黄芩素联合 U0126 诱导人乳腺癌细胞株MCF-7 凋亡的分子机制研究

M echanism sofapoptosisofhum an breastcancercelllineM CF-7 induced by baicalein and U0126

摘要:

探讨黄芩素和 U0126 诱导人乳腺癌细胞株 MCF-7 凋亡的分子机制。方法 单独及联合使用 20μmol 黄芩素、10 μmol U0126 处理 MCF-7 细胞 24 h；光学显微镜观察细胞数量变化，流式细胞术检测 MCF-7 细胞周期变化，CCK8 法检测细胞增殖变化，TUNEL 法和流式细胞术检测细胞凋亡，实时聚合酶链反应（Real Time PCR）和 Western blot 检测凋亡相关因子 mRNA 和蛋白的表达水平。结果 与黄芩素单独刺激 MCF-7 细胞相比，黄芩素联合 U0126 刺激 MCF-7 细胞时，S 期细胞比例降低更明显；MCF-7 细胞经不同浓度黄芩素或 U0126 处理后，增殖抑制，且具有浓度依赖性（ <0.05）；与黄芩素单独处理 MCF-7 细胞相比，黄芩素与 U0126 联合处理时，细胞凋亡更加显著（ <0.05），早期凋亡和晚期凋亡均增多（ <0.05）；与黄芩素或 U0126 单独处理 MCF-7 细胞相比，黄芩素和 U0126 联合处理 MCF-7 细胞时，凋亡抑制因子 Bcl-2 的mRNA 水平降低（ <0.05），凋亡促进因子 Bax 的 mRNA 水平增高（ <0.05），ERK1/2、GSK-3β 和 P38 的磷酸化水平降低（ <0.05），凋亡促进因子 Bax 表达量增高（ <0.05），凋亡抑制因子 Bcl-2 表达量降低（ <0.05）。结论 黄芩素或 U0126 通过调控 Bcl-2/Bax 的表达水平以及 ERK1/2、GSK-3β 和 P38 的磷酸化水平抑制 MCF-7 细胞增殖，促进细胞凋亡，且具有协同效应。因此，黄芩素和 U0126 可联合用于临床治疗乳腺癌，具有重要临床意义。

Abstract:

To investigate the molecular mechanisms ofapoptosis ofhuman breastcancer celllineMCF-7 induced bybaicalein and U0126.M ethodsThehuman breastcancercelllineMCF-7 cellsweretreated by20 μM baicalein,10μM U0126,and 20 μM baicalein combined with 10 μM U0126 respectively.Flow cytometrywasused totestthechangesofMCF-7 cellcycle.CCK8 assaywasapplied tomeasureproliferation ofMCF-7 cells.The numberofcells was observed underlightmicroscope.Flow cytometry and TUNEL were used to evaluate the apoptosis ofMCF-7 cells.RT-PCR and W estern blotwere adopted to detectthe mRNA and protein levels of apoptosis-related proteins.ResultsCompared totheMCF-7 cellsstimulated with 20 μM baicalein alone,theratio of S phase MCF-7 cells decreased aftertreatmentby baicalein combined with U0126 ( < 0.05).W hen the MCF-7 cellsweretreated bydifferentconcentrationsofbaicalein orU0126,the proliferation wasinhibited dramatically in a dose-dependentway ( < 0.05).Compared to the MCF-7 cells treated with baicalein alone,the early and late apoptoticcellswere augmented dramatically when they were treated by both baicalein and U0126 ( < 0.05).W hen theMCF-7 cellsweretreated bybaicalein combined with U0126,thelevelofBcl-2 decreased ( < 0.05),thelevel ofBaxgreatlyincreased ( < 0.05),and the phosphorylation levelsofERK1/2,GSK-3β and P38 obviously reduced ( < 0.05)compared with the cellswith single treatmentofbaicalein orU0126.ConclusionsBaicalein and U0126 can dramatically inhibitthe proliferation and increase the apoptosisofMCF-7 cellsthrough decreasing the levelof Bcl-2,reducing the phosphorylation levelsofERK1/2,GSK-3β and P38,and increasing the levelofBax.Hence, baicalein and U0126 can beused totreatbreastcancer,both areveryimportantin clinic.