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

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。
- PUMA基因 /
- 乳腺癌 /
- MCF-7细胞 /
- 凋亡
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.
- epirubicin up-regulated modulator of apoptosis(PUMA) /
- breast cancer /
- epirubicin /
- apoptosis

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图 1 PUMA基因转染增强MCF-7细胞对表柔比星细胞毒作用的敏感性

Figure 1. PUMA gene transfection in enhancing the sensitivity of MCF-7cells on pirarubicin cytotoxicity

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图 2 流式细胞仪检测表柔比星诱导的MCF-7/PUMA细胞的凋亡

Figure 2. Flow cytometry of pirarubicin-induced apoptosis in MCF-7/PUMA cells

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图 3 表柔比星诱导的MCF-7/PUMA细胞PUMA蛋白的表达

1:MCF-7；2：MCF-7/PUMA+表柔比星（EPI）；3：MCF-7/pCDNA3+表柔比星（EPI）；4：MCF-7+表柔比星（EPI）

Figure 3. Pirarubicin-induced PUMA protein expression in MCF-7/PUMA cells

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参考文献(10)

[1]	李媛媛, 张斌, 赵洪猛, 等. PXD101对人乳腺癌细胞MCF-7增殖及凋亡影响的机制探讨[J]. 中国肿瘤临床, 2012, 39(5): 249-253. doi: 10.3969/j.issn.1000-8179.2012.05.003
[2]	马斌林, 耿中利, 阿力比亚提. 艾尼, 等. pcDNA3.0-PTEN质粒转染15dPGJ2对乳腺癌MCF-7细胞体外培养的影响[J]. 新疆医科大学学报, 2009, 32(7): 833-838. doi: 10.3969/j.issn.1009-5551.2009.07.001
[3]	Wang R, Wang X, Li B, et al. Tumor-specific adenovirus-mediated PUMA gene transfer using the survivin promoter enhances radiosensitivity of breast cancer cells in vitro and in vivo[J]. Breast Cancer Res Treat, 2009, 117(1): 45-54. doi: 10.1007/s10549-008-0163-6
[4]	Sun QH, Sakaida T, Yue W, et al. Chemosensitization of head andneck cancer cells by PUMA[J]. Mol Cancer Ther, 2007, 6(12): 3180-3188. doi: 10.1158/1535-7163.MCT-07-0265
[5]	Chen Y, Qian H, Wang H, et al. Ad-PUMA sensitizesdrug-resistant choriocarcinoma cells to chemotherapeutic agents[J]. Gynecol Oncol, 2007, 107(3): 505-512. doi: 10.1016/j.ygyno.2007.08.007
[6]	孙燕, 周继昌, 主编. 临床肿瘤内科手册[M]. 第3版. 北京: 人民卫生出版社, 1996: 348-353.
[7]	李昆, 李世正, 张蕴莉, 等. 槲皮素对人乳腺癌细胞MDA-MB-435S增殖及侵袭力的影响[J]. 肿瘤防治研究, 2009, 36(7): 549-551. https://www.cnki.com.cn/Article/CJFDTOTAL-ZLFY200907004.htm
[8]	宋俊颖, 张丽娜, 郑红, 等. 凋亡抑制基因AVEN mRNA在乳腺癌中的表达差异[J]. 中国肿瘤临床, 2012, 39(4): 197-200. doi: 10.3969/j.issn.1000-8179.2012.04.005
[9]	刘锋, 孙根, 何川, 等. siRNA沉默c-FLIP基因表达与表柔比星联合应用对MCF-7细胞凋亡的影响[J]. 实用肿瘤学杂志, 2010, 24(4): 337-340.
[10]	周云海, 周士福. 乳腺癌COX-2表达与紫杉醇脂质体联合表柔比星新辅助化疗疗效关系的研究[J]. 临床肿瘤学杂志, 2010, 15(5): 435-437. https://www.cnki.com.cn/Article/CJFDTOTAL-LCZL201005014.htm