4±1.6） μmol/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 μmol/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 μmol/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〈0.01） . FCM and electrophoresis showed the same results. Expression of PUMA protein in AsPC-1/PUMA cells was significantly higher than those in AsPC-1 and AsPC-1pCEP4 cells. Conclusion： PUMA gene transfection greatly enhances the sensitivity of AsPC-1 cells to 5-FU-induced apoptosis.