PUMA基因转染乳腺癌MCF-7细胞增强表柔比星致凋亡敏感性的研究

Effect of PUMA gene transfection in breast cancer cells (MCF-7) on enhancing the sensitivity of epirubicin-induced apoptosis

    摘要
  • 摘要:
      目的  探讨PUMA基因转染是否增强乳腺癌MCF-7细胞对表柔比星致凋亡的敏感性。
      方法  应用脂质体介导重组真核表达载体PUMA-pCDNA3和空载体pCDNA3质粒瞬时转染至乳腺癌MCF-7细胞中, G418筛选阳性细胞。将系列浓度(0.01~100μmol/L)的表柔比星分别作用于MCF-7、MCF-7/PUMA和MCF-7/pCDNA3细胞72 h, MTT法测定各组细胞的存活率并计算IC50, FCM、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  MCF-7、MCF-7/PUMA和MCF-7/pCDNA3细胞的表柔比星IC50分别为(13±1.4)、(1.8±0.2)和(10.7±1.3)μmol/L, MCF-7/PUMA细胞对表柔比星作用的敏感性增加了7.2倍。表柔比星以剂量依赖方式诱导MCF-7细胞凋亡, 但对MCF-7/PUMA细胞所诱导的凋亡比MCF-7和MCF-7pCDNA3更明显。低浓度表柔比星(0.1μmol/L)轻微引起MCF-7/pCDNA3(1.15±0.26)%和MCF-7细胞凋亡(0.9±0.24)%, 但能诱导MCF-7/PUMA细胞明显凋亡(6.44±1.46)%; 高浓度表柔比星(1μmol/L)诱导各组细胞凋亡, 但表柔比星MCF-7/PU MA细胞凋亡率(35.47±9.36)%明显高于MCF-7(12.6±3.73)%和MCF-7/pCDNA3细胞(15.2±5.17)%, 差异均有统计学意义(P < 0.01);FCM和TUNEL方法检测显示相同的结果。PUMA蛋白在MCF-7/PUMA细胞中的表达明显高于MCF-7和MCF-7/pCD NA3细胞。
      结论  PUMA基因稳定转染明显地增强了乳腺癌MCF-7细胞增强表柔比星致凋亡作用的敏感性。

     

    Abstract:
      Objective  To investigate if PUMA gene transfection could increase the sensitivity of breast cancer cells(MCF-7) on Epirubicin-induced apoptosis.
      Methods  PUMA-pCDNA3, which contains full length PUMA cDNA or-pCDNA3, was transfected into the human breast cancer cell line MCF-7 by using lipofectamine.G418 selection was used to select positive cells.MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells were respectively treated with serial concentrations of Epirubicin(0.01 μmol/L to 100 μmol/L).MTT assay was performed to determine the cell survival rate in each group.IC50 of Epirubicin was then calculated.TUNEL and FCM were employed to investigate cell apoptosis.Western blot was performed to detect PUMA protein expression.
      Results  The Epirubicin IC50 values of MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells were(13±1.4), (1.8±0.2), and(10.7±1.3) μmol/L, respectively.The sensitivity of MCF-7/PUMA cells had a 7.2-fold increase.The Epirubicin-induced apoptosis of MCF-7 cells behaved in a dose-dependent manner.The apoptosis of MCF-7/ PUMA cells was more prominent than that of MCF-7 and MCF-7/ pCDNA3 cells.At a low concentration(0.1 μmol/L), Epirubicin could only slightly induce MCF-7/ pCDNA3 cells (1.15±0.26)% and MCF-7 cells (0.9±0.24)%, as well as the apoptosis in MCF-7/PUMA cells (6.44±1.46)%.At a high concentration, Epirubicin(1.0 μmol/L) could induce apoptosis in all groups.The apoptotic rate was significantly higher in MCF-7/PUMA cells (35.47±9.36)% than in MCF-7/ pCDNA3 cells (15.2±5.17)% and in MCF-7 cells (12.6±3.73)%, both P < 0.01).Resultsobtained from FCM and TUNELwere the same.PUMAprotein expression was significantly higher in MCF-7/PUMAcells than in MCF-7 and MCF-7/pCDNA3 cells.
      Conclusion  PUMA gene transfection greatly enhances the sensitivity of MCF-7 cells during Epirubicin-induced apoptosis.

     

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