Dexamethasone-tamoxifen combination exerts synergistic therapeutic effects in tamoxifen-resistance breast cancer cells

Abstract

Tamoxifen (TAM) is a key player in estrogen receptor-positive (ER+) breast cancer (BC); however, ∼30% of patients experience relapse and a lower survival rate due to TAM resistance. TAM resistance was related to the over expression of SOX-2 gene, which is regulated by the E2F3 transcription factor in the Wnt signaling pathway. It was suggested that SOX-2 overexpression was suppressed by dexamethasone (DEX), a glucocorticoid commonly prescribed to BC patients. The aim of the present study is to explore the effect of combining DEX and TAM on the inhibition of TAM-resistant LCC-2 cells (TAMR-1) through modulating the E2F3/SOX-2-mediated Wnt signaling pathway. The effect of the combination therapy on MCF-7 and TAMR-1 cell viability was assessed. Drug interactions were analyzed using CompuSyn and SynergyFinder softwares. Cell cycle distribution, apoptotic protein expression, gene expression levels of SOX-2 and E2F3, and cell migration were also assessed. Combining DEX with TAM led to synergistic inhibition of TAMR-1 cell proliferation and migration, induced apoptosis, reduced SOX-2 and E2F3 expression and was also associated with S and G2-M phase arrest. Therefore, combining DEX with TAM may present an effective therapeutic option to overcome TAM resistance, by targeting the E2F3/SOX-2/Wnt signaling pathway, in addition to its anti-inflammatory effect.

Keywords: E2F3; SOX-2; breast cancer; dexamethasone; synergistic effect; tamoxifen-resistance.

Figure 1. Anticancer activity of DEX and TAM on MCF-7 and TAMR-1 cells

MCF-7 cells were seeded into 96-well plate and treated with 39.25–117.74 μg/ml of DEX and 0.93–11.15 μg/ml of TAM (A). TAMR-1 was treated with 39.25–117.74 μg/ml DEX and 12.38–37.15 μg/ml TAM (B). Cells were then incubated for 48 h and stained with SRB. The inhibitory effect of each drug was assessed through determining the percentage inhibition, and IC50 values were identified. Percentage inhibition = 100 - (100 × optical density (treated cells)/optical density [untreated cells]).

Figure 2. Inhibitory effect of DEX and TAM combination on TAMR-1 cells

Percentage inhibition of TAMR-1 cells treated with increasing concentrations of DEX and TAM in a constant 3:1 ratio (A) for 48 h. The shade of red represents the amount of inhibition of TAMR-1 cells. (B,C) 2D and 3D synergism visualization showing the presence of synergy between DEX and TAM combination therapy. SynergyFinder 3.0 software was used to visualize the percentage inhibition of DEX and TAM combination therapy as a dose-response matrix, and the 2D and 3D visualization of synergy between DEX and TAM. The synergy score was calculated using the HSA reference model. (D) A Normalized Isobologram represents the fraction affected (Fa) and the CI values of different DEX and TAM combinations. CompuSyn software was used to confirm the presence of synergy between DEX and TAM through calculating the CI. CI < 1 suggests a synergistic effect, CI = 1 suggests an additive effect, and CI > 1 suggests an antagonistic effect.

Figure 3. Western blotting assay of TAMR-1 cells

Western blotting assay of TAMR-1 cells treated with 39.25 μg/ml DEX, 12.38 μg/ml TAM, or their combination for 48 h. (A) Western blotting images of Bax and BCL-xL expression levels in TAMR-1 cells. (B) Bar graph comparing the expression levels of Bax and BCL-xL. Data were normalized against β-actin and expressed as a fold change compared with control. (C) Bar graph representing Bax to BCL-xL ratio. Data is presented as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by a Tukey’s post-hoc test. **** Significant at P<0.0001.

Figure 4. Cell cycle distribution of TAMR-1 cells

Cell cycle distribution of TAMR-1 cells exposed to 39.25 DEX, 12.38 TAM, or their combination for 48 h. (A) Flow cytometry results showing percentage TAMR-1 cells arrested at each cell cycle phase (G0-G1, purple; S, green; G2-M, red). (B) Representative bar graph comparing percentage of cell cycle distribution in control and treated cells.

Figure 5. Wound healing assay of TAMR-1 cells

Wound healing assay of TAMR-1 cells treated with 39.25 μg/ml DEX, 12.38 μg/ml TAM, or their combination for 48 h (A). Crystal violet was used as a staining agent. Representative bar graph showing the quantitative analysis of TAMR-1 cell migration presented as mean ± SD (B). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by a Tukey’s post-hoc test. **** Significant at P<0.0001, ***P<0.001, **P<0.01, ns = no significance.

Figure 6. RT-qPCR analysis of SOX-2 and E2F3 mRNA expression in TAMR-1 cells

RT-qPCR analysis of SOX-2 and E2F3 mRNA expression in TAMR-1 cells treated with 39.25 μg/ml DEX, 12.38 μg/ml TAM, or their combination for 48 h. Quantitative PCR results were normalized against GAPDH and expressed as a fold change. Data are acquired from three independent experiments and are presented as mean ± SD. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by a Tukey’s post-hoc test. (a, b, c, and d represent significance from TAMR-1, MCF-7, DEX, and TAM, respectively, at P<0.01).