苦参碱逆转人乳腺癌MCF-7/ADR细胞株耐药性及对PI3K/AKT信号通路下游因子的影响

The matrine reverse multidrug resistance on breast cancer MCF-7/ADR cell line and have an influence on downstream factors of PI3K/AKT signal pathway

目的:探索苦参碱对人乳腺癌耐药株MCF-7/ADR多药耐药性逆转的作用机制及对PI3K/AKT信号通路下游因子的影响。方法:采用MTT法检测不同浓度苦参碱和MK2206作用MCF-7/ADR细胞24h后的生长抑制率,以抑制率为10%左右为检测标准;用荧光定量RT-PCR、Western blot检测不同浓度苦参碱作用MCF-7/ADR细胞24h后Bcl-2、Bax、Caspase-3、PARP、GSK-3β、p65六种基因mRNA和蛋白的相对表达量。结果:荧光定量RT-PCR结果显示0.6、1.2g/L的苦参碱处理组与空白对照组相比,随着苦参碱浓度的提高,PI3K/AKT信号通路下游作用因子Bcl-2基因和p65基因的相对表达量逐渐降低,Caspase-3基因的相对表达量变化不显著,而Bax基因、GSK-3β基因及PARP基因的相对表达量逐渐增高;相同抑制率的苦参碱组(0.6g/L)和MK2206组(0.05μmol/L)相比,两组降低基因表达量的差异不明显,苦参碱组更能增加Bax、PARP和GSK-3β基因的相对表达量并且能够明显减低p65基因的相对表达量。Western blot结果显示0.3、0.6、1.2g/L三组不同浓度苦参碱干预组与空白对照组相比,随着苦参碱浓度的提高Bcl-2和p65蛋白表达量逐渐降低,Bax、PARP和GSK-3β蛋白表达量逐渐增高,Caspase-3蛋白表达量变化不明显;相同抑制率的苦参碱组(0.6g/L)和MK2206组(0.05μmol/L)相比,苦参碱组更明显。结论:苦参碱可能是通过作用AKT靶基因启动PI3K/AKT信号转导通路中下游相关凋亡因子而发挥逆转乳腺癌多药耐药的作用。

Objective： To explore the mechanism of matrine in reversaling multidrug resistance on breast cancer cells and have an influence on regulating downstream factors of PI3K / AKT signal pathway. Methods： MTT assay was used to measure the inhibition rate of Matrine on MCF-7 / ADR cells after 24 hours,Real-time RT-PCR and Western blotting were used to evaluate the mRNA expression and the protein level of Bcl-2,Bax,Caspase-3,PARP,GSK-3β and p65 in MCF-7 / ADR cells. Results： Real-time RT-PCR showed that 0. 3,0. 6 and 1. 2g / L groups of matrine were compared with control group,the multidrug resistance genes expression amount of Bcl-2,p65 reduced gradually,but the gene expression differences of Bax,GSK-3β and PARP increased gradually and gene expression differences of Caspase-3 was not obvious. The gene of Bcl-2,p65 which were intervened by 0. 6g / L matrine group and the difference of 0. 05μmol / L MK2206 group had no significant difference at the same inhibition radio. Western blotting showed when 0. 3,0. 6 and 1. 2g / L groups of matrine were compared with control group,the protein expression of Bcl-2 and p65 reduced gradually. But that of Bax,GSK-3β and PARP increased gradually and that of Caspase-3 didn＇t changed obviously. Matrine group was more likely to reduce the expression of Bcl-2 and p65 and increase the expression of Bax,GSK-3β and PARP at the same inhibition radio of matrine（ 0. 6g / L） and MK2206（ 0. 05μmol / L）. Conclusion： The effects of matrine reversaling multidrug resistance is probably related to regulating the downstream apoptosis factors of PI3 K / AKT signal pathway through AKT target gene.