[AKBA combined with doxorubicin inhibits proliferation and metastasis of triple-negative breast cancer MDA-MB-231 cells and xenograft growth in nude mice]

Abstract

Objectives: 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.

Conclusions: 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.

Keywords: AKBA; acetyl-11-keto‑β‑boswellic acid; doxorubicin; triple-negative breast cancer.

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.