Table 1.
Half inhibitory concentration (IC50) values of IVM in breast cancer cell lines at 24-h treatment.
Fig 1.
IVM reduced ERα and HER2 in ER⁺ and endocrine-resistant cell lines.
Additionally, IVM decreased cyclin D1 levels in ER+ cells: MCF-7, MCF-7/LCC2, MCF-7/LCC9. (A) Protein levels were evaluated by Western blotting after treating cells with various concentrations of IVM for 24 h. The graphs demonstrated the inhibitory effect of IVM on the protein levels of (B–D) ERα, (E–G) HER2, (H-I) pHER2, and (K-L) Cyclin D1. The data were shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001 compared with the nontreatment control (n = 3)..
Fig 2.
IVM inhibited estrogen-induced cell proliferation in ER⁺ breast cancer cell lines: MCF-7 and T-47D.
MTT assay measured cell viability of ER-positive breast cancer cells: (A) MCF-7 and (B) T-47D, were treated with (+E2) or without (−E2) 17β-estradiol at physiologic level (10 nM E2) in the presence of nontoxic concentrations of IVM (1, 3, 5 μM) or 4-OHT (5, 10 μM) as a positive control for 5 days. (C–E) The inhibitory effect of IVM on ERα and HER2 protein levels of ER-positive breast cancer was performed after the treatments with various concentrations of IVM with (+E2) or without (−E2) 17β-estradiol (10 nM E2) for 24 h in MCF-7. (F) mRNA expression of the ESR1 gene (ERα) was determined by qPCR analysis in MCF-7 cells. The graphs showed the mean ± SEM. The blue and red asterisks highlight the significant differences in comparison to non-treatment without (−E2) and with (+E2), respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus the non-treatment control (n = 3).
Fig 3.
Combined treatment of IVM and 4-OHT further reduced cell viability and ERα and HER2 levels in ER⁺ and endocrine-resistant breast cancer cell lines.
IC25 values of IVM and/or 4-OHT-treated concentrations were obtained from MTT assays. Cell viability was evaluated in three MCF-7 breast cancer cell lines: (A) MCF-7, (B) MCF-7/LCC2, and (C) MCF-7/LCC9 at 24 h. The graphs showed the mean ± SEM at each treated concentration. **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with nontreatment control by one-way ANOVA (n = 3). (D) Western blots were performed in three MCF-7 breast cancer cell lines after the treatments with concentrations at IC25 values of IVM and/or 4-OHT for 24 h. The fold change on the protein level of ERα was assessed in (E) MCF-7, (F) MCF-7/LCC2, and (G) MCF-7/LCC9 cells and HER2 in (H) MCF-7, (I) MCF-7/LCC2, and (J) MCF-7/LCC9 cells. The graphs displayed the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, comparing between two groups (n = 3).
Fig 4.
IVM inhibited pSMAD2 and pERK involved in the TGF-β signaling pathway in ER-positive and endocrine-resistant breast cancer cell lines: MCF-7, MCF-7/LCC2, MCF-7/LCC9.
(A) The protein levels were determined by Western blot after the treatment with various concentrations of IVM for 24 h. The graphs demonstrated the inhibitory effect of IVM as the fold change in protein levels of (B–D) pSMAD2/SMAD2 and (E–G) pERK/ERK in MCF-7, MCF-7/LCC2, and MCF-7/LCC9 cells, respectively. The data were shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001 compared with the nontreatment control (n = 3).