ZLM-7 Blocks Breast Cancer Progression by Inhibiting MDM2 via Upregulation of 14-3-3 Sigma

Abstract

Breast cancer is one of the most prevalent malignancies with poor prognosis. Inhibition of angiogenesis is becoming a valid and evident therapeutic strategy to treat cancer. Recent studies uncovered the antiangiogenic activity of ZLM-7 (a combretastain A-4 derivative), but the regulatory mechanism is unclear. ZLM-7 treatment was applied in estrogen receptor-positive cell MCF-7, triple-negative breast cancer cell MDA-MB-231 and xenograft models. Transfections were conducted to overexpress or knockdown targeted genes. The gene and protein expressions were measured by qPCR and Western blotting assay, respectively. Cell proliferation and apoptosis were evaluated using the CCK8 method, clone formation assay and flow cytometry. We found that ZLM-7 upregulated 14-3-3 sigma expression but downregulated MDM2 expression in breast cancer cells. ZLM-7 delayed cell proliferation, promoted apoptosis and blocked cell-cycle progression in human breast cancer cells in vitro, while those effects were abolished by 14-3-3 sigma knockdown; overexpression of 14-3-3 sigma reproduced the actions of ZLM-7 on the cell cycle, which could be reversed by MDM2 overexpression. In xenograft models, ZLM-7 treatment significantly inhibited tumor growth while the inhibition was attenuated when 14-3-3 sigma was silenced. Collectively, ZLM-7 could inhibit MDM2 via upregulating 14-3-3 sigma expression, thereby blocking the breast cancer progression.

Keywords: 14-3-3 sigma; MDM2; ZLM-7; breast cancer; cell cycle.

Figure 1

ZLM-7 upregulated 14-3-3 sigma expression but downregulated MDM2 expression in breast cancer cells. (A,B). Relative gene expressions of 14-3-3 sigma and MDM2 in MCF-10A, MCF-7 and MDA-MB-231 cells measured by qPCR. (C,D). Relative gene expression of 14-3-3 sigma and MDM2 with or without ZLM-7 treatment. Gene expressions were measured by qPCR. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

ZLM-7 suppressed cell proliferation and promoted apoptosis by upregulating 14-3-3 sigma in breast cancer cells. (A) Relative gene expression of 14-3-3 sigma in MCF-7 and MDA-MB-231 cells transfected with either sh-NC or sh-14-3-3 sigma measured by qPCR. (B) Relative protein level of 14-3-3 sigma in MCF-7 and MDA-MB-231 cells transfected with either sh-NC or sh-14-3-3 sigma measured by Western blotting. (C) Cell viability at 24 h, 48 h and 72 h measured by CCK8 assay. (D) Cell proliferation ability assessed by clone-formation assay in MCF-7 and MDA-MB-231 cells transfected with either sh-NC or sh-14-3-3 sigma. (E) Cell apoptosis analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

ZLM-7 blocked cell-cycle progression via upregulating 14-3-3 sigma. (A) Cell-cycle analyzed via flow cytometry. (B) Relative protein levels of cell-cycle regulatory proteins measured by Western blotting. MCF-7 and MDA-MB-231 cells transfected with either sh-NC or sh-14-3-3 sigma were treated with ZLM-7 for 8 h. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

14-3-3 sigma could negatively regulate the expression of MDM2 in breast cancer cells. (A) Relative gene expression of 14-3-3 sigma in MCF-7 and MDA-MB-231 cells transfected with either pcDNA3.1 or p-14-3-3 sigma measured by qPCR. (B) Relative protein level of 14-3-3 sigma in MCF-7 and MDA-MB-231 cells transfected with either pcDNA3.1 or p-14-3-3 sigma measured by Western blotting. (C) Relative gene expression of MDM2 in MCF-7 and MDA-MB-231 cells transfected with either pcDNA3.1 or p-14-3-3 sigma measured by qPCR. (D) Relative protein level of MDM2 in MCF-7 and MDA-MB-231 cells transfected with either pcDNA3.1 or p-14-3-3 sigma measured by Western blotting. * p < 0.05, ** p < 0.01.

Figure 5

Overexpressed 14-3-3 sigma inhibited cell proliferation and promoted apoptosis by reducing MDM2 expression in breast cancer cells. (A) Relative gene expression of MDM2 in MCF-7 and MDA-MB-231 cells transfected with either pcDNA3.1 or p-MDM2 measured by qPCR. (B) Relative protein level of MDM2 in MCF-7 and MDA-MB-231 cells transfected with either pcDNA3.1 or p-MDM2 measured by Western blot. (C) Cell viability at 24 h, 48 h and 72 h measured by CCK8 assay. (D) Cell proliferation ability assessed by clone formation assay in MCF-7 and MDA-MB-231 cells transfected with pcDNA3.1 or p-14-3-3 sigma or co-transfected with p-14-3-3 sigma and p-MDM2. (E) Cell apoptosis analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

14-3-3 sigma blocked cell-cycle progression by suppressing MDM2 expression in breast cancer cells. (A) Cell-cycle analyses via flow cytometry. (B) Relative protein levels of cell-cycle regulatory proteins measured by Western blot. MCF-7 and MDA-MB-231 cells transfected with pcDNA3.1 or p-14-3-3 sigma or co-transfected with p-14-3-3 sigma and p-MDM2. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

ZLM-7 suppressed tumor growth via regulating 14-3-3 sigma/MDM2 axis in vivo. Mice were inoculated subcutaneously with breast cancer cells or cells transfected with either sh-14-3-3 sigma or sh-NC. (A) Xenografts harvested from the animals; (B,C) Tumor volume and weight with ZLM-7 treatment for 28 days. (D) Relative gene expressions of 14-3-3 sigma and MDM2 in xenografts measured by qPCR. (E) Relative protein levels of 14-3-3 sigma, MDM2 and cell-cycle regulatory proteins in xenografts detected by Western blotting. * p < 0.05, ** p < 0.01, *** p < 0.001.