Pomegranate sensitizes Tamoxifen action in ER-α positive breast cancer cells

Abstract

It is estimated that one in eight women will be affected with cancer during their lives, which means over 1 million women worldwide will be diagnosed with breast cancer in the year of 2011. Roughly, 70% of breast cancer will be estrogen receptor-alpha (ER-α) positive. The presence of ER-α is associated with better prognosis and is able to determine if tumors will respond to the estrogen-blocking/ER-antagonist drug Tamoxifen (TAM). However, a significant fraction of ER-positive tumors respond with minimal or no response to TAM. It is unclear why some breast cancer cells resist TAM and how to make these cells respond. Early evidence suggests Pomegranate fruit extracts (PFEs) exhibit an anticancer effect against some cancers. The objective of the study was to determine whether PFEs may able to enhance/sensitize the TAM's effect in ER-positive MCF-7 breast cancer cells. To test the hypothesis, we determined the effect of PFEs on sensitive and TAM-resistant-MCF-7 cell viability and cell death in the presence or absence of TAM under estrogenic or non-estrogenic culture environment. The present studies demonstrated that PFEs enhance the TAM action in both sensitive and TAM-resistant MCF-7 cells through the inhibition of cell viability (regular or estrogen-induced) by inducing cell-death machinery. Collectively, the results showed for the first time that pomegranate combined with TAM may represent a novel and a powerful approach to enhance and sensitize TAM action.

Fig. 1

The additive effect of PFEs on TAM-induced inhibition of mitogenic action of estrogen on MCF-7 cells. The bar graph represents the cell proliferation of MCF-7 cells before and after the treatment of 17β-E2 (10 nM), TAM (1 μM) or PFEs (300 μM) alone or in combination for 48 h. Untreated cells were exposed to vehicle controls (DMSO). Data are displayed as means ± SE of at least four independent experiments. P values were determined by a Student’s t test

Fig. 2

Time-dependent effect of PFEs on TAM-induced inhibition of mitogenic action of estrogen on MCF-7 cells. MCF-7 cells were treated with or 17β-E2 (10 nM), TAM (1 μM) or PFEs (300 μg/ml) alone or in combination for different times and cell counts were determined using automated cell counter (Nexcelom) as described in the Materials and methods section. Data are shown as the means of three separate experiments; bars represent SE. *Significantly different from treatment with 17β-E2 (p < 0.001 by a Student’s t test.). ^#significantly different from treatment with Untreated (p < 0.05 by a Student’s t test)

Fig. 3

The additive effect of PFEs on TAM-induced regulation of cell cycle phases in estrogen treated MCF-7 cells. The bar graph illustrates the effect of TAM (1 μM) or PFEs (300 μg/ml) or combination of TAM and PFEs on cell-cycle distribution in 17β-E₂-treated (10nM) and untreated MCF-7 cells. The cell cycle phases were analyzed in DAPI-stained MCF-7 cells using flow cytometry. Data are shown as the means of three separate experiments; bars represent SE. *Significantly different from treatment with 17β-E2 (p < 0.01 by a Student’s t test)

Fig. 4

The additive effect of PFEs on TAM-induced apoptosis in estrogen treated MCF-7 cells. a. Annexin V: FITC assay for the detection of early (green) and late apoptosis (red) in MCF-7 cells. The upper panel (left and right side) represents without or with 17β-E2 treated cells respectively. The lower panel (left and right side) represents the cells treated with 17β-E2 and Tam and 17β-E2, Tam and PFE together, respectively. b. Immunoblot analysis represents the expression of Bcl2 and Bax protein. β-actin was used for loading control. The left bar graph represents the Bcl-2/β-actin or BAX/β-actin ratio while right bar graph exhibits the ration of Bcl-2 and BAX in treated and untreated samples. Data are shown as the means of three separate experiments; bars represent SE. *Significantly different from treatment with Untreated (p < 0.05 by a Student’s t test.), ^#significantly different from treatment with 17β-E2 (p < 0.005 by a Student’s t test.) and ** significantly different from treatment with 17β-E2 (p < 0.001 by a Student’s t test)

Fig. 5

PFEs sensitize TAM action on TAM-resistance MCF-7 cells. a. Photographs are showing the morphological difference between Tamoxifen sensitive and resistant MCF-7 cells. b. A diagrammatic representation of the experiments and c. The bar graph represents the cell proliferation assay in Tamoxifen-resistant MCF-7 cells treated with TAM in the presence or absence of PFEs for 72 h. Data are shown as the means of three separate experiments; bars represent SE. p values were determined by a Student’s t test. * indicates a p of <0.01 vs untreated or control; ** indicate a p value of <0.001 vs PFE