【摘要】 目的探讨藤梨根正丁醇提取物体外对乳腺癌细胞MDA-MB-231增殖、凋亡和自噬的影响及可能分子机制。方法 MTT法、Annexin V/PI双染法、Hoechst33342染色法检测藤梨根正丁醇提取物对MDA-MB-231细胞增殖和凋亡活性的影响。转染pEGFP-LC3自噬指示质粒观察细胞内自噬流。Western blotting法检测藤梨根正丁醇提取物对MDA-MB-231细胞自噬相关蛋白微管相关蛋白1轻链3-β(LC3-Ⅱ)、UNC-51样激酶1(ULK1)、Beclin 1蛋白以及磷脂酰肌醇激酶(PI3K)、蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)蛋白磷酸化水平。设置空白对照组、自噬抑制剂3-甲基腺嘌呤(3-MA)组和mTOR抑制剂雷帕霉素组,MTT法检测藤梨根正丁醇提取物对3组细胞增殖抑制作用。结果藤梨根正丁醇提取物可抑制MDA-MB-231细胞增殖,并呈浓度和时间依赖性。经Annexin V/PI双染法检测,藤梨根正丁醇提取物作用于MDA-MB-231细胞24 h后,细胞凋亡率为(37.88±5.16)%,高于空白对照组(P<0.05)。与空白对照组比较,藤梨根正丁醇提取物作用组p-Beclin1/Beclin1、p-ULK1/ULK1、LC3-Ⅱ/LC3-Ⅰ蛋白表达量升高,p-PI3K/PI3K、p-Akt/Akt、mTOR/p-mTOR蛋白表达量却明显降低,差异有统计学意义(P<0.05)。与空白对照组比较,500μg/ml藤梨根正丁醇提取物对3-MA组增殖抑制率降低,对雷帕霉素组细胞增殖抑制率升高(P<0.05)。结论藤梨根正丁醇提取物可通过抑制PI3K/Akt-mTOR信号通路磷酸化水平,上调MDA-MB-231细胞的自噬活性和凋亡活性,并抑制细胞增殖。更多还原

【Abstract】 Objective To discuss the effect of N-butanol extract of Radix Actinidiae on autophagy and apoptosis of breast cancer cells and its impossible mechanism. Methods MTT assay,Annexin V/PI staining and Hoechst33342 staining were used to detect the proliferation and apoptosis of breast cancer cell MDA-MB-231 treated with N-butanol extract of Radix Actinidiae. Changes of autophagy flux in MDA-MB-231 cells were assessed by the formation of pEGFP-LC3 puncta. The phosphorylations of LC3-Ⅱ,ULK1,Beclin1,PI3K,Akt,mTOR were detected by Western blotting. MTT assay was also detected the inhibition rate of N-butanol extract of Radix Actinidiae on the MDA-MB-231 cells pretreated with autophagy inhibitor 3-MA or mTOR inhibitor rapamycin. Results Nbutanol extract of Radix Actinidiae had obviously inhibition effect on the proliferation of MDA-MB-231 cells,which was doseindependent and time-independent. After treatment with N-butanol extract of Radix Actinidiae for 24 h,the apoptosis rate of MDA-MB-231 cells was(37. 88±5. 16) %,higher than that without N-butanol extract of Radix Actinidiae treatment(P<0. 05),while the levels of p-Beclin1/Beclin1,p-ULK1/ULK1,LC3-Ⅱ/LC3-Ⅰ proteins were higher,and the levels of p-PI3K/PI3K,p-Akt/Akt,mTOR/p-mTOR were lower(P<0. 05). Compared with control group,the inhibition rate of N-butanol extract of Radix Actinidiae on MDA-MB-231 cells pretreated with 3-MA was lower,meanwhile the inhibition rate of N-butanol extract of Radix Actinidiae on MDA-MB-231 cells pretreated with rapamycin was higher(P< 0. 05). Conclusion N-butanol extract of Radix Actinidiae can inhibit the proliferation of MDA-MB-231 cells,induce the apoptosis and autophagy of MDA-MB-231 cells,which would be related to inhibition of PI3K/Akt-mTOR pathway.更多还原