AKBA联合阿霉素抑制三阴性乳腺癌细胞MDA-MB-231的增殖、迁移和裸鼠移植瘤生长

doi:10.12122/j.issn.1673-4254.2024.12.22

摘要/Abstract

摘要：

目的探究AKBA联合阿霉素对三阴性乳腺癌细胞MDA-MB-231在增殖、迁移、侵袭和凋亡上的协同抑制作用，并通过网络药理学分析AKBA作用乳腺癌的下游信号通路。方法 MDA-MB-231细胞体外培养，CCK-8法分别检测AKBA和阿霉素（ADR）作用MDA-MB-231细胞48 h的半抑制浓度（IC₅₀），SynergyFinder在线网站（https：//synergyfinder.fimm.fi）分析AKBA联合阿霉素的协同指数和最佳协同浓度。设置实验分组，对照组：MDA-MB-231正常培养；AKBA组：MDA-MB-231+AKBA（22.5 μmol/L）处理48 h；ADR组：MDA-MB-231+（0.84 μmol/L）处理48 h；AKBA+ADR组：MDA-MB-231+AKBA（22.5 μmol/L）+ADR（0.84 μmol/L）处理48 h，使用克隆形成实验、Transwell迁移和侵袭、划痕实验检测AKBA联合阿霉素抑制细胞迁移和侵袭的能力，采用Western blotting和qPCR实验检测凋亡相关基因的表达情况，将4~5周龄Balb/c nude小鼠随机分为4组（6只/组），建立裸鼠异种移植瘤模型，对照组：腹腔注射200 μL 0.9%生理盐水；AKBA组：腹腔注射200 μL50 mg/kg的AKBA；ADR组：腹腔注射200 μL 2.5 mg/kg的ADR；AKBA+ADR组：腹腔注射200 μL50 mg/kg的AKBA和2.5 mg/kg的ADR等量混合溶液，检测各组抑瘤率，HE染色检测组织病理情况，并通过网络药理学分析预测AKBA作用于乳腺癌的下游靶点和信号通路。结果 AKBA对MDA-MB-231细胞作用48 h的IC₅₀为45.15±0.97 μmol/L，ADR的IC₅₀为0.42±0.99 μmol/L；SynergyFinder证实AKBA与ADR联合具有协同作用（ZIP>10）；相较于对照组、AKBA组和阿霉素组，AKBA+ADR组明显抑制TNBC细胞增殖活性（P<0.0001），集落形成数量减少更多（P<0.05）；相较对照组、AKBA和ADR组，AKBA+ADR组明显减弱细胞划痕愈合率（P<0.01）；较对照组、AKBA组和ADR组，AKBA+ADR组所致细胞迁移至下室的数量减少更多（P<0.01）；与对照组和AKBA或ADR单独使用相比，AKBA+ADR组抑制MDA-MB-231细胞侵袭基质胶能力增强（P<0.05）；较AKBA或ADR单独作用，AKBA+ADR组在mRNA和蛋白水平上调Bax、Caspase-3剪切体和下调Bcl-2的表达更多（P<0.05）；AKBA组、ADR组和AKBA+ADR组的抑瘤率分别为23.80%、52.73%和81.83%（P<0.01），AKBA+ADR组相比于ADR组对小鼠脏器造成的毒性损伤更小；网络药理学预测AKBA通过PTGS2影响乳腺癌进展。结论 AKBA联合阿霉素能够显著抑制TNBC细胞MDA-MB-231的增殖、迁移和侵袭，并促进细胞凋亡，在裸鼠体内抑制移植瘤生长并减轻阿霉素的毒性作用。

关键词: AKBA, 乙酰基-11-酮基-β-乳香酸, 三阴性乳腺癌, 阿霉素

Abstract:

Objective To investigate the synergistic inhibitory effects of AKBA and doxorubicin on malignant phenotype of triple-negative breast cancer (TNBC) MDA-MB-231 cells. Methods CCK-8 assay was used to determine the 48-h IC₅₀ of AKBA and doxorubicin in MDA-MB-231 cells, and SynergyFinder was employed to calculate the synergistic index and the optimal concentrations of the two agents. MDA-MB-231 cells treated with AKBA (22.5 μmol/L), doxorubicin (0.84 μmol/L) or their combination were examined for changes in cell proliferation, migration, invasion and apoptosis using Transwell migration, scratch assay, clone generation, RT-qPCR and Western blotting. Network pharmacology analysis was conducted to identify the downstream targets of AKBA in TNBC. In nude mouse models bearing subcutaneous MDA-MB-231 cell xenografts, the effects of normal saline, AKBA (50 mg/kg), doxorubicin (2.5 mg/kg), and AKBA combined with doxorubicin on xenograft growth and histopathology were observed. Results The IC₅₀ of AKBA and doxorubicin in MDA-MB-231 cells at 48 h was 45.15±0.97 μmol/L and 0.42±0.99 μmol/L, respectively. SynergyFinder confirmed the synergistic effect of AKBA and ADR with a ZIP>10. The combined treatment with AKBA and doxorubicin significantly inhibited the proliferation, migration and invasion, promoted apoptosis of MDA-MB-231 cells, and effectively suppressed xenograft growth in nude mice. Network pharmacology analysis predicted that AKBA affects the progression of TNBC through its downstream target AKBA. Conclusion AKBA combined with doxorubicin inhibits proliferation, migration and invasion, promotes apoptosis of MDA-MB-231 cells and suppresses MDA-MB-231 cell xenograft growth in nude mice. The combined use of AKBA can attenuate the toxic effects of doxorubicin in nude mice.

Key words: AKBA, acetyl-11-keto?β?boswellic acid, triple-negative breast cancer, doxorubicin

曾佑琴, 陈思雨, 刘燕, 刘奕彤, 张玲, 夏姣, 吴心语, 魏常友, 冷平. AKBA联合阿霉素抑制三阴性乳腺癌细胞MDA-MB-231的增殖、迁移和裸鼠移植瘤生长[J]. 南方医科大学学报, 2024, 44(12): 2449-2460.

Youqin ZENG, Siyu CHEN, Yan LIU, Yitong LIU, Ling ZHANG, Jiao XIA, Xinyu WU, Changyou WEI, Ping LENG. AKBA combined with doxorubicin inhibits proliferation and metastasis of triple-negative breast cancer MDA-MB-231 cells and xenograft growth in nude mice[J]. Journal of Southern Medical University, 2024, 44(12): 2449-2460.

图/表 6

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequence 5'-3'
Caspase-3	Forward: TATTCCACAGCACCTGGTTA Reverse: CAATACATGGAATCTGTTTCTT
Bax	Forward: CCTCAGGATGCGTCCACCAAGA Reverse: TGTGTCCACGGCGGCAATCA
Bcl-2	Forward: GTGTGTGGAGAGCGTCAACC Reverse: TCTTCAGAGACAGCCAGGAGAA
GAPDH	Forward: GGAGCGAGATCCCTCCAAAAT Reverse: GGCTGTTGTCATACTTCTCATGG
PTGS2	Forward: CTGGCGCTCAGCCATACAG Reverse: CGCACTTATACTGGTCAAATCCC

图1 CCK-8和Synergy Finder检测AKBA联合ADR对MDA-MB-231细胞增殖活性的影响以及药物联合协同指数

Fig.1 CCK-8 assay and SynergyFinder detection of the effect of AKBA combined with ADR on proliferation of MDA-MB-231 cells and drug combination synergy indices. A: Viability of AKBA-treated MDA-MB-231 and MCF-10A cells at 24 and 48 h. B: Viability of ADR-treated MDA-MB-231 and MCF-10A cells at 24 and 48 h. C: Synergistic indices of the combination of AKBA and ADR determined with Synergy Finder. D: Low concentrations of AKBA combined with different concentrations of ADR inhibit proliferation of MDA-MB-231 cells. E: Proliferation of MDA-MB-231 cells treated with saline, AKBA, ADR and AKBA+ADR for 72 h. *P<0.05, **P<0.01, ****P<0.0001.

图2 AKBA联合ADR对TNBC细胞增殖和迁移的影响

Fig.2 Effect of AKBA combined with ADR on TNBC cell proliferation and migration. A: Clone formation assay in each group. B: Scratch assay for assessing migration ability of TNBC cells (Original magnification: ×40). C: Transwell migration assay of TNBC cells treated with AKBA combined with ADR (×100). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs CTL; #P<0.05, ##P<0.01, ###P<0.001.

图3 AKBA联合ADR对TNBC细胞侵袭和凋亡的影响

Fig.3 Effect of AKBA combined with ADR on TNBC cell invasion and apoptosis. A: Transwell invasion assay for assessing the ability of TNBC cells to invade stromal gel (×100). B: RT-qPCR for detecting mRNA expressions of apoptosis-related genes in TNBC cells. C: Western blotting for detecting expressions of apoptosis-related proteins in TNBC cells. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs CTL; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001.

图4 AKBA联合ADR对TNBC细胞裸鼠移植瘤生长的影响以及脏器毒性作用

Fig.4 Effect of AKBA combined with ADR on TNBC xenograft growth and its organ toxicity in nude mice. A: Observation of the tumor-bearing mice and the dissected tumors on day 27 and changes of body weight of the mice and tumor volume over time (black arrows indicate the time points of drug administration). B: HE staining for examining tumor histopathology and evaluating toxic effects of AKBA combined with ADR in the heart, liver, kidney, and lungs of the nude mice (×10). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs CTL; ##P<0.01, ###P<0.001, ####P<0.0001 vs AKBA group; †P<0.05, ††P<0.01 vs ADR group

图5 网络药理学分析筛选AKBA作用乳腺癌的下游通路和靶点

Fig.5 Network pharmacological analysis of downstream pathways and targets of AKBA in breast cancer. A: Venn diagram of intersected targets of AKBA and breast cancer. B: Protein interaction network obtained by STRING analysis of the intersected targets. C: Protein-protein interaction (PPI) network created was by Cytoscape for identifying the key targets of AKBA in breast cancer. D, E: Bubble diagram of KEGG pathway enrichment analysis of the biological process (BP), cellular composition (CC), molecular function (MF), GeneRatio, ratio of differential genes in KEGG pathway to the total differential genes. F: RT-qPCR of mRNA expression of the predicted target PTGS2 in each group. ****P<0.0001 vs CTL; #P<0.05.

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