Oridonin impedes breast cancer growth by blocking cells in S phase and inhibiting the PI3K/AKT/mTOR signaling pathway

Abstract

Breast cancer is one of the most common cancers. Oridonin, a traditional Chinese medicine, is believed to inhibit tumor growth, but its particular effects on breast cancer remain unknown. In this study, we examined oridonin's effects on 4T1, MCF-7, and MDAMB-231 cellular activity using CCK8. Scratch assays were used to detect oridonin's effects on cellular migration. Oridonin's effects on the breast cancer cell cycle were studied using flow cytometry, and expression of cell cycle related proteins p53, CDK2, and p21 was detected using Western blot assays. Metabolomics assays were used to detect changes in small molecule metabolites and metabolic pathways in breast cancer cells after treatment with oridonin. Oridonin's effects on breast cancer growth were also studied using xenograft mice. Metabolomics assays were used to detect changes in metabolites and metabolic pathways in xenograft mouse plasma in a control group, model group, and drug administration group. Experimental results showed that oridonin could significantly inhibit breast cancer growth both in vivo and in vitro. Scratch experiments showed that oridonin could inhibit breast cancer cell migration. Oridonin was also able to arrest cells in S phase by affecting several cell cycle-related proteins, including p53, CDK2, and p21. Metabolomic analysis of 4T1 cells identified a total of 33 differential metabolites, including multiple amino acids (such as l-Glutamic acid, l-Asparagine, l-Histidine, l-Valine, and l-Isoleucine). KEGG pathway enrichment analysis showed significant changes in aminoacyl-tRNA biosynthesis, and in multiple amino acid metabolic pathways. Plasma metabolomic analyses of xenograft mice revealed 28 differentially-expressed metabolites between the different animal model groups, including multiple amino acids. KEGG pathway analysis showed significant alterations in multiple amino acid metabolic pathways in oridonin-treated mice. Additionally, changes in the expression of PI3K, AKT and mTOR proteins, as well as in branched amino acids, suggest that oridonin affects the PI3K/AKT/mTOR signaling pathway by inhibiting the biosynthesis of valine, leucine and isoleucine. Taken together, our results suggest that oridonin has strong anti-tumor activity in vitro and in vivo, and has potential as an adjuvant to breast cancer treatment regimens.

Keywords: Amino acid metabolism; Breast cancer; Metabolomics; Oridonin; PI3K/AKT/mTOR signaling pathway.

Fig. 1

Oridonin's effects on the proliferation of 4T1,MCF-7 and MDAMB-231 cells. A. 4T1 cell survival rates. B. MCF-7 cell survival rates. C. MDAMB-231 cell survival rates.

Fig. 2

A. 4T1 cell migration assay results after oridonin treatment. B. Wound area of cells at different concentrations and times. (ns, no significant difference, *P < 0.05, ***P < 0.001, ****P < 0.0001).

Fig. 3

Effects of oridonin on the cell cycle and cycle-related proteins of 4T1 cells. A and B. Graphical representation of the cell cycle distribution of 4T1 cells after treatment with oridonin. C. Western blot analysis of the expression of the S phase arrest-related proteins p53, p-p53, p21 and CDK2. Relative expression levels of p53, p-p53, p21 and CDK2 proteins (ns, no significant difference,**P < 0.01,***P < 0.001****P < 0.0001). D. Schematic diagram of oridonin-induced cell cycle arrest in S phase.

Fig. 4

Tumor growth curves in xenograft mice (Nor-normal group, Mod-model group, Ori-oridonin group). A. Changes in body weights of mice. Over time, mouse body weight gradually increased, but the average body weight of the oridonin group was lower than that of the model group (Mod vs. Ori group. P = 0.0087). B. Mouse tumor volume also gradually increased in both groups, but the overall growth rate was significantly lower in the oridonin group (Mod vs. Ori. P = 0.0065). C, Tumor quality after two weeks of was significantly lower in the oridonin group than in the untreated mice (Mod vs. Ori group. **P < 0.01).

Fig. 5

Results of metabolomic analyses in cells and plasma of mice treated with oridonin. A. Cluster plot of differentially expressed metabolites in 4T1 cells. Yellow represents an elevated level of metabolites, and blue represents decreased levels of metabolites. (Con-control group, Ori- oridonin group) B. Cluster plot of differentially expressed metabolites in mice serum. Yellow represents increased metabolites, and blue represents decreased metabolites (Nor-normal group, Mod-model group, Ori-oridonin group). C. Enrichment of metabolites in various metabolic pathways in 4T1 cells. The darker the color, the smaller the P-value, and the larger the circle, the greater the number of differentially expressed metabolites. D. Overview of the enriched metabolome in mice serum.

Fig. 6

Effects of oridonin on branched-chain amino acid metabolism and the mTOR pathway. A. Changes in l-Isoleucine, l-Valine, and Leucine in 4T1 cells after oridonin treatment. B. Western blot analysis of the expression of BCAT1, and relative expression levels of BCAT1 (**P < 0.01 versus the control group). C. Western blot analysis of mTOR, PI3K and AKT expression, and the relative expression levels of mTOR, PI3K and AKT proteins (**P < 0.01,****P < 0.0001).

Fig. 7

Mechanistic diagram of oridonin's effects on breast cancer cells.