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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2013, Vol. 07 ›› Issue (05) : 322 -328. doi: 10.3877/cma.j.issn.1674-0807.2013.05.003

论著

金雀异黄素诱导乳腺癌MDA-MB-231 细胞凋亡的研究
王欢1, 卢陈嘉2, 马威2, 毛俊2, 范盼红2, 赵文月2, 王露1, 李连宏2,†()   
  1. 1.116044 大连医科大学临床医学系
    2.116044 大连医科大学病理教研室和辽宁省肿瘤干细胞研究重点实验室
  • 收稿日期:2013-07-09 出版日期:2013-10-01
  • 通信作者: 李连宏
  • 基金资助:
    国家自然科学基金(81272430)

Genistein-induced apoptosis in breast cancer MDA-MB-231 cells

Huan WANG1, Chen-jia LU1, Wei MA1, Jun MAO1, Pan-hong FAN1, Wen-yue ZHAO1, Lu WANG1, Lian-hong LI1,†()   

  1. 1.Department of Clinical Medicine,Dalian Medical University, Dalian 116044, China
  • Received:2013-07-09 Published:2013-10-01
  • Corresponding author: Lian-hong LI
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王欢, 卢陈嘉, 马威, 毛俊, 范盼红, 赵文月, 王露, 李连宏. 金雀异黄素诱导乳腺癌MDA-MB-231 细胞凋亡的研究[J/OL]. 中华乳腺病杂志(电子版), 2013, 07(05): 322-328.

Huan WANG, Chen-jia LU, Wei MA, Jun MAO, Pan-hong FAN, Wen-yue ZHAO, Lu WANG, Lian-hong LI. Genistein-induced apoptosis in breast cancer MDA-MB-231 cells[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2013, 07(05): 322-328.

目的

探讨金雀异黄素(genistein,GEN)诱导乳腺癌MDA-MB-231 细胞凋亡的分子机制。

方法

用0、5、10、20 μmol/L GEN 处理MDA-MB-231 细胞24 h。采用CCK-8、Hoechst 33342 染色和流式细胞仪测定不同浓度GEN 对MDA-MB-231 细胞增殖和凋亡的影响。采用Western blotting 检测不同浓度GEN 处理前后MDA-MB-231 细胞中Fas 相关死亡域蛋白(FADD)、活性半胱天冬酶8(cleaved caspase-8)、Fas、 FasL 蛋白表达水平。采用实时RT-PCR 分析不同浓度GEN 处理前后MDA-MB-231 细胞中Fas、FasL 基因表达水平。多组均数比较采用方差齐性检验后进行单因素方差分析。

结果

在GEN 作用24 h后,0、5、10 和20 μmol/L 组对MDA-MB-231 细胞增殖的抑制率分别为(3.00±1.41)%、(14.02±1.57)%、(27.5±1.52)%、 (48.90±1.44)%。与0 μmol/L 组相比,5、10 和20 μmol/L 组呈浓度依赖性增加(F=528.119, P=0.000)。两两比较显示:各浓度组之间差异均有统计学意义(P＜0.05)。0、5、10和20 μmol/L 组诱导MDA-MB-231 细胞的早期凋亡率分别为(3.40±0.40)%、(9.34±1.34)%、(19.26±0.93)%、(27.41±1.12)%。与0 μmol/L 组相比,5、10 和20 μmol/L 组呈浓度依赖性增加(F=379.573,P=0.000)。两两比较显示:各浓度组之间差异均有统计学意义(P＜0.05)。Western blotting 结果显示,与0 μmol/L 组相比,其他浓度组经GEN 处理的MDA-MB-231 细胞FADD、cleaved caspase-8、FasL 蛋白表达升高(F=368.621、456.744、419.129,P 均=0.000),Fas 蛋白表达差异无统计学意义(F=0.800,P=0.528);与10 μmol/L 组相比,20 μmol/L 组FasL 蛋白表达降低有统计学意义(F=92.235,P=0.001)。实时RT-PCR 结果显示,与0 μmol/L 组相比,其他浓度组经GEN 处理的MDA-MB-231 细胞FasL mRNA表达升高(F=646.983,P=0.000),Fas mRNA 表达差异无统计学意义(F= 1.556,P = 0.274);与10 μmol/L 组相比,20 μmol/L 组FasL mRNA 表达降低有统计学意义(F=52.562,P=0.020)。

结论

GEN通过上调Fas/FasL 途径中FasL 基因表达诱导乳腺癌MDA-MB-231 细胞凋亡。

关键词: 乳腺肿瘤, 细胞凋亡, 金雀异黄素, MDA-MB-231 细胞, Fas/FasL 途径

Objective

To explore the molecular mechanism of genistein-induced apoptosis in breast cancer MDA-MB-231 cells.

Methods

MDA-MB-231 cells were treated with 0, 5, 10, 20 μmol/L genistein for 24 h, respectively.CCK-8,Hoechst 33342 staining and flow cytometry were used to determine the effects of genistein on proliferation and apoptosis of MDA-MB-231 cells.The expressions of FADD, cleaved caspase-8,Fas, FasL in the protein level were measured by Western blotting.The expressions of Fas and FasL in the mRNA level were detected by real-time RT-PCR.The one-way ANOVA was conducted after homogeneity for variance was tested.

Results

The cell proliferation inhibition rate of MDA-MB-231 cells were (3.00±1.41)%,(14.02±1.57)%,(27.5±1.52)%,(48.90±1.44)% after the treatment of 0, 5, 10, 20 μmol/L genistein for 24 h respectively.Compared with 0 μmol/L group, the inhibition rate was increased in a concentration-dependent manner in the other 3 groups(F=528.119, P=0.000).The pairwise comparison showed the difference was statistically significant(all P＜0.05).The early apoptotic rate of MDA-MB-231 cells were (3.40±0.40)%,(9.34±1.34)%,(19.26±0.93)%,(27.41±1.12)% in each concentration group,respectively, after genistein treatment for 24 h.Compared with 0 μmol/L group, the early apoptotic rate was increased in a concentration-dependent manner in the other 3 groups(F=379.573,P=0.000).The pairwise comparison showed the difference was statistically significant(all P ＜0.05).Western blotting showed that genistein increased the protein expression levels of FADD, cleaved caspase-8 and FasL(F=368.621,456.744,419.129;all P=0.000), with no alteration in Fas protein level(F=0.800,P=0.528).Compared with 10 μmol/L group, FasL protein expression decreased in 20 μmol/L group(F=92.235,P=0.001).Real-time RT-PCR showed that genistein increased the mRNA expression of FasL(F= 646.983,P = 0.000), with alteration in Fas mRNA level(F = 1.556,P = 0.274).Compared with 10 μmol/L group, FasL mRNA expression also decreased in 20 μmol/L group(F=52.562,P=0.020).

Conclusion

Genistein induces apoptosis of breast cancer MDA-MB-231 cells by up-regulating FasL gene expression in Fas/FasL pathway.

Key words: Breast neoplasms, Apoptosis, Genistein, MDA-MB-231 cells, Fas/FasL pathway
图1 不同浓度的金雀异黄素作用24 h 后MDA-MB-231细胞的增殖抑制率 a:P＜0.05,与0 μmol/L 组相比;两两比较显示:各浓度组之间差异均有统计学意义(P＜0.05)
图2 AV-PI 流式细胞仪分析不同浓度金雀异黄素作用于MDA-MB-231 细胞的早期凋亡率
图3 不同浓度的金雀异黄素作用24 h 后MDA-MB-231细胞的早期凋亡率 a:P＜0.05, 与0 μmol/L 组相比;两两比较显示:各浓度组之间差异均有统计学意义(P＜0.05)
图4 不同浓度的金雀异黄素作用24 h 后的MDA-MB-231 细胞在荧光显微镜下的图片(Hoechst 33342 ×200) a:0 μmol/L;b:5 μmol/L;c:10 μmol/L;d:20 μmol/L;红箭头:早期凋亡细胞;凋亡细胞发出强烈的蓝色荧光,随着金雀异黄素处理浓度的增加,发出强烈蓝色荧光的凋亡细胞数量也逐渐增加
图5 Western blotting 法检测MDA-MB-231 细胞中各蛋白表达
图6 不同浓度的金雀异黄素作用24 h 后MDA-MB-231细胞中各蛋白的表达水平 a:P＜0.05,与0 μmol/L 组相比;b:P＜0.05, 与10 μmol/L 组相比
图7 不同浓度的金雀异黄素作用24 h 后MDA-MB-231细胞中各蛋白的mRNA 表达水平 a:P＜0.05,与0 μmol/L 组相比;b:P＜0.05, 与10 μmol/L 组相比
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