Synergistic Anti-Cancer Effects of ERB-041 and Genistein through Estrogen Receptor Suppression-Mediated PI3K/AKT Pathway Downregulation in Canine Mammary Gland Tumor Cells

Abstract

Canine-mammary-gland tumors (CMTs) are prevalent in female dogs, with approximately 50% of them being malignant and often presenting as inoperable owing to their size or metastasis. Owing to poor outcomes, effective alternatives to conventional chemotherapy for humans are necessary. Two estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), which act in opposition to each other, are involved, and CMT growth involves ERα through the phosphoinositide 3-kinases (PI3K)/AKT pathway. In this study, we aimed to identify the synergistic anti-cancer effects of ERB-041, an ERβ agonist, and genistein, an isoflavonoid from soybeans known to have ERβ-specific pseudo-estrogenic actions, on CMT-U27 and CF41.Mg CMT cell lines. ERB-041 and genistein synergistically inhibited cell proliferation and increased the number of annexin V-positive cells in both cell lines. Furthermore, we observed a synergistic increase in the Bax/Bcl-2 ratio and cleaved caspase-3 expression. Additionally, cell-cycle arrest occurred through the synergistic regulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). We also found a synergistic decrease in the expression of ERα, and the expression of proteins involved in the PI3K/AKT pathway, including p-PI3K, phosphatase and tensin homolog (PTEN), AKT, and mechanistic target of rapamycin (mTOR). In conclusion, ERB-041 and genistein exhibited a synergistic anticancer effect on CMTs, suggesting that cotreatment with ERB-041 and genistein is a promising treatment for CMTs.

Keywords: ERB-041; PI3K/AKT pathway; canine-mammary-gland tumors; estrogen receptor; genistein.

Figure 1

ERB-041 and genistein synergistically suppress the proliferation of CMT cells. (A,B) Cell viability at indicated concentrations of ERB-041 and genistein in CMT-U27 (A) and CF41.Mg (B); (C,D) morphological images of CMT-U27 (C) and CF41.Mg (D); (E,F) synergistic cell viability reduction in CMT-U27 (E) and CF41.Mg (F) following treatment with ERB-041 and genistein at the indicated concentrations. Magnification, 100×. Values are mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001 versus untreated cells by one-way ANOVA followed by Bonferroni post hoc test.

Figure 2

ERB-041 and genistein synergistically induced apoptosis in CMT cells. (A,B) Images of Annexin-V/PI staining results of CMT-U27 (A) and CF41.Mg (B) cells treated with ERB-041 and genistein at the indicated concentrations.

Figure 3

ERB-041 and genistein are involved in the regulation of Bcl-2, Bax, and caspase-3. (A,B) Western blot images and quantification of the expression of Bcl-2, Bax (A), caspase-3, and cleaved caspase-3 (B) in CMT-U27 upon ERB-041 and genistein treatment at the indicated concentrations and the expression of Bcl-2, Bax (C), caspase-3, and cleaved caspase-3 (D) in CF41.Mg.; (E–H) immunocytochemistry images and quantitative analysis of activated caspase-3 in CMT-U27 (E,F) and CF41.Mg (G,H). Cas-3, caspase-3. Scale bar, 50 µm. Values are mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001 versus untreated cells by one-way ANOVA followed by Bonferroni post hoc test.

Figure 4

ERB-041 and genistein induce cell-cycle arrest in CMT cells through synergistic regulation of CDK4 and cyclin D1. (A–C) Western blotting images and quantification of CDK4 (A,B) and cyclin D1 (A,C) following treatment with indicated concentrations of ERB-041 and genistein in CMT-U27; (D,E) cell-cycle-arrest assay data by flow cytometry (D) and quantitative analysis (E) in CMT-U27; (F–H) Western blotting images and quantification of CDK4 (F,G) and cyclin D1 (F,H) after treatment with ERB-041 and genistein at the concentrations indicated in CF41.Mg; (I,J) cell-cycle-arrest assay data by flow cytometry (I) and quantitative analysis (J) in CF41.Mg. CDK4, cyclin-dependent kinase 4. Values are mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001 versus untreated cells by one-way ANOVA followed by Bonferroni post hoc test.

Figure 5

Effects of ERB-041 and genistein treatment on the expression of ERβ and ERα. (A,B) Western blot images and quantitative analysis of ERβ and ERα upon treatment with ERB-041 and genistein at the concentrations indicated in CMT-U27 (A) and CF41.Mg (B); (C,D) immunocytochemistry images and quantification of ERβ and ERα in CMT-U27 (C) and CF41.Mg (D). Scale bar, 50 µm. ERβ, estrogen receptor beta; ERα, estrogen receptor alpha. Values are mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001 versus untreated cells by one-way ANOVA followed by Bonferroni post hoc test.

Figure 6

Western blotting results demonstrate that ERB-041 and genistein synergistically regulate the expression of the PI3K/AKT pathway. (A–D) Western blot images and quantification of p-PI3K (A), PTEN, p-PTEN (B), AKT, p-AKT (C), mTOR, and p-mTOR (D) expression in CMT-U27 following ERB-041 and genistein treatment; (E–H) Western blot images and quantification of the expression of PI3K/AKT-pathway-related proteins in CF41.Mg of ERB-041 and genistein at the indicated concentrations. PI3K, phosphoinositide 3-kinases; PTEN, phosphatase and tensin homolog; mTOR, mechanistic target of rapamycin. Values are mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001 versus untreated cells by one-way ANOVA followed by Bonferroni post hoc test.

Figure 7

Immunocytochemistry results demonstrate that ERB-041 and genistein synergistically reduce the expression of the PI3K and PTEN. (A–D) Immunocytochemistry images and quantitative analysis of the p-PI3K expression in CMT-U27 (A) and CF41.Mg (B) when treated with ERB-041 and genistein at the indicated concentrations and PTEN expression in CMT-U27 (C) and CF41.Mg (D). PI3K, phosphoinositide 3-kinases; PTEN, phosphatase and tensin homolog. Scale bar, 50 µm. Values are mean ± SD. ** p < 0.01; *** p < 0.001 versus untreated cells by one-way ANOVA followed by Bonferroni post hoc test.