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. 2017 Feb 16;17(1):115.
doi: 10.1186/s12906-017-1621-7.

BreastDefend enhances effect of tamoxifen in estrogen receptor-positive human breast cancer in vitro and in vivo

Shujie Cheng  1   2 , Victor Castillo  1 , Matt Welty  1 , Mark Alvarado  1 , Isaac Eliaz  3 , Constance J Temm  4 , George E Sandusky  4 , Daniel Sliva  5   6   7
Affiliations

BreastDefend enhances effect of tamoxifen in estrogen receptor-positive human breast cancer in vitro and in vivo

Shujie Cheng et al. BMC Complement Altern Med. .

Abstract

Background: Tamoxifen (TAM) has been widely used for the treatment of estrogen receptor (ER)-positive breast cancer and its combination with other therapies is being actively investigated as a way to increase efficacy and decrease side effects. Here, we evaluate the therapeutic potential of co-treatment with TAM and BreastDefend (BD), a dietary supplement formula, in ER-positive human breast cancer.

Methods: Cell proliferation and apoptosis were determined in ER-positive human breast cancer cells MCF-7 by MTT assay, quantitation of cytoplasmic histone-associated DNA fragments and expression of cleaved PARP, respectively. The molecular mechanism was identified using RNA microarray analysis and western blotting. Tumor tissues from xenograft mouse model were analyzed by immunohistochemistry.

Results: Our data clearly demonstrate that a combination of 4-hydroxytamoxifen (4-OHT) with BD lead to profound inhibition of cell proliferation and induction of apoptosis in MCF-7 cells. This effect is consistent with the regulation of apoptotic and TAM resistant genes at the transcription and translation levels. Importantly, TAM and BD co-treatment significantly enhanced apoptosis, suppressed tumor growth and reduced tumor weight in a xenograft model of human ER-positive breast cancer.

Conclusion: BD sensitized ER-positive human breast cancer cells to 4-OHT/TAM treatment in vitro and in vivo. BreastDefend can be used in an adjuvant therapy to increase the therapeutic effect of tamoxifen in patients with ER-positive breast cancer.

Keywords: Apoptosis; BreastDefend; Estrogen receptor; MCF-7; Polybotanical supplement; Tamoxifen; Xenograft model.

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Figures

Fig. 1
Fig. 1
Effect of BD on the normal MCF-10A and breast cancer MCF-7 and MDA-231 cells. a MCF-10A, MCF-7 and MDA-231 cells were seeded and treated with BD (0–50 μg/ml) for 3 days. b, c MCF-7 cells were stripped of steroids for 3 days before seeding by culturing in steroid-free medium. After 24 h seeded into 96-well plates, cells were treated with E2 (10 nM) plus 4-OHT (1 μM), BD (0–50 μg/ml) or combination of 4-OHT and BD for b 3 days and c 6 days, respectively. Cell proliferation was determined by MTT assay. Each bar represents the mean ± SD of triplicate. Similar results were obtained in three independent experiments. Statistical analysis by ANOVA and Holm-Sidak. a * P < 0.05 BD vs control (0 μg/ml) for different cell lines. b, c * P < 0.05 BD vs control (0 μg/ml) in E2 group, # P < 0.05 BD vs control (0 μg/ml) in E2 + 4-OHT group, different letters above bars indicate significant differences P < 0.05 for the same concentration of BD
Fig. 2
Fig. 2
Effect of 4-OHT and BD on apoptosis of MCF-7 human breast cancer cells. MCF-7 cells were seeded and treated as described in Fig. 1c for 6 days. Apoptosis was evaluated by Cell Death Detection ELISA (a) and western blotting for the expression of c-PARP (b). Representative blots show expression of c-PARP and β-actin was used as loading control. Three independent experiments were done for the western blot studies and quantitative data with statistical analysis were shown below the representative blot image (c). Statistical analysis by ANOVA and Holm-Sidak. Different letters above bars indicate significant differences P < 0.05. The graphical data represent mean +/− SD
Fig. 3
Fig. 3
Combination of 4-OHT with BD regulates expression of apoptosis related proteins. a MCF-7 cells were treated for 6 days as described in Fig. 1. Whole protein extracts isolated from cells were prepared and western blot analysis with anti-Raf-B, anti-p21, anti-Bcl-2, anti-Fibronectin and anti-β-actin antibodies were performed as described in Materials and Methods. β-actin was used as loading control and representative blots from three experiments were shown. b Quantitative data composed of all the experiments in MCF-7 cells with statistical analysis were on the right of the representative blot image. Statistical analysis by ANOVA and Holm-Sidak. Different letters above bars indicate significant differences P < 0.05. The graphical data represent mean +/− SD
Fig. 4
Fig. 4
Inhibition of human breast tumor growth by TAM, BD, and TAM and BD combination in vivo. a Xenograft experiments were performed as described in Materials and Methods. During the treatment period, tumor sizes were measured 3 times per week. Statistical analysis by ANOVA and Holm-Sidak. *P < 0.05: control vs TAM, control vs. BD, control vs, BD + TAM. (n = 16-24 tumors per group). b Tumor sizes at the beginning (Day1) and the end (Day 29) of the treatment. Statistical analysis by ANOVA and Holm-Sidak. Different letters above bars indicate significant differences P < 0.05. Box plots represent 5th/10th percentiles, mean (white dotted line), horizontal bars represent median values, whiskers indicate minimum to maximum values and triangles represent outliers. c At the end of the experiment (Day 29), tumors were harvested and weighed. Statistical analysis by ANOVA and Holm-Sidak. Different letters above bars indicate significant differences P < 0.05. Box plots represent 5th/10th percentiles, mean (white dotted line), horizontal bars represent median values, whiskers indicate minimum to maximum values and triangles represent outliers
Fig. 5
Fig. 5
Induction of apoptosis in human breast tumor xenografts. a Representative H&E staining of apoptotic bodies in MCF-7 human breast tumors, b quantification was determined as described in Materials and Methods. Statistical analysis by ANOVA and Holm-Sidak. Different letters above bars indicate significant differences P < 0.05. The graphical data represent mean +/− SD. (n = 5–10)
Fig. 6
Fig. 6
Combination of TAM with BD regulates expression of apoptosis and TAM resistance related proteins in human breast tumors. Animal experiments were performed as described in Fig. 3. Paraffin-embedded tumor tissue sections were analyzed by immunohistochemistry using antibodies against BRAF, p21, Bcl-2 and Fibronectin (FN). Representative localization and intensity of immunoreactivities against all primary antibodies are shown. Immunohistochemical quantification of BRAF, p21, Bcl-2 and FN were determined as described in Materials and Methods. Statistical analysis by ANOVA and Holm-Sidak. Different letters above bars indicate significant differences P < 0.05. The graphical data represent mean (white dotted line) +/− SD, triangles represent outliers. (n = 10)
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