Cell Metabolomics Reveals the Potential Mechanism of Aloe Emodin and Emodin Inhibiting Breast Cancer Metastasis

Abstract

Metastasis is one of the main obstacles for the treatment and prognosis of breast cancer. In this study, the effects and possible mechanisms of aloe emodin (AE) and emodin (EMD) for inhibiting breast cancer metastasis were investigated via cell metabolomics. First, a co-culture model of MCF-7 and HUVEC cells was established and compared with a traditional single culture of MCF-7 cells. The results showed that HUVEC cells could promote the development of cancer cells to a malignant phenotype. Moreover, AE and EMD could inhibit adhesion, invasion, and angiogenesis and induce anoikis of MCF-7 cells in co-culture model. Then, the potential mechanisms behind AE and EMD inhibition of MCF-7 cell metastasis were explored using a metabolomics method based on UPLC-Q-TOF/MS multivariate statistical analysis. Consequently, 27 and 13 biomarkers were identified in AE and EMD groups, respectively, including polyamine metabolism, methionine cycle, TCA cycle, glutathione metabolism, purine metabolism, and aspartate synthesis. The typical metabolites were quantitatively analyzed, and the results showed that the inhibitory effect of AE was significantly better than EMD. All results confirmed that AE and EMD could inhibit metastasis of breast cancer cells through different pathways. Our study provides an overall view of the underlying mechanisms of AE and EMD against breast cancer metastasis.

Keywords: aloe emodin (AE); breast cancer; cell metabolomics; emodin (EMD); metastasis.

Figure 1

Heat map of single-culture and co-culture MCF-7 cells to visualize the abundance of biomarkers in each group. Each row represents a potential biomarker, each column represents a sample, and the change of color represents the change of biomarker content (a). Metabolic pathways affected by the co-culture model of MCF-7 cells. The meaning of numbers is as follows: 1. phenylalanine, tyrosine and tryptophan biosynthesis; 2. purine metabolism; 3. aminoacyl-tRNA biosynthesis; 4. glutathione metabolism; 5. phenylalanine metabolism; 6. D-glutamine and D-glutamate metabolism; 7. arginine biosynthesis; 8. alanine, aspartate and glutamate metabolism; 9. cysteine and methionine metabolism; 10. arginine and proline metabolism; 11. glyoxylate and dicarboxylate metabolism; 12. vitamin B6 metabolism; 13. citrate cycle (TCA cycle); 14. tyrosine metabolism; 15. nitrogen metabolism; 16. ubiquinone and other terpenoid–quinone biosynthesis; 17. butanoate metabolism; 18. histidine metabolism; 19. pantothenate and CoA biosynthesis; 20. porphyrin and chlorophyll metabolism (b). Correlation networks of potential biomarkers. The red-marked metabolites were regarded as the potential metabolites (c).

Figure 2

Quantitative analysis of biomarkers. The data are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations are as follows: M represents MCF-7 cell single-culture group, M + H represents the co-culture group of MCF-7 and HUVEC cells. GSH, glutathione; GSSG, oxidized glutathione; Meth, methionine; SAM, S-adenosine methionine; SAH, S-adenosine homocysteine; Cys, cysteine; Hypoxan, hypoxanthine; Homocys, homocysteine; Glu, glutamate; Keto, α-ketoglutarate.

Figure 3

The cytotoxicity of AE (a) and EMD (b) at different concentrations (40, 20, 10 μM) on HUVEC and MCF-7 cells.

Figure 4

The effects of AE and EMD at different concentrations (40, 20, 10 μM) on the adhesion. The data are expressed as the mean ± SD (n = 3). ** p < 0.01, **** p < 0.0001 compared with control group.

Figure 5

The effects of AE and EMD at different concentrations (40, 20, 10 μM) on the invasion (a), soft agar clone (b), VEGF secretion (c), MMP-2 and MMP-9 secretion (d) of MCF-7 cells. The data are expressed as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control group.

Figure 6

PCA score plots of aloe emodin (AE), emodin (EMD), doxorubicin (DOX), and control group (M + H) in positive (left) and negative (right) ion mode (a). S-polts of metabolic profiling between aloe emodin (AE)/emodin (EMD)/doxorubicin (DOX) and control group (M + H) in positive (left) and negative (right) ion modes. The orange and green cubes represent metabolites of VIP>1 and VIP<1, respectively (b).

Figure 7

Quantitative analysis of biomarkers. The data are expressed as the mean ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with control group.