β-Boswellic Acid Suppresses Breast Precancerous Lesions via GLUT1 Targeting-Mediated Glycolysis Inhibition and AMPK Pathway Activation

Abstract

Breast carcinoma is a multistep progressive disease. Precancerous prevention seems to be crucial. β-Boswellic acid (β-BA), the main component of the folk medicine Boswellia serrata (B. serrata), has been reported to be effective in various diseases including tumors. In this work, we demonstrated that β-BA could inhibit breast precancerous lesions in rat disease models. Consistently, β-BA could suppress proliferation and induce apoptosis on MCF-10AT without significantly influencing MCF-10A. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that β-BA may interfere with the metabolic pathway. Metabolism-related assays showed that β-BA suppressed glycolysis and reduced ATP production, which then activated the AMPK pathway and inhibited the mTOR pathway to limit MCF-10AT proliferation. Further molecular docking analysis suggested that GLUT1 might be the target of β-BA. Forced expression of GLUT1 could rescue the glycolysis suppression and survival limitation induced by β-BA on MCF-10AT. Taken together, β-BA could relieve precancerous lesions in vivo and in vitro through GLUT1 targeting-induced glycolysis suppression and AMPK/mTOR pathway alterations. Here, we offered a molecular basis for β-BA to be developed as a promising drug candidate for the prevention of breast precancerous lesions.

Keywords: AMPK; GLUT1; breast precancerous lesions; glycolysis; β-boswellic acid.

Figure 1

β-BA inhibits cell proliferation in MCF-10AT and MDA-MB-231. (A) Chemical structure of β-BA. (B) Cell viability was detected by CCK8 assay following treatment with different doses of β-BA. Cells were treated with 0, 10, 20, 40, 80, 160, and 320 μM of β-BA for 24, 36 and 48 h, respectively. IC₅₀ values for each cell line at different time points were determined. (C) Colony formation assay for MCF-10A, MCF-10AT, and MDA-MB-231 treated with 0, 20, 40, and 60 μM of β-BA, respectively. Numbers of colonies were counted and shown below. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2

β-BA promotes apoptosis in MCF-10AT. (A) FACS analysis Annexin V/PI staining was used to determine apoptosis induced by β-BA. (B) The population of early apoptosis and that of late apoptosis were counted. (C) The expression levels of cleaved-caspase 3, pro-caspase 3, and Bax were analyzed in MCF-10AT following a 48-h treatment with β-BA at designated concentrations. Protein levels were quantified by Image J and normalized with that of β-actin in the right panel. Data were shown as the mean ± S.D from independent experiments. ***p < 0.001.

Figure 3

β-BA suppresses glycolysis and activates the AMPK pathway in MCF-10AT. (A) KEGG pathway enrichment analysis was performed with predicted targets derived from BATMAN-TCM. (B) The cellular ECAR was measured in MCF-10A and MCF-10AT cells with or without β-BA treatment. Statistics of glycolysis ECAR values are shown in the lower panel. The lactate production (C) and ATP levels (D) were measured in MCF-10AT cells with or without β-BA treatment. (E) The expression levels of AMPK, p-AMPK, ACC, p-ACC, mTOR, p-mTOR, p70S6k, and p-p70S6k were analyzed in MCF-10A and MCF-10AT following a 48-h treatment with 40 μM β-BA. Protein levels were quantified by ImageJ and normalized with that of β-actin in the lower panel. Data were shown as the mean ± S.D from independent experiments. ***p < 0.001.

Figure 4

β-BA interacts with GLUT1 to block glucose uptake in MCF-10AT. (A) Visualization of 3D interaction of docked complex composed of GLUT1 and β-BA. (B) Ligand interactions between β-BA and GLUT1. (C) The protein expression of GLUT1 in MCF-10AT cell with or without β-BA treatment was measured by Western blot. (D) The gene expression of GLUT1 in MCF-10AT cell with or without β-BA treatment was measured by real-time PCR. (E) The 2-NBDG glucose uptake in GLUT1 overexpression MCF-10AT and its corresponding control cells was measured by FACS. Statistics are shown in the right panel as mean fluorescent intensity ± S.D.

Figure 5

GLUT1 mediates β-BA regulated glycolysis suppression. (A) The overexpression level of GLUT1 protein was detected by Western blot. (B) The 2-NBDG glucose uptake levels of GLUT1 overexpressing and its corresponding control cells were detected by FACS. Statistics are shown in the right panel as mean fluorescent intensity ± S.D. (C) Cell viability was detected by CCK8 assay in GLUT1 overexpressing and its corresponding control cells. The glucose uptake level (D) ATP levels (E), lactate production (F), and cell viability (G) were measured in the group of MCF-10AT+pcDNA3.1, MCF-10AT+pcDNA3.1+BA, and MCF-10AT+GLUT1+BA, respectively. Data were shown as the mean ± S.D from independent experiments. **p < 0.01 and ***p < 0.001.

Figure 6

β-BA could inhibit the breast precancerous lesions in vivo This panel shows the hematoxylin–eosin staining of the control group, the disease model group, the tamoxifen-treated group, and the β-BA-treated group, respectively.