山奈素对乳腺癌细胞增殖、凋亡的影响及其机制

Effect of kaempferol on proliferation and apoptosis of breast cancer cells and its mechanism

目的 探讨山奈素对乳腺癌细胞增殖和凋亡的影响及其可能的作用机制。方法 将不同浓度(25、50、75和100μg/mL)的山奈素作用乳腺癌MCF7细胞12、24、48、72 h,MTT法检测细胞增殖抑制率,筛选山奈素的最佳作用浓度为75μg/mL、作用时间为48 h。将乳腺癌MCF7细胞分为山奈素组、索拉菲尼(sorafenib)组、ML-099+山奈素组、空白对照组,分别加入含75μg/mL山奈素的培养基、含10μmol/L有丝分裂原活化蛋白激酶(Ras/Raf/MEK/ERK)信号通路抑制剂sorafenib的培养基、含5μmol/L Ras/Raf/MEK/ERK信号通路激活剂ML-099与75μg/mL山奈素的培养基、等量PRMI1640培养基。培养48 h,采用MTT法检测细胞增殖抑制率,流式细胞术检测细胞凋亡率,实时荧光定量PCR法检测丝氨酸/苏氨酸蛋白激酶1(Raf-1)、细胞外信号调节激酶(ERK)、细胞周期蛋白D1(Cyclin D1)、半胱氨酸蛋白酶3(Caspase-3)mRNA表达,Western blotting法检测p-Raf-1、p-ERK、Cyclin D1、Caspase-3蛋白表达。结果 山奈素组和sorafenib组细胞增殖抑制率、细胞凋亡率、Caspase-3 mRNA相对表达量均高于ML-099+山奈素组和空白对照组,Raf-1、ERK、Cyclin D1 mRNA相对表达量均低于ML-099+山奈素组和空白对照组(P均<0.05)。山奈素组和sorafenib组细胞p-Raf-1、p-ERK、Cyclin D1蛋白相对表达量均低于ML-099+山奈素组和空白对照组,Caspase-3蛋白相对表达量均高于ML-099+山奈素组和空白对照组(P均<0.05)。ML-099+山奈素组细胞p-Raf-1、p-ERK、Cyclin D1蛋白相对表达量均低于空白对照组,Caspase-3蛋白相对表达量高于空白对照组(P均<0.05)。山奈素组、sorafenib组上述指标比较P均>0.05。结论 山奈素可抑制乳腺癌细胞增殖并促进其凋亡,其机制可能与抑制Ras/Raf/MEK/ERK信号通路有关。

Objective To investigate the effects of kaempferol on the proliferation and apoptosis of breast cancer cells and its mechanism.Methods Different concentrations(25,50,75 and 100μg/mL)of kaempferol acted on MCF7 cells for 12,24,48,72 h.The cell proliferation inhibition rate was detected by MTT.The optimal concentration of capsaicin was 75μg/mL and the action time was 48 h.MCF7 cells were divided into the kaempferin group(75μg/mL kaempferin),sorafenib group(10μmol/L sorafenib),ML-099+kaempferin group(5μmol/LML-099 and 75μg/mL kaempferol),and blank control group(normal culture).Cell proliferation was detected by MTT,and apoptosis was detected by flow cytometry,and the levels of Serine/threonine protein kinase 1(Raf-1),extracellular signal-regulated kinase(ERK),Cyclin D1 and Caspase-3 mRNA were detected by qRT-PCR,and the levels of phosphorylated Raf-1(p-Raf-1),phosphorylated ERK(p-ERK),Cyclin D1 and Caspase-3 proteins were detected by Western blotting.Results The cell proliferation inhibitory rate,apoptosis rate,and relative expression of Caspase-3 mRNA in the kaempferin group and sorafenib group were higher than those in the ML-099+kaempferin group and blank control group,but the relative expression levels of Raf-1,ERK,and Cyclin D1 mRNA were lower than those of the ML-099+kaempferol group and blank control group(all P<0.05).The relative expression levels of p-Raf-1,p-ERK,and Cyclin D1 proteins in the kaempferin group and sorafenib group were lower than those in the ML-099+kaempferin group and the blank control group,but the relative expression levels of Caspase-3 protein were higher than those of the ML-099+kaempferol group and blank control group(all P<0.05).The relative expression levels of p-Raf-1,p-ERK and Cyclin D1 protein in the ML-099+kaempferol group were lower than those in the blank control group,but the relative expression level of Caspase-3 protein was higher than that in the blank control group(all P<0.05).There was no significant difference in the above indexes between the kaempferin group and the sorafenib group(all P>0.05).Conclusion Kaempferol may inhibit the proliferation of breast cancer cells and promote its apoptosis by inhibiting the activation of Ras/Raf/MEK/ERK signaling pathway.