多柔比星对乳腺癌MCF-7细胞表达BRCA1和PARP-1蛋白的影响

Effects of doxorubicin on the expressions of BRCA1 and PARP-1 proteins in breast cancer MCF-7 cells

目的:探讨多柔比星(doxorubicin)对乳腺癌MCF-7细胞表达DNA损伤修复相关蛋白乳腺癌易感蛋白1(breast cancer-associated protein 1,BRCA1)和多聚腺苷二磷酸核糖聚合酶-1[poly (ADP-ribose) polymerase-1,PARP-1]的影响。方法:蛋白质印迹法检测不同浓度的多柔比星干预并恢复不同时间后,MCF-7细胞表达BRCA1和PARP-1的水平及其PARP-1活性的动态变化。FCM法检测多柔比星和PARP-1抑制剂3-氨基苯酰胺(3-aminobenzamide,3-ABA)单独或联合干预后MCF-7细胞及SKBR3.0细胞(BRCA1突变型乳腺癌细胞)的凋亡情况。结果:随着多柔比星浓度的提高,PARP-1的活性产物聚腺苷二磷酸核糖[poly(ADP-ribose),PAR]逐渐增加(P<0.01),然而其全长表达(相对分子质量为1.13×105的片段)略有降低,断裂(相对分子质量为8.9×104的片段)有所增加;而BRCA1表达却呈现先增加后减少的趋势(P<0.01)。PARP-1活性随着恢复时间的延长逐渐升高(P<0.01),但全长PARP-1表达变化不大,断裂略有减弱;BRCA1表达却逐渐减少(P<0.01)。3-ABA能抑制PARP-1活性(P<0.01),诱导PARP-1断裂,但不影响BRCA1表达。多柔比星及3-ABA都能诱导MCF-7细胞凋亡,与对照组相比较,其差异均有统计学意义(P<0.05);多柔比星和3-ABA两者联合作用时能进一步增加BRCA1野生型乳腺癌细胞MCF-7的凋亡,与多柔比星及3-ABA单独使用相比较,差异具有统计学意义(P<0.05);多柔比星和3-ABA两者联合作用时能诱导BRCA1突变型乳腺癌细胞SKBR3.0发生凋亡,与两者联合作用MCF-7细胞的影响相比较,差异具有统计学意义(P<0.05)。结论:多柔比星干预能影响DNA损伤修复蛋白PARP-1的活性及BRCA1的表达。多柔比星和PARP-1抑制剂3-ABA都能诱导MCF-7细胞凋亡,两者联合应用能增加这种凋亡诱导效应。

Objective： To investigate the effects of doxorubicin on the expressions of DNA-damage/ repair-related proteins BRCA1 （breast cancer-associated protein 1）and PARP-1 [poly（ADP-ribose） polymerase-1] in BRCA1 wild-type breast cancer MCF-7 cells. Methods： The MCF-7 cells were treated with doxorubicin, then the expressions of BRCA1 and PARP-1 and the activity of PARP-1 in the cells recovering after different time peroids were detected by Western blotting. The apoptotic rates of MCF-7 cells and SKBR3.0 cells （BRCA1 mutant-type breast cancer cells） after intervention with doxorubicin and PARP-1 inhibitor 3-ABA （3-aminobenzamide） were detected by FCM （flow cytometry）. Results： After MCF-7 cells were treated with different concentrations of doxorubicin for 24 h and then recovered for 12 h, the expression of PAR [poly（ADP-ribose）], an active product of PARP-1, was increased in a dose-dependent manner （P 〈 0.01）, but the expression of full-length PARP-1 （the molecular mass is 1.13-10s） was in aslightly decrease accompanied by more cleavage fragments of PARP-1 （the molecular mass is 8.9）〈104）, while the expression of BRCA1 was firstly increased and then decreased （P 〈 0.01）. After MCF-7 cells were treated with 1 IJmol/L doxorubicin for 24 h and then recovered for different time peroids, the activity of PARP-1 （PAR） was increased gradually over time peroid of recovery （P 〈 0.01）, but the expression of full- length PARP-1 had no significant change with less cleavage fragments of PARP-1, while the expression of BRCA1 was decreased gradually （P 〈 0.01）. The activity of PARP-1 was significantly inhibited by 3-ABA （P 〈 0.01） via inducing its cleavage, which didn＇t affect BRCA1 expression （P 〉 0.05）. Both doxorubicin and 3-ABA alone could significantly induce the apoptosis of MCF-7 cells （P 〈 0.05）, and the combination of the two could further increase the apoptosis of BRCA1 wild-type breast MCF-7 cells （P 〈 0.05）. Doxorubicin in combination with 3-ABA could also induce the apoptosis of BRCA1 mutant-type SKBR3.0 cells, and this effect was stronger than that in MCF-7 cells （P 〈 0.05）. Conclusion： Doxorubicin intervention can affect the activity of DNA-damage/repair-related protein PARP-1 and the expression of BRCA1. Both doxorubicin and PARP-1 inhibitor 3-ABA can induce the apoptosis of MCF-7 cells, and the combination of the two can increase this apoptosis-inducing effect.