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

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

Abstract

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

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.

Figures/Tables 7

References 28

[1]	Kashyap D, Pal D, Sharma R, et al. Global increase in breast cancer incidence: risk factors and preventive measures[J]. Biomed Res Int, 2022, 2022: 9605439. DOI: 10.1155/2022/9605439.
[2]	Thorat MA, Balasubramanian R. Breast cancer prevention in high-risk women[J]. Best Pract Res Clin Obstet Gynaecol, 2020, 65: 18-31. DOI: 10.1016/j.bpobgyn.2019.11.006. pmid: 31862315
[3]	王敏, 李习平, 杨梅, 等. Caspase信号途径及其在乳腺癌治疗中的作用研究进展[J]. 陕西医学杂志, 2021, 50(3): 377-380. DOI: 10.3969/j.issn.1000-7377.2021.03.032.
[4]	张丽娜, 张迪迪, 赵雷, 等. 乳腺癌细胞与巨噬细胞融合对PI3K/AKT和MAPK/ERK信号通路的影响[J]. 生物学杂志, 2021, 38(6): 25-30. DOI: 10.3969/j.issn.2095-1736.2021.06.025.
[5]	Li W, Qian C, Ma F, et al. MAPK/ERK-CBP-RFPL-3 mediates adipose-derived stem cell-induced tumor growth in breast cancer cells by activating telomerase reverse transcriptase expression[J]. Stem Cells Int, 2022, 2022: 8540535. DOI: 10.1155/2022/8540535.
[6]	赵婵, 耿良, 刘艳丽. 调气消痞汤结合消痞五穴针刺法对三阴性乳腺癌化疗相关性消化不良患者的疗效分析[J]. 实用癌症杂志, 2023, 38(7): 1110-1113. DOI: 10.3969/j.issn.1001-5930.2023.07.017.
[7]	Singh SK, Srivastav S, Castellani RJ, et al. Neuroprotective and antioxidant effect of Ginkgo biloba extract against AD and other neurological disorders[J]. Neurotherapeutics, 2019, 16(3): 666-674. DOI: 10.1007/s13311-019-00767-8. pmid: 31376068
[8]	李珊. 银杏叶提取物(EGb761)对人脂肪间充质干细胞神经向分化的影响[D]. 大连: 大连医科大学, 2012.
[9]	Zhang A, Wang X, Fan C, et al. The role of Ki67 in evaluating neoadjuvant endocrine therapy of hormone receptor-positive breast cancer[J]. Front Endocrinol (Lausanne), 2021, 12: 687244. DOI: 10.3389/fendo.2021.687244.
[10]	Shen Q, Reedijk M. Notch signaling and the breast cancer microenvironment[J]. Adv Exp Med Biol, 2021, 1287: 183-200. DOI: 10.1007/978-3-030-55031-8_12. pmid: 33034033
[11]	Tian J, Liu Y, Chen K. Ginkgo biloba extract in vascular protection: molecular mechanisms and clinical applications[J]. Curr Vasc Pharmacol, 2017, 15(6): 532-548. DOI: 10.2174/1570161115666170713095545. pmid: 28707602
[12]	刘鹏程, 张晔, 陈晓宇, 等. MitoQ通过NIX介导的线粒体自噬对肺腺癌A549细胞迁移及凋亡的影响[J]. 安徽医科大学学报, 2021, 56(7): 1037-1041. DOI: 10.19405/j.cnki.issn1000-1492.2021.07.007.
[13]	Lai CP, Chen YS, Ying TH, et al. Melatonin acts synergistically with pazopanib against renal cell carcinoma cells through p38 mitogen-activated protein kinase-mediated mitochondrial and autophagic apoptosis[J]. Kidney Res Clin Pract, 2023, 42(4): 487-500. DOI: 10.23876/j.krcp.22.114.
[14]	Mazumder K, Aktar A, Ramasamy S, et al. Attenuating colorectal cancer using nine cultivars of Australian lupin seeds: apoptosis induction triggered by mitochondrial reactive oxygen species generation and caspases-3/7 activation[J]. Cells, 2023, 12(21): 2557. DOI: 10.3390/cells12212557.
[15]	Chen P, Wang T, Chen Q. Ginkgo biloba golden leaf extract (GGLE) inhibits melanoma cell invasion and angiogenesis through inhibition of angiogenin[J]. Integr Cancer Ther, 2023, 22: 15347354221134513. DOI: 10.1177/15347354221134513.
[16]	Kim JK, Choi MS, Kim JY, et al. Ginkgo biloba leaf extract suppresses intestinal human breast cancer resistance protein expression in mice: correlation with gut microbiota[J]. Biomed Pharmacother, 2021, 140: 111712. DOI: 10.1016/j.biopha.2021.111712.
[17]	王苗苗. 银杏酸通过抑制线粒体生物合成同时促进线粒体自噬从而导致线粒体功能损伤[D]. 广州: 暨南大学, 2020.
[18]	Zhou D, Jiang C, Fu C, et al. Antiproliferative effect of 2-hydroxy-6-tridecylbenzoic acid from Ginkgo biloba sarcotestas through the aryl hydrocarbon receptor pathway in triple-negative breast cancer cells[J]. Nat Prod Res, 2020, 34(6): 893-897. DOI: 10.1080/14786419.2018.1508144. pmid: 30445863
[19]	陈剑靖, 徐美丽, 何子夜, 等. 银杏叶提取物金纳多对敌草快诱导大鼠急性肾损伤的改善作用[J]. 山东医药, 2023, 63(18): 49-53. DOI: 10.3969/j.issn.1002-266X.2023.18.011.
[20]	秦明明, 浦春, 冯钢, 等. let-7i通过抑制K-Ras、Bcl2的表达降低乳腺癌细胞化疗耐药性[J]. 细胞与分子免疫学杂志, 2019, 35(11): 992-999. DOI: 10.13423/j.cnki.cjcmi.008888.
[21]	Guo YJ, Pan WW, Liu SB, et al. ERK/MAPK signalling pathway and tumorigenesis[J]. Exp Ther Med, 2020, 19(3): 1997-2007. DOI: 10.3892/etm.2020.8454.
[22]	Lev S. Targeted therapy and drug resistance in triple-negative breast cancer: the EGFR axis[J]. Biochem Soc Trans, 2020, 48(2): 657-665. DOI: 10.1042/BST20191055.
[23]	Lou JS, Zhao LP, Huang ZH, et al. Ginkgetin derived from Ginkgo biloba leaves enhances the therapeutic effect of cisplatin via ferroptosis-mediated disruption of the Nrf2/HO-1 axis in EGFR wild-type non-small-cell lung cancer[J]. Phytomedicine, 2021, 80: 153370. DOI: 10.1016/j.phymed.2020.153370.
[24]	董超. 银杏叶总黄酮EGb761在膀胱癌细胞T24化疗中的增效性及其作用机制的研究[D]. 桂林: 桂林医学院, 2011. DOI: 10.7666/d.D421893.
[25]	覃楠, 覃琳, 诸葛春凤, 等. EGb761逆转NF-κB介导的胃癌细胞耐药及协同增敏的机制研究[J]. 中药新药与临床药理, 2016, 27(6): 768-774. DOI: 10.19378/j.issn.1003-9783.2016.06.006.
[26]	Su CC. Tanshinone ⅡA inhibits gastric carcinoma AGS cells through increasing p-p38, p-JNK and p53 but reducing p-ERK, CDC2 and cyclin B1 expression[J]. Anticancer Res, 2014, 34(12): 7097-7110.
[27]	Sun Q, Liang Y, Zhang T, et al. ER-α36 mediates estrogen-stimulated MAPK/ERK activation and regulates migration, invasion, proliferation in cervical cancer cells[J]. Biochem Biophys Res Commun, 2017, 487(3): 625-632. DOI: 10.1016/j.bbrc.2017.04.105.
[28]	Yeh HT, Tsai YS, Chen MS, et al. Flavopereirine induces cell cycle arrest and apoptosis via the AKT/p38 MAPK/ERK1/2 sig-naling pathway in human breast cancer cells[J]. Eur J Pharmacol, 2019, 863: 172658. DOI: 10.1016/j.ejphar.2019.172658.

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

组别	P62	LC3Ⅱ	caspase-3
对照组	3.34±0.52	0.24±0.05	0.25±0.03
EGB低浓度组	2.85±0.47^a	1.02±0.14^a	0.68±0.21^a
EGB中浓度组	2.02±0.18^ab	1.47±0.26^ab	1.12±0.17^ab
EGB高浓度组	1.08±0.21^abc	1.95±0.21^abc	1.65±0.23^abc
F值	41.55	94.82	68.09
P值	<0.001	<0.001	<0.001

组别	MRP1 mRNA	MDR1 mRNA	BCRP mRNA
对照组	1.06±0.14	1.14±0.17	1.09±0.11
EGB低浓度组	0.83±0.18^a	0.75±0.13^a	0.88±0.13^a
EGB中浓度组	0.71±0.11^ab	0.60±0.09^ab	0.69±0.07^ab
EGB高浓度组	0.52±0.08^abc	0.48±0.06^abc	0.57±0.05^abc
F值	17.41	34.40	34.13
P值	<0.001	<0.001	<0.001

组别	ERK	MAPK	p-ERK	p-MAPK
对照组	2.54±0.38	2.47±0.34	2.03±0.29	2.26±0.47
EGB低浓度组	1.89±0.25^a	1.96±0.29^a	1.74±0.21^a	1.90±0.41^a
EGB中浓度组	1.55±0.21^ab	1.63±0.27^ab	1.45±0.11^ab	1.61±0.33^ab
EGB高浓度组	1.12±0.16^abc	1.20±0.24^abc	1.18±0.24^abc	1.35±0.16^abc
F值	31.18	20.90	16.31	7.01
P值	<0.001	<0.001	<0.001	0.002