活性氧抑制剂NAC对人乳腺癌细胞化疗的增敏作用观察及机制初探

Sensitization and mechanism of reactive oxygen inhibitor NAC for human breast cancer cells chemotherapy

目的:观察活性氧抑制剂N-乙酰半胱氨酸(NAC)对人乳腺癌细胞化疗的增敏作用,并初步探讨其调控机制。方法:人乳腺癌细胞株MCF-7培养至对数期后,分为5组,每孔5个复孔,多柔比星(Dox)组、低、中、高剂量组和对照组分别用0. 4μg/ml Dox、0. 4μg/ml Dox+(10、30、60 mmol/L) NAC、磷酸盐缓冲液(PBS)进行处理。对比48 h后细胞凋亡率及细胞内活性氧(ROS)水平,并对比48 h后含半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA及蛋白相对表达量。结果:细胞凋亡率组间、时间及交互比较差异均有显著统计学意义(P<0. 05);细胞凋亡率、Caspase-3、Bax mRNA及蛋白相对表达量组间比较,对照组最低,Dox组其次,低、高剂量组稍高,中剂量组最高;ROS荧光强度组间比较,对照组最低,中剂量组其次,低、高剂量组稍高,Dox组最高;Bcl-2 mRNA及蛋白相对表达量组间比较,对照组最高,Dox组其次,低、高剂量组稍低,中剂量组最低;细胞凋亡率、Bax、Caspase-3、Bcl-2 mRNA及蛋白相对表达量降低、高剂量组比较差异无统计学意义(P>0. 05),其他每2组间比较差异有统计学意义(P<0. 05);组内比较,除对照组细胞凋亡率随时间延长差异无统计学意义(P>0. 05),其他各组细胞凋亡率均随时间延长显著增加(P<0. 05)。结论:在一定范围,NAC可增强DOX的化疗敏感性,促进人乳腺癌细胞凋亡、调节细胞内ROS含量,其中30 mmol/L的NAC效果最佳,推测是通过降低Bcl-2mRNA及蛋白表达,增强Caspase-3、Bax mRNA及蛋白表达,且与Bcl-2/Bax信号通路有关。

Objective:To observe the sensitization effect of N-acetylcysteine(NAC)on human breast cancer cells chemotherapy,and to explore its regulatory mechanism.Methods:When the human breast cancer cell line MCF-7was cultured to the logarithmic stage,and it was divided into5groups,with5multiple pores in each hole.Doxorubicin(Dox)group,low,medium and high dose group and control group was treated with0.4g/ml Dox,0.4g/ml Dox with(10,30,60mmol/L)NAC and phosphate buffered saline(PBS)respectively.The apoptosis rates and the reactive oxygen species(ROS)levels of the cells cultured to48h were compared.The mRNA and protein levels of cysteinyl aspartate specific proteinase-3(Caspase-3),B-cell lymphoma-2(Bcl-2),Bcl-2associated X protein(Bax)of the cells cultured to48h were compared.Results:There were significant differences in the apoptosis rates among the groups,moments and interact(P<0.05).The apoptosis rates,the mRNA and protein levels of Caspase-3and Bax were compared between groups,and the results showed that the control group was the lowest,the Dox group the second,the low and high dose groups slightly higher,and the medium dose group was the highest.The fluorescence intensity of ROS were compared between groups,and the results showed that the control group was the lowest,the medium dose group the second,the low and high dose groups slightly higher,and the Dox group was the highest.The mRNA and protein levels of Bcl-2were compared between groups,and the results showed that the control group was the highest,the Dox group the second,the low and high dose groups slightly lower,and the medium dose group was the lowest.There were significant differences between each two groups(P<0.05),without differences between the low and high dose groups(P>0.05).There were no significant differences in the apoptosis rates of the control group with time(P>0.05),while the apoptosis rates in other groups showed a significant increase with time(P<0.05).Conclusion:In a certain range,NAC can enhance the chemosensitivity of Dox,promote the apoptosis of breast cancer cells and regulate the content of ROS in cells,of which30mmol/L NAC best,and it is presumed to decrease the mRNA and protein expression of Bcl-2and increase the protein and mRNA expression of Caspase-3and Bax,which is related to the Bcl-2/Bax signaling pathway.