银杏叶提取物诱导乳腺癌MCF-7细胞线粒体自噬的机制研究  

The mechanism of extract of ginkgo biloba inducing mitochondrial autophagy in breast cancer cells MCF-7

作　　者：邵建强 王鹏[1] 白杰[1] 李会欣 王遵义[1] 徐志宏 Shao Jianqiang;Wang Peng;Bai Jie;Li Huixin;Wang Zunyi;Xu Zhihong(Department of Thyroid and Breast Surgery,Cangzhou Central Hospital,Hebei Province,Cangzhou 061000,China)

机构地区：[1]河北省沧州市中心医院甲状腺乳腺外科,沧州061000

出　　处：《国际肿瘤学杂志》2024年第2期65-72,共8页Journal of International Oncology

基　　金：国家癌症中心攀登基金(NCC201803B004);河北省医学科学研究基金(20220400)。

摘　　要：目的探讨银杏叶提取物(EGB)对乳腺癌MCF-7细胞线粒体自噬的作用机制。方法将乳腺癌MCF-7细胞株分为4组,使用质量浓度为40、80、120 mg/L的EGB分别作用于乳腺癌MCF-7细胞株,培养24或48 h,作为EGB低浓度组、EGB中浓度组、EGB高浓度组,另取未干预的乳腺癌MCF-7细胞作为对照组。MTT法测定细胞增殖;流式细胞仪检测细胞凋亡;免疫荧光法测定核孔蛋白(P62)、微管相关蛋白轻链3Ⅱ(LC3Ⅱ)、胱天蛋白酶-3(caspase-3)含量;PCR仪检测多药耐药相关蛋白1(MRP1)、多药耐药基因1(MDR1)及乳腺癌耐药蛋白(BCRP)mRNA水平;蛋白质印迹法检测细胞胞外信号激酶(ERK)、丝裂原活化蛋白激酶(MAPK)、p-ERK、p-MAPK蛋白表达。结果MTT法测定细胞增殖结果表明,对照组,EGB低、中、高浓度组24 h细胞增殖分别为0.95±0.14、0.65±0.09、0.51±0.07、0.37±0.04,差异有统计学意义(F=43.13,P<0.001);48 h细胞增殖分别为1.32±0.19、0.54±0.08、0.32±0.05、0.15±0.02,差异有统计学意义(F=141.30,P<0.001);与24 h比较,EGB低、中、高浓度组48 h细胞增殖均降低(均P<0.05)。两两比较发现,EGB处理显著降低了MCF-7细胞活力,对照组,EGB低、中、高浓度组24、48 h细胞增殖均依次降低(均P<0.05)。流式细胞术检测发现,对照组,EGB低、中、高浓度组乳腺癌MCF-7细胞凋亡率分别为2.12%±0.23%、9.28%±0.45%、15.17%±1.28%、22.21%±2.32%,差异有统计学意义(F=128.80,P<0.001)。两两比较发现,对照组,EGB低、中、高浓度组细胞凋亡率依次升高(均P<0.05)。免疫荧光法测定结果显示,对照组,EGB低、中、高浓度组乳腺癌MCF-7细胞P62蛋白相对表达量分别为3.34±0.52、2.85±0.47、2.02±0.18、1.08±0.21,差异有统计学意义(F=41.55,P<0.001);LC3Ⅱ蛋白相对表达量分别为0.24±0.05、1.02±0.14、1.47±0.26、1.95±0.21,差异有统计学意义(F=94.82,P<0.001);caspase-3蛋白相对表达量分别为0.25±0.03、0.68±0.21、1.12±0.17、1.65±0.23,差异有统Objective To investigate the mechanism of extract of ginkgo biloba(EGB)on mitochondrial autophagy in breast cancer cells MCF-7.Methods Breast cancer MCF-7 cells were divided into four groups.EGB with mass concentrations of 40,80,120 mg/L was used to incubate breast cancer MCF-7 cells for 24 h or 48 h,as a low concentration group of EGB,a medium concentration group of EGB,and a high concentration group of EGB.Breast cancer MCF-7 cells without intervention were taken as control group.Cell proliferation was measured using MTT assay;Flow cytometry was used to detect cell apoptosis;Immunofluorescence assay was used to determine the contents of prostacyclin(P62),microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ),and caspase-3;The levels of multidrug resistance-associated protein 1(MRP1),multidrug resistance gene 1(MDR1)and breast cancer resistance protein(BCRP)were identified by PCR;Western blotting was used to detect the expression of extracellular signal-regulated kinase(ERK),mitogen-activated protein kinase(MAPK),p-ERK,and p-MAPK proteins in cells.Results The results of MTT assay for cell proliferation showed that cell proliferation at 24 h in control group,EGB low,medium and high concentration groups were 0.95±0.14,0.65±0.09,0.51±0.07,0.37±0.04,respectively,with a statistically significant difference(F=43.13,P<0.001),cell proliferation at 48 h were 1.32±0.19,0.54±0.08,0.32±0.05,0.15±0.02,respectively,with a statistically significant difference(F=141.30,P<0.001).Compared with 24 h,cell proliferation was decreased in EGB low,medium and high concentration groups at 48 h(all P<0.05).Pairwise comparison showed that EGB treatment significantly decreased MCF-7 cell viability and cell proliferation was decreased in turn at 24 and 48 h in control group,low,medium,high EGB groups(all P<0.05).Flow cytometry analysis revealed that the apoptosis rates of MCF-7 cells in control group,EGB low,medium and high concentration groups were 2.12%±0.23%,9.28%±0.45%,15.17%±1.28%and 22.21%±2.32%,respectively,with a statist

关 键 词：银杏叶提取物 乳腺癌细胞 线粒体自噬 胞外信号调节激酶 丝裂原活化蛋白激酶 

分 类 号：R737.9[医药卫生—肿瘤]