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

doi:10.3760/cma.j.cn371439-20230901-00009

摘要/Abstract

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，差异有统计学意义（F=68.09，P<0.001）。两两比较发现，对照组，EGB低、中、高浓度组LC3Ⅱ、caspase-3蛋白表达量依次升高，而P62蛋白表达量依次降低（均P<0.05）。PCR实验结果显示，对照组，EGB低、中、高浓度组乳腺癌MCF-7细胞MRP1 mRNA水平分别为1.06±0.14、0.83±0.18、0.71±0.11、0.52±0.08，差异有统计学意义（F=17.41，P<0.001）；MDR1 mRNA水平分别为1.14±0.17、0.75±0.13、0.60±0.09、0.48±0.06，差异有统计学意义（F=34.40，P<0.001）；BCRP mRNA水平分别为1.09±0.11、0.88±0.13、0.69±0.07、0.57±0.05，差异有统计学意义（F=34.13，P<0.001）。两两比较发现，对照组，EGB低、中、高浓度组MRP1、MDR1、BCRP mRNA水平均依次降低（均P<0.05）。蛋白质印迹法检测结果表明，对照组，EGB低、中、高浓度组乳腺癌MCF-7细胞ERK表达分别为2.54±0.38、1.89±0.25、1.55±0.21、1.12±0.16，差异有统计学意义（F=31.18，P<0.001）；MAPK表达分别为2.47±0.34、1.96±0.29、1.63±0.27、1.20±0.24，差异有统计学意义（F=20.90，P<0.001）；p-ERK表达分别为2.03±0.29、1.74±0.21、1.45±0.11、1.18±0.24，差异有统计学意义（F=16.31，P<0.001）；p-MAPK表达分别为2.26±0.47、1.90±0.41、1.61±0.33、1.35±0.16，差异有统计学意义（F=7.01，P=0.002）。两两比较发现，对照组，EGB低、中、高浓度组细胞ERK、MAPK、p-ERK、p-MAPK表达均依次降低（均P<0.05）。结论 EGB可抑制乳腺癌MCF-7细胞增殖，促进MCF-7细胞凋亡，降低P62蛋白表达，升高LC3Ⅱ、caspases-3蛋白表达，诱导细胞线粒体自噬。

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

Abstract:

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 statistically significant difference （F=128.80，P<0.001）. Pairwise comparison showed that the apoptosis rate of control group，EGB low，medium and high concentration groups were increased in turn （all P<0.05）. The results of immunofluorescence assay showed that the protein relative expression levels of P62 protein in MCF-7 cells of control group，EGB low，medium and high concentration groups were 3.34±0.52，2.85±0.47，2.02±0.18 and 1.08±0.21，respectively，with a statistically significant difference （F=41.55，P<0.001）. LC3Ⅱ protein relative expression levels were 0.24±0.05，1.02±0.14，1.47±0.26，1.95±0.21，respectively，with a statistically significant difference （F=94.82，P<0.001）. The relative expression levels of caspase-3 protein were 0.25±0.03，0.68±0.21，1.12±0.17 and 1.65±0.23，respectively，with a statistically significant difference （F=68.09， P<0.001）. Pairwise comparison showed that LC3Ⅱ and caspase-3 protein expression levels were increased in turn in control group，EGB low，medium and high concentration groups，while P62 protein expression levels were decreased in turn （all P<0.05）. The PCR experiment results showed that the MRP1 mRNA level of MCF-7 cells in control group， EGB low，medium and high concentration groups were 1.06±0.14， 0.83±0.18， 0.71±0.11， 0.52±0.08， respectively，with a statistically significant difference （F=17.41，P<0.001）. The mRNA levels of MDR1 were 1.14±0.17， 0.75±0.13， 0.60±0.09， 0.48±0.06，respectively，with a statistically significant difference （F=34.40，P<0.001）. BCRP mRNA levels were 1.09±0.11， 0.88±0.13， 0.69±0.07， 0.57±0.05，respectively，with a statistically significant difference （F=34.13，P<0.001）. Pairwise comparison showed that the levels of MRP1，MDR1 and BCRP mRNA were decreased in turn in control group，EGB low，medium and high concentration groups （all P<0.05）. The results of Western blotting showed that the expression of ERK in MCF-7 cells in control group，EGB low，medium and high concentration groups were 2.54±0.38， 1.89±0.25， 1.55±0.21， 1.12±0.16， respectively，with a statistically significant difference （F=31.18，P<0.001）. MAPK expression were 2.47±0.34， 1.96±0.29， 1.63±0.27， 1.20±0.24， respectively，with a statistically significant difference （F=20.90，P<0.001）. p-ERK expression were 2.03±0.29， 1.74±0.21， 1.45±0.11， 1.18±0.24， respectively，with a statistically significant difference （F=16.31，P<0.001）. p-MAPK expression were 2.26±0.47， 1.90±0.41， 1.61±0.33， 1.35±0.16， respectively，with a statistically significant difference （F=7.01，P=0.002）. Pairwise comparison showed that the expressions of ERK，MAPK，p-ERK and p-MAPK in control group，EGB low，medium and high concentration groups were decreased in turn （all P<0.05）. Conclusion EGB can inhibit the proliferation of breast cancer MCF-7 cells，promote the apoptosis of MCF-7 cells，decrease the expression of P62 protein，increase the expression of LC3Ⅱ and caspase-3 protein，induce mitochondrial autophagy.

Key words: Extract of ginkgo biloba, Breast cancer cells, Mitochondrial autophagy, Extracellular signal regulated kinase, Mitogen activated protein kinase

邵建强, 王鹏, 白杰, 李会欣, 王遵义, 徐志宏. 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[J]. 国际肿瘤学杂志, 2024, 51(2): 65-72.

Shao Jianqiang, Wang Peng, Bai Jie, Li Huixin, Wang Zunyi, Xu Zhihong. The mechanism of extract of ginkgo biloba inducing mitochondrial autophagy in breast cancer cells MCF-7[J]. Journal of International Oncology, 2024, 51(2): 65-72.

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组别	24 h	48 h	t值	P值
对照组	0.95±0.14	1.32±0.19	3.84	0.003
EGB低浓度组	0.65±0.09^a	0.54±0.08^a	2.24	0.048
EGB中浓度组	0.51±0.07^ab	0.32±0.05^ab	5.41	<0.001
EGB高浓度组	0.37±0.04^abc	0.15±0.02^abc	16.63	<0.001
F值	43.13	141.30
P值	<0.001	<0.001