藤梨根正丁醇提取物通过抑制PI3K/Akt/mTOR通路诱导乳腺癌细胞自噬和凋亡的实验研究  

Experimental study onautophagy and apoptosis of breast cancer cells induced by N-butanol extract of Radix Actinidiae by inhibiting PI3K/Akt/mTOR pathway

作　　者：叶惠荣[1] 袁惠玲[1] 曹茵[1] 王西跃[1] 吴丽华[1] 陈桂林[1] 陈丽娟[1] 张玉娟[1] YE Huirong;YUAN Huiling;CAO Yin;WANG Xiyue;WU Lihua;CHEN Guilin;CHEN Lijuan;ZHANG Yujuan(Department of Breast, Dongguan People's Hospital, Dongguan 523000, China)

机构地区：[1]东莞市人民医院乳腺科,广东东莞523000

出　　处：《临床肿瘤学杂志》2021年第2期104-109,共6页Chinese Clinical Oncology

基　　金：国家自然科学基金资助项目(81802625);东莞市社会科技发展(重点)资助项目(201850715001557)。

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。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 N-butanol extract of Radix Actinidiae had obviously inhibition effect on the proliferation of MDA-MB-231 cells,which was dose-independent and time-independent.After treatment with N-butanol extract of Radix Actinidiae for 24h,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-Ⅰproteins 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.

关 键 词：乳腺癌 藤梨根正丁醇提取物 自噬 PI3K/Akt-mTOR通路 凋亡 

分 类 号：R737.9[医药卫生—肿瘤]