Individual and combined soy isoflavones exert differential effects on metastatic cancer progression

Abstract

To investigate the effects soy isoflavones in established cancers, the role of genistein, daidzein, and combined soy isoflavones was studied on progression of subcutaneous tumors in nude mice created from green fluorescent protein (GFP) tagged-MDA-MB-435 cells. Following tumor establishment, mice were gavaged with vehicle or genistein or daidzein at 10 mg/kg body weight (BW) or a combination of genistein (10 mg/kg BW), daidzein (9 mg/kg BW), and glycitein (1 mg/kg BW) three times per week. Tumor progression was quantified by whole body fluorescence image analysis followed by microscopic image analysis of excised organs for metastases. Results show that daidzein increased while genistein decreased mammary tumor growth by 38 and 33% respectively, compared to vehicle. Daidzein increased lung and heart metastases while genistein decreased bone and liver metastases. Combined soy isoflavones did not affect primary tumor growth but increased metastasis to all organs tested, which include lung, liver, heart, kidney, and bones. Phosphoinositide-3-kinase (PI3-K) pathway real time PCR array analysis and western blotting of excised tumors demonstrate that genistein significantly downregulated 10/84 genes, including the Rho GTPases RHOA, RAC1, and CDC42 and their effector PAK1. Daidzein significantly upregulated 9/84 genes that regulate proliferation and protein synthesis including EIF4G1, eIF4E, and survivin protein levels. Combined soy treatment significantly increased gene and protein levels of EIF4E and decreased TIRAP gene expression. Differential regulation of Rho GTPases, initiation factors, and survivin may account for the disparate responses of breast cancers to genistein and daidzein diets. This study indicates that consumption of soy foods may increase metastasis.

Fig. 1

Effect of soy isoflavones on the growth of MDA-MB-435 mammary fat pad tumors. One week following injection of MDA-MB-435 cells, mice (10/treatment) were fed vehicle (Veh), 10 mg/kg-BW genistein (Gen), or daidzein (Daid), or soy isoflavones (10 mg/kg-BW genistein, 9 mg/kg-BW daidzein, and 1 mg/kg-BW glycitein) 3× a week. Whole body fluorescence images were acquired 2× a week. a Average relative tumor area with days post injection, calculated as the area of fluorescence on each day of imaging as a function of the fluorescence intensity of the same tumor on d 01. b Representative digital images of GFP-MDA-MB-435 tumors following vehicle, genistein, or daidzein diets at d 77. c Average mammary tumor growth as quantified from digital images acquired on d 77, made relative to same tumor image on d 01. Values are mean ± SEM (n = 8). Asterisk denotes statistical significance at P < 0.05 compared to vehicle control

Fig. 2

Effect of genistein and daidzein on metastasis. a Images of representative lungs and fluorescence image analysis of lungs from mice following vehicle genistein, or daidzein treatment. The average area of fluorescent metastatic foci was calculated from excised lungs after vehicle, genistein, or daidzein treatment. b Fluorescence image analysis of hearts from mice following vehicle, genistein, or daidzein. c Fluorescence image analysis of bones from mice following vehicle genistein, or daidzein. d Fluorescence image analysis of livers from mice following vehicle genistein, or daidzein. Average area or integrated density of fluorescent metastatic foci/mouse ± SEM for n = 8/treatment is shown. Asterisk denotes statistical significance (P < 0.05) compared to vehicle controls. e The percentage of mice with metastases for each organ (bone, heart, kidney, liver, lung) after vehicle, genistein, or daidzein treatment, as detected from fluorescence images (n = 8)

Fig. 3

Effect of soy isoflavones on metastasis. a Images of representative lungs and fluorescence image analysis of lungs from mice following vehicle or soy isoflavones treatment. The average area of fluorescent metastatic foci was calculated from excised lungs after vehicle or soy isoflavone (genistein:daidzein:glycitein, 5:4:1 ratio) treatment (images of representative lungs are shown). b Fluorescence image analysis of hearts from mice following vehicle or soy isoflavone treatment. c Fluorescence image analysis of bones from mice following vehicle or soy isoflavone treatment. d Fluorescence image analysis of livers from mice following vehicle or soy isoflavone treatment. e Fluorescence image analysis of kidneys from mice following vehicle or soy isoflavone treatment. Integrated density of fluorescent metastatic foci/mouse ± SEM for n = 8/treatment is shown. Asterisk denotes statistical significance (P < 0.05) compared to vehicle controls. f The percentage of mice with metastases for each organ (bone, heart, kidney, liver, lung) after soy isoflavone treatment, as detected from fluorescence images (n = 8)

Fig. 4

Effect of dietary soy isoflavones on PI3-K pathway gene expression. Total RNA was extracted from mammary tumors excised from mice that received vehicle, genistein, daidzein, or soy isoflavone combination (genistein, daidzein, glycitein) diets. RT² PCR array designed to profile the expression of PI3-K pathway-specific genes was used, according to manufacturer’s instructions (SA Biosciences). Volcano plots show a genistein; b daidzein; c soy combination effects on gene expression analyzed at −1.3 ≥ 1.3 log₂-fold change (dashed line). Down-regulated genes are to the left of the vertical black line while up-regulated genes are to the right. Statistically significant regulated genes are above the horizontal black line at P < 0.05 (n = 3)

Fig. 5

Effect of genistein and daidzein on gene and protein expression of Rho GTPases and Pak1. Total lysates of mammary tumors were western blotted with anti Pak1, RhoA, Rac, Cdc42, or GFP antibodies. a Representative western blot for tumor extracts from vehicle, genistein or daidzein treatments. b Fold changes in Rho GTPase gene or protein expression for tumors from genistein- or daidzein-treated mice compared to vehicle. log₂-fold changes in gene expression were calculated from PCR arrays. Fold changes in protein expression were calculated from integrated density of positive bands from western blots. Equal total protein content as well as confirmation of human cancer cells in the mouse tumor extracts was maintained by expressing the integrated density of each band for Pak1, RhoA, Rac1, or Cdc42 as a function of the integrated density of the GFP band from the same tumor extract. Values show mean ± SEM (n = 6). An asterisk indicates statistical significance of P < 0.05

Fig. 6

Effect of soy isoflavones on protein expression. Total lysates of mammary tumors from mice treated with vehicle, genistein, daidzein, or soy (genistein:daidzein:glycitein, 5:4:1 ratio) were western blotted with anti-eIF4G, anti-eIF4E, or anti-survivin antibodies. a Representative western blots for tumor extracts from vehicle, genistein, or daidzein. b Representative western blots for tumor extracts from vehicle, or combined soy treatments. c Fold changes in eIF4G, eIF4E, and survivin protein expression for tumors from genistein, daidzein, or soy treated mice compared to vehicle as calculated from the integrated density of positive bands from western blots and normalized with actin expression. Values show mean ± SEM (n = 4). An asterisk indicates statistical significance of P < 0.05