摘要
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、20μmol/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20μmol/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0μmol/L组相比,5、10和20μmol/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20μmol/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0μmol/L组相比,5、10和20μmol/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0μmol/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10μmol/L组相比,20μmol/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0μmol/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10μmol/L组相比,20μmol/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。
Objective To explore the molecular mechanism of genistein-induced apoptosis in breast cancer MDA-MB-231 cells. Methods MDA-MB-231 ceils 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 aconcentration-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 eoneentration group, respectively, after genistein treatment for 24 h. Compared with 0 μmol/L group, the early apoptotie 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 ~xmol/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 deereased in 20 μmol/L group （ F = 52. 562, P = 0. 020 ）. Conclusion Genistein induees apoptosis of breast cancer MDA-MB-231 cells by up-regulating FasL gene expression in Fas/FasL pathway.
出处
《中华乳腺病杂志（电子版）》
CAS
2013年第5期7-11,共5页
Chinese Journal of Breast Disease(Electronic Edition)
基金
国家自然科学基金(81272430)
