Marine-Derived Stichloroside C2 Inhibits Epithelial-Mesenchymal Transition and Induces Apoptosis through the Mitogen-Activated Protein Kinase Signalling Pathway in Triple-Negative Breast Cancer Cells

Abstract

Background: Triterpenoid saponins from sea cucumbers exhibit significant antitumour, antifungal, and antibacterial activities. However, the associated molecular mechanisms have yet to be elucidated. In this study, we screened and explored the antitumour activity and underlying mechanisms of triterpenoid saponins isolated from Thelenota ananas.

Methods: We isolated and purified sea cucumber saponins, determined their chemical structures, and confirmed their function in vitro. We also screened and explored the antitumour activity and underlying mechanisms of triterpenoid saponins isolated from Thelenota ananas.

Results: Four saponins were discovered from sea cucumber Thelenota ananas collected from the South China Sea. We found that stichloroside C2 (STC2) inhibited the proliferation and clonogenesis of the human triple-negative breast cancer (TNBC) cell line MDA-MB-231 and mouse TNBC cell line 4 T1 in a dose-dependent manner and induced apoptosis and cycle arrest in these two TNBC cell lines. STC2 induced DNA damage in two TNBC cell lines and significantly increased the protein expression level of the DNA double-strand break marker γ-H2AX. STC2 downregulated the protein expression levels of phosphorylated cyclin-dependent kinase 1 (CDK1), cyclin B1, CDK2, and cyclin A2 in MDA-MB-231 and 4 T1 cells. STC2 upregulated Bax and cleaved PARP protein expression in two types of breast cancer cells. In addition, STC2 promoted E-cadherin expression; inhibited vimentin expression; upregulated the phosphorylation levels of the mitogen-activated protein kinase (MAPK) signalling pathway-related proteins p38, JNK, and ERK1/2; and downregulated Akt phosphorylation.

Conclusions: STC2 exerts anti-TNBC activity, inhibits epithelial-mesenchymal transition (EMT), and induces apoptosis by regulating the cell cycle, EMT-related proteins, and MAPK signalling pathway.

Figure 1

Structures of the four detected sea cucumber saponins.

Figure 2

Stichloroside C2 (STC2) can inhibit the proliferation of breast cancer cells. (a) Three breast cancer cell lines, comprising a human breast cancer cell line (MCF-7), human triple-negative breast cancer (TNBC) cell line (MDA-MB-231), and mouse TNBC cell line (4 T1), and a normal epithelial cell line (IOSE-80) were selected and treated with a series of different concentrations of STC2 after treatment for 24 h, the cell viability was detected using the Cell Counting Kit-8 (CCK-8) method. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, compared to the control group. (b, c) Inhibition of TNBC cell clone formation by sea cucumber saponin monomer. Human MDA-MB-231 cells and mouse 4 T1 cells were treated with STC2 at different concentrations (0.25, 0.5, and 1 μM) for 24 h. The cells were cultured for 1–2 weeks and stained with crystal violet. The number of clones formed was detected. ∗∗P < 0.01, ∗∗∗P < 0.001, compared to the control group.

Figure 3

Cell cycle arrest of triple-negative breast cancer (TNBC) induced by sea cucumber saponins. (a) The human TNBC cell line MDA-MB-231 and the mouse TNBC cell line 4 T1 were treated with 0.5 and 1 μM of stichloroside C2 (STC2) for 24 h. The cells were digested and collected, and the cycle changes of the two cells were detected by flow cytometry with propidium iodide (PI). (b) Prism 8.0 was used for statistical analysis of the experimental data, which were expressed as the mean ± SEM of three independent experiments. ∗∗∗P < 0.001, compared to the control group. (c) MDA-MB-231 and 4 T1 cells were treated with different concentrations of STC2 (0.25, 0.5, and 1 μM) for 6 h, and the expression of cycle-related proteins in MDA-MB-231 and 4 T1 cells was detected by western blot. (d) Data are presented as the mean ± SEM of three independent experiments, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, compared to the control group.

Figure 4

Sea cucumber saponins induced DNA damage and apoptosis of triple-negative breast cancer (TNBC) cells. (a) Western blot was used to detect the phosphorylation of H2AX in both cells. (c) MDA-MB-231 and 4 T1 cells were treated with 0.5 and 1 μM stichloroside C2 (STC2) for 24 h. The cells were digested and collected. The Annexin V-FITC apoptosis kit was used to treat the cells. The average fluorescence intensity of Annexin V/PI was determined by flow cytometry. (e) Bax and cleaved PARP protein expression levels were detected by western blot. (b, d, and f) Prism 8.0 was used for the statistical analysis of experimental data, which were expressed as the mean ± SEM of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, compared to the control group.

Figure 5

Stichloroside C2 (STC2) inhibited epithelial–mesenchymal transition (EMT) in triple-negative breast cancer (TNBC) cells. (a) MDA-MB-231 and 4 T1 cells were treated with different concentrations of STC2 (0.25, 0.5, and 1 μM) for 6 h, and the protein expression levels of E-cadherin and vimentin were detected by western blot. (b) Data are presented as the mean ± SEM of three independent experiments, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, compared to the control group.

Figure 6

Effect of stichloroside C2 (STC2) on MAPK pathway-related protein expression in triple-negative breast cancer (TNBC) cells. (a) MDA-MB-231 and 4 T1 cells were treated with different concentrations of STC2 (0.25, 0.5, and 1 μM) for 6 h. Western blotting was used to detect the protein expression and phosphorylation levels of p38, JNK, ERK1/2, and Akt, the main subfamilies of the MAPK signalling pathway family. (b) Data are presented as the mean ± SEM of three independent experiments ∗∗P < 0.01, ∗∗∗P < 0.001, compared to the control group.

Figure 7

Schematic diagram of STC2 inhibiting EMT and inducing apoptosis of TNBC cells through MAPK signalling pathway.