Genistein sensitizes inhibitory effect of tamoxifen on the growth of estrogen receptor-positive and HER2-overexpressing human breast cancer cells

Abstract

Although tamoxifen (TAM) is used for the front-line treatment and prevention of estrogen receptor-positive (ER+) breast tumors, nearly 40% of estrogen-dependent breast tumors do not respond to TAM treatment. Moreover, the positive response is usually of short duration, and most tumors eventually develop TAM-resistance. Overexpression of HER2 gene is associated with TAM-resistance of breast tumor, and suppression of HER2 expression enhances the TAM activity. Soy isoflavone genistein has been shown to have anti-cancer activities and suppress expression of HER2 and ERalpha. The objective of this study was to test the hypothesis that genistein may sensitize the response of ER+ and HER2-overexpressing breast cancer cells to TAM treatment. The combination treatment of TAM and genistein inhibited the growth of ER+/HER2-overexpressing BT-474 human breast cancer cells in a synergistic manner in vitro. Determination of cellular markers indicated that this synergistic inhibitory effect might be contributed in part from combined effects on cell-cycle arrest at G(1) phase and on induction of apoptosis. Further determination of the molecular markers showed that TAM and genistein combination synergistically induced BT-474 cell apoptosis in part by synergistic downregulation of the expression of survivin, one of the apoptotic effectors, and downregulation of EGFR, HER2, and ERalpha expression. Our research may provide a novel approach for the prevention and/or treatment of TAM insensitive/resistant human breast cancer, and warrants further in vivo studies to verify the efficacy of genistein and TAM combination on the growth of ER+/HER2-overexpressing breast tumors and to elucidate the in vivo mechanisms of synergistic actions.

Figure 1

Effects of TAM and genistein on the growth of BT-474 cells in vitro. Cells were treated with the indicated concentrations of TAM and/or genistein dissolved in 1:1 of DMSO and ethanol for 3 d; the cells were counted with Z1 Coulter Particle Counter. Theoretic additive inhibitory values in % of control = % of control for TAM treatment multiplied by % of control for genistein treatment. Results are expressed as mean ± SD, n = 3.

Figure 2

Dose-dependent responses of BT-474 cell at G₁ (A), S (B), and G2/M (C) phases to the treatments in vitro. Cells were treated and cycle distributions were measured by flow cytometry. Results are expressed as mean ± SD, n = 3.

Figure 3

Effects of TAM and genistein on apoptosis of BT-474 cells in vitro. Apoptosis was determined by DNA ladder (A) and DNA fragmentation (B, C) of BT-474 cells following the treatments with TAM and genistein alone or in combination. BT-474 cells were treated with 5 μM TAM, 25 μM genistein, and the TAM (5 μM) and genistein (25 μM) combination for 3 d and genomic DNA was isolated and subject to 1% agarose gel electrophoresis analysis (A). The SD of each data point (less than 10% of the mean) in B and C are not added in the figure for clear presentation.

Figure 4

Effects of TAM and genistein on survivin expression. Both mRNA and protein levels of surviving were measured, and the relative folds of change were the means of signal log ratios (SLR, log₂[treatment expression level/control expression level]) ± standard deviation after being normalized to GAPDH (A). The experiments were duplicated and repeated at least once. Within each method in subpart A, the means without a common superscript letter are significantly different with P values at least <0.05. A representative of protein expression is shown in panel B.

Figure 5

Effects of TAM and genistein treatments on the protein expression of EGFR (A), HER2 (B), and HER3 (C) in BT-474 cells in vitro. BT-474 cells were treated with TAM and genistein alone, or in combination. The proteins were detected by Western blot analysis and quantitated by image analysis. Results are expressed as mean ± SD. Each experiment was duplicated and repeated at least twice. Within each panel, the means without a common superscript letter are significantly different with P values at least <0.05. A representative of protein expression for EGFR, HER2, and HER3 with indicated treatments for 3 d is shown in panel D.

Figure 6

Effects of TAM and genistein treatments on ERα expression in BT-474 cells in vitro. Gene expression was quantitated by real-time PCR. Results are expressed as mean ± SD (A). Each experiment was duplicated and repeated at least twice. Within each time point, the means without a common superscript letter are significantly different with P values at least <0.05. A representative of protein expression for ERα with indicated treatments for 3 d is shown in panel B. [Color figure can be viewed in the online issue, which is available at www.interscience. wiley.com.]