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, 149 (11), 5366-73

Low Concentrations of the Soy Phytoestrogen Genistein Induce Proteinase Inhibitor 9 and Block Killing of Breast Cancer Cells by Immune Cells

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Low Concentrations of the Soy Phytoestrogen Genistein Induce Proteinase Inhibitor 9 and Block Killing of Breast Cancer Cells by Immune Cells

Xinguo Jiang et al. Endocrinology.

Abstract

The risks and benefits of diets and supplements containing the estrogenic soy isoflavone genistein are not well established. We report that 10 nm genistein potently induces the granzyme B inhibitor, proteinase inhibitor 9 (PI-9) in MCF-7 human breast cancer cells. By inducing PI-9, genistein inhibits the ability of human natural killer (NK) cells to lyse the target breast cancer cells. In ERalphaHA cells, stably transfected MCF-7 cells, which contain elevated levels of estrogen receptor-alpha (ERalpha), 100 pm genistein or 17beta-estradiol potently induce PI-9 and prevent NK cells from killing the target breast cancer cells. The concentrations of genistein that fully induce PI-9 in MCF-7 cells, and in ERalphaHA cells, are far lower than those previously reported to elicit estrogenic responses through ERalpha. Because 4-hydroxytamoxifen, raloxifene, and ICI 182,780/Faslodex all block genistein induction of PI-9 and elevated levels of ERalpha enhance induction of PI-9, genistein acts via ERalpha to induce PI-9. Increasing levels of ERalpha in breast cancer cells results in a progressive increase in induction of PI-9 by genistein and in the cell's ability to evade killing by NK cells. Moderate levels of dietary genistein and soy flour effectively induce PI-9 in human breast cancers grown in ovariectomized athymic mice. A significant population consumes levels of genistein in soy products that may be high enough to induce PI-9, perhaps potentiating the survival of some preexisting breast cancers by enabling them to evade immunosurveillance.

Figures

Figure 1
Figure 1
Low concentrations of genistein induce PI-9 mRNA in MCF-7 cells. A, Dose-response curves for phytoestrogen induction of PI-9 and pS2 mRNAs. For the PI-9 study, RNA was isolated 4 h after addition of the indicated concentrations of E2, genistein (Gen), equol (−), and coumestrol to the cell culture medium. To analyze pS2 induction (dashed line), RNA was isolated 24 h after genistein treatment. The level of mRNA in cells maintained in ethanol vehicle was set equal to 1. B, Effect of SERMs on induction of PI-9 mRNA. The medium contained ethanol vehicle (control) or 10 nm genistein (Gen). The SERMs OHT, raloxifene (RAL), and ICI 182,780 (ICI) were present at 1000 nm in the presence or absence of 10 nm genistein. Cells were harvested at 4 h, and RNA was isolated and analyzed for PI-9 mRNA by quantitative RT-PCR. The data represent the mean ± sem for at least three separate experiments.
Figure 2
Figure 2
Increasing levels of ERα in breast cancer cells enhance genistein’s potency and efficacy as an inducer of PI-9. A and B, In ERαHA cells, increased concentrations of DOX induce increased levels of ERα mRNA and protein. The cells were maintained in medium lacking DOX (Con) or containing 0.5 μg/ml (Dox 0.5) or 1.0 μg/ml (Dox 1) DOX . RNA was isolated at 48 h, and quantitative RT-PCR was carried out (A) or cell extracts were prepared and analyzed for ERα protein level by Western blotting using calnexin as internal standard (B). C and D, Dose-response curves for induction of PI-9 mRNA in cells expressing different levels of ERα. RNA was isolated 24 h after addition of 0.5 μg/ml DOX alone (C) or 1.0 μg/ml DOX alone (D) plus the indicated concentrations of E2, genistein, Equol (−), and coumestrol. The cells in C and D contain 2- to 3- and 7- to 8-fold more ERα than MCF-7 cells, respectively. E, Breast cancer cells expressing very high levels of ERα produce large quantities of PI-9 protein. Cells were maintained in medium containing 100 pm genistein and the indicated concentrations of DOX for 24 h (MCF-7) or 48 h (ERαHA), which maximizes PI-9 production, and analyzed for PI-9 protein level by Western blotting. The data in A, C, and D represent the mean ± sem for three separate experiments.
Figure 3
Figure 3
Induction of increasing levels of PI-9 progressively block NK cell-mediated cell death. A, Genistein protects MCF-7 cells against NK92 cell-mediated killing. B, Genistein does not protect MCF-7 cells against NKL cell-mediated killing. MCF-7 target (T) cells were maintained in medium containing ethanol vehicle or 10 nm genistein for 24 h, followed by incubation with NK92 effector (E) cells (A) or NKL effector cells (B) at different effector cell/target cell ratios. Cytotoxicity assays were performed as described in Materials and Methods and Ref. . C and D, Genistein protects ERαHA cells expressing high levels of ERα against NK92 cell and NKL cell-mediated killing. ERαHA cells were treated with ethanol vehicle or with 0.5 μg/ml DOX plus 100 pm genistein for 48 h, followed by the cytotoxicity assay. E and F, In ERαHA cells expressing very high levels of ERα, genistein nearly abolishes NK92 and NKL cell-induced cell death. ERαHA cells were treated with ethanol vehicle or with 1.0 μg/ml DOX plus 100 pm genistein for 48 h, followed by the cytotoxicity assay. Data in A–F represent the mean ± sem for three separate experiments. For each panel, we used the single-tailed t test to compare apoptosis in control cells and cells treated with three ratios of NK cells (P < 0.05 for control vs. genistein-treated cells in A, C, and D–F; B, P > 0.05).
Figure 4
Figure 4
Dietary genistein induces PI-9 in MCF-7 tumors in mice. A, Growth rates of MCF-7 tumors in ovariectomized athymic mice. At wk 0, the estrogen pellets were removed, and the mice were divided into three treatment groups: PC (positive control, E2) (13 mice; n = 51 tumors), GEN 500 (genistein 500 ppm, 14 mice; n = 56 tumors), and NC (negative control, regressing tumor) that were fed AIN-93G diet alone (13 mice; n = 52 tumors). Tumor size was then measured weekly for 23 wk. Data are expressed as means ± sem cross-sectional tumor area for all tumors in each group. B, Dietary genistein and soy induce PI-9 in MCF-7 solid tumors. Mice were exposed to high (2 mg) E2 in implanted cholesterol pellets and tumors harvested (n = 4) at about 11 wk (black bar) and frozen. Mice were fed diets containing genistein (500 ppm; n = 5) or soy flour (diet containing 20% soy protein, ∼400 ppm genistein; n = 3) (black bars). Tumors were harvested at about 23 wk when they reached the same size as the E2 tumors (average cross-sectional area = 38 mm2). Control MCF-7 cells were maintained in medium lacking hormones or in 10 nm E2 or 10 nm genistein for 24 h and harvested (n ≥ 3; white bars). RNA was extracted and analyzed for PI-9 mRNA by quantitative RT-PCR as we describe. Data represent the mean ± sem for at least three samples.

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