Biochanin A Modulates Cell Viability, Invasion, and Growth Promoting Signaling Pathways in HER-2-Positive Breast Cancer Cells

Abstract

Overexpression of HER-2 receptor is associated with poor prognosis and aggressive forms of breast cancer. Scientific literature indicates a preventive role of isoflavones in cancer. Since activation of HER-2 receptor initiates growth-promoting events in cancer cells, we studied the effect of biochanin A (an isoflavone) on associated signaling events like receptor activation, downstream signaling, and invasive pathways. HER-2-positive SK-BR-3 breast cancer cells, MCF-10A normal breast epithelial cells, and NIH-3T3 normal fibroblast cells were treated with biochanin A (2-100 muM) for 72 hours. Subsequently cell viability assay, western blotting and zymography were carried out. The data indicate that biochanin A inhibits cell viability, signaling pathways, and invasive enzyme expression and activity in SK-BR-3 cancer cells. Biochanin A did not inhibit MCF-10A and NIH-3T3 cell viability. Therefore, biochanin A could be a unique natural anticancer agent which can selectively target cancer cells and inhibit multiple signaling pathways in HER-2-positive breast cancer cells.

Figure 1

Effect of biochanin A on MCF-10A, NIH-3T3, and SK-BR-3 cell viability. SK-BR-3/NIH-3T3 and MCF-10A cells were seeded at 2000 and 5000 cells/well in a 96-well plate. After 8 hours of cell adhesion period, the cells were treated with biochanin A (2–100 μM) for 72 hours. The data indicate a biphasic effect of biochanin A on SK-BR-3 breast cancer cell viability. Biochanin A did not inhibit MCF-10A and NIH-3T3 cell viability. C: control or no treatment; DMSO: vehicle (Statistical analysis: One-way Anova, n = 3, *P < .05).

Figure 2

Effect of biochanin A on HER-2 and p-HER-2 protein expression. (a) SK-BR-3 breast cancer cells were seeded in a T-75 flask in RPMI-1640 cell culture media (10% FBS). Following 72 hours of treatment with biochanin A (2–50 μM) HER-2 and p-HER-2 protein expression was evaluated. The data indicate reduced HER-2 phosphorylation with 50 μM biochanin A (7.93 ± 5.27; P < .05). C: control or no treatment; DMSO: vehicle (Statistical analysis: One-way Anova, n = 3, *P < .05). (b) Representative blot for HER-2 and p-HER-2 protein expression.

Figure 3

Effect of biochanin A on Erk1/2 and p-Erk1/2 protein expression. (a) SK-BR-3 breast cancer cells were seeded in a T-75 flask in RPMI-1640 cell culture media (10% FBS). Following 72 hours of treatment with biochanin A (2–50 μM) Erk1/2 and p-Erk1/2 protein expression was evaluated. The data indicate reduced Erk1/2 phosphorylation at 50 μM biochanin A (31.27 ± 16.71; P < .05). C: control or no treatment; DMSO: vehicle (Statistical analysis: One-way Anova, n = 3, *P < .05). (b) Representative blot for Erk1/2 and p-Erk1/2 protein expression.

Figure 4

Effect of biochanin A on Akt and p-Akt protein expression. (a) SK-BR-3 breast cancer cells were seeded in a T-75 flask in RPMI-1640 cell culture media (10% FBS). Following 72 hours of treatment with biochanin A (2–50 μM) Akt and p-Akt protein expression was evaluated. The data indicate reduced Akt phosphorylation at 50 μM (10.17 ± 7.89; P < .05) biochanin A. C: control or no treatment; DMSO: vehicle (Statistical analysis: One-way Anova, n = 3, *P < .05). (b) Representative blot for Akt and p-Akt protein expression.

Figure 5

Effect of biochanin A on mTOR and p-mTOR protein expression. (a) SK-BR-3 breast cancer cells were seeded in a T-75 flask in RPMI-1640 cell culture media (10% FBS). Following 72 hours of treatment with biochanin A (2–50 μM) mTOR and p-mTOR protein expression was evaluated. The data indicate reduced mTOR phosphorylation at 50 μM biochanin A (42.26 ± 12.18; P < .05). C: control or no treatment; DMSO: vehicle (Statistical analysis: One-way Anova, n = 3, *P < .05). (b) Representative blot for mTOR and p-mTOR protein expression.

Figure 6

Effect of biochanin A on NFκB protein expression. (a) SK-BR-3 breast cancer cells were seeded in a T-75 flask in RPMI-1640 cell culture media (10% FBS). Following 72 hours of treatment with biochanin A (2–50 μM) NFκB protein expression was evaluated. The data indicate reduced NFκB expression at 20 and 50 μM biochanin A (Biochanin A: 20 μM  → 49.93 ± 5.41; 50 μM  → 44.53 ± 6.44; P < .05). C: control or no treatment; DMSO: vehicle (Statistical analysis: One-way Anova, n = 3, *P < .05). (b) Representative blot for NFκB protein expression.

Figure 7

Effect of biochanin A on MMP-2 and MMP-9 activity. (a) SK-BR-3 breast cancer cells were seeded in a T-75 flask in RPMI-1640 cell culture media (10% FBS) for 24 hours. Subsequently they were treated for 72 hours with biochanin A (20 and 50 μM) in serum free conditions and MMP-2 and MMP-9 gelatinase enzyme activity was evaluated. The data indicate reduced MMP-9 activity with 50 μM biochanin A (2.28 ± 2.28; P < .05). C: control or no treatment; DMSO: vehicle; PL: protein ladder (Statistical analysis: One-way Anova, n = 3, *P < .05). (b) Representative gelatin gel for MMP-2 and MMP-9 activity.

Figure 8

Effect of biochanin A on MT-MMP1 protein expression. (a) SK-BR-3 breast cancer cells were seeded in a T-75 flask in RPMI-1640 cell culture media (10% FBS). Following 72 hours of treatment with biochanin A (2–50 μM) MT-MMP1 protein expression was evaluated. Our data indicate reduced MT-MMP-1 expression at 10 and 50 μM biochanin A (Biochanin A: 10 μM  → 19.22 ± 9.93; 50 μM  → 2.39 ± 2.30; P < .05). C: control or no treatment; DMSO: vehicle (Statistical analysis: One-way Anova, n = 3, *P < .05). (b) Representative blot for MT-MMP-1 protein expression.

Figure 9

Pathways inhibited by biochanin A in HER-2+ SK-BR-3 breast cancer cells. The protein expression data indicate that biochanin A treatment inhibits growth (p-HER-2, p-Erk), survival (Akt, mTOR, and NFκB), and invasion (MMP-9 and MMP-14) promoting pathways in SK-BR-3 cells.