青蒿素对乳腺癌细胞增殖、凋亡的影响及其作用机制

Effect of artemisinin on proliferation and apoptosis of breast cancer cells and relevant mechanism

目的探究青蒿素(artemisinin,ART)对乳腺癌细胞(MDA-MB-231和MCF-7)增殖、凋亡的影响及其作用机制。方法实验分为对照组(加入1μL/mL DMSO)和ART处理组(加入终浓度为300μmol/L的ART)。ART作用48 h后,采用平板克隆试验检测乳腺癌细胞与乳腺上皮细胞(MCF-10A)的克隆形成能力,免疫荧光染色法检测细胞增殖标记Ki-67蛋白的表达,流式细胞术分析3种细胞的凋亡变化,RT-qPCR检测乳腺癌细胞中DNMTs家族与抑癌基因FHIT、CDH1、PTEN等的表达变化,Western blot分析BAX、BCL-2、DNMT1和DNMT3A蛋白的表达水平,亚硫酸氢盐测序法分析FHIT和CDH1基因的甲基化水平。结果 ART显著抑制乳腺癌细胞的增殖,并诱导乳腺癌细胞的凋亡(P <0. 01),同时对正常人上皮细胞有明显的诱导凋亡作用(P <0. 01);ART抑制DNMT1的表达,促进DNMT3A的表达(P <0. 05);ART处理乳腺癌细胞后,抑癌基因FHIT和CDH1的甲基化水平降低,表达水平增加。结论 ART通过改变DNMT1和DNMT3A的表达,降低某些抑癌基因如FHIT、CDH1的甲基化水平,促进这些抑癌基因的表达,从而发挥抑制乳腺癌细胞增殖与诱导凋亡的作用。此外,ART用于乳腺癌治疗的安全性需进一步评估。

Objective To explore the effect of artemisinin(ART)on the proliferation and apoptosis of human breast cancer MDA-MB-231 and MCF-7 cells as well as the relevant mechanism.Methods The cells were divided into control and test groups,and treated with 1μL/mL DMSO and ART at a final concentration of 300μmol/L respectively.The colony formation abilities of human breast cancer cell lines and human breast epithelial cell line MCF-10 A after treatment with ART for 48 h were determined by plate cloning test.The expression of Ki-67 protein as a marker of cell proliferation was determined by immunofluorescent staining,while the apoptosis of human breast cancer and human breast epithelial cell lines by flow cytometry.The expressions of DNMTs and cancer suppressor genes FHIT,CDH1 and PTEN in human breast cancer cell lines were determined by RT-qPCR,while those of BAX,BCL-2,DNMT1 and DNMT3 A proteins by Western blot.The DNA methylation levels of FHIT and CDH1 genes were determined by bisulfite genomic sequencing method.Results ART significantly inhibited the proliferation and induced the apoptosis of breast cancer cells(P<0.01).ART also significantly induced the apoptosis of normal human epithelial cells(P<0.01).However,ART decreased DNMT1 expression and increased DNMT3 A expression(P<0.05).The DNA methylation levels of FHIT and CDH1 genes decreased in breast cancer cells after treatment with ART.Conclusion ART inhibits the proliferation and induces the apoptosis of breast cancer cells by changing the expressions of DNMT1 and DNMT3 A,resulting in reduced methylation and increased expression level of some anti-cancer genes such as FHIT and CDH1.The safety of ART in the treatment of breast cancer should be further evaluated.