Inhibition of mammary tumor growth and metastases to bone and liver by dietary grape polyphenols

Abstract

The cancer preventive properties of grape products such as red wine have been attributed to polyphenols enriched in red wine. However, much of the studies on cancer preventive mechanisms of grape polyphenols have been conducted with individual compounds at concentrations too high to be achieved via dietary consumption. We recently reported that combined grape polyphenols at physiologically relevant concentrations are more effective than individual compounds at inhibition of ERalpha(-), ERbeta(+) MDA-MB-231 breast cancer cell proliferation, cell cycle progression, and primary mammary tumor growth (Schlachterman et al., Transl Oncol 1:19-27, 2008). Herein, we show that combined grape polyphenols induce apoptosis and are more effective than individual resveratrol, quercetin, or catechin at inhibition of cell proliferation, cell cycle progression, and cell migration in the highly metastatic ER (-) MDA-MB-435 cell line. The combined effect of dietary grape polyphenols (5 mg/kg each resveratrol, quercetin, and catechin) was tested on progression of mammary tumors in nude mice created from green fluorescent protein-tagged MDA-MB-435 bone metastatic variant. Fluorescence image analysis of primary tumor growth demonstrated a statistically significant decrease in tumor area by dietary grape polyphenols. Molecular analysis of excised tumors demonstrated that reduced mammary tumor growth may be due to upregulation of FOXO1 (forkhead box O1) and NFKBIA (IkappaBalpha), thus activating apoptosis and potentially inhibiting NfkappaB (nuclear factor kappaB) activity. Image analysis of distant organs for metastases demonstrated that grape polyphenols reduced metastasis especially to liver and bone. Overall, these results indicate that combined dietary grape polyphenols are effective at inhibition of mammary tumor growth and site-specific metastasis.

Fig. 1

Effect of grape polyphenols on MDA-MB-435 cell proliferation and cell cycle progression. Cells in 5% serum and phenol red-free media were treated with vehicle, 0.5, 5, or 20 μM resveratrol, quercetin, or catechin, or a combination of 0.5, 5, or 20 μM each (RQC) every 48 h for 96 h. Data was quantified from PI-stained intact (non-apoptotic) nuclei. a Cell proliferation. Percentage of viable cells ± SEM for 20 microscopic fields/triplicate treatments is presented. b Cell cycle progression. Cell cycle stage following 5 μM treatment with individual resveratrol, quercetin, or catechin or combined RQC. An asterisk indicates statistical significance of P < 0.05 and three asterisks indicate P < 0.001 when compared to vehicle

Fig. 2

Effect of grape polyphenols on apoptosis of MDA-MB-435 cells. Apoptosis of MDA-MB-435 cells was detected by caspase 3 activity assays (a) or fluorescence microscopy for Annexin V staining (b) following 48 h incubation with vehicle or RQC. a Average caspase 3 activity in μmol of pNA min⁻¹ ml⁻¹ relative to vehicle (n = 4 ± SEM) as quantified from absorbance at 405 nm of the pNA released by caspase 3 activity. b Percentage of cells undergoing apoptosis was calculated by Image J analysis of brightfield (total number of cells) and red fluorescence (apoptotic cells stained with Annexin V-Cy3) from ten random microscopic fields/coverslip. Averages ± SEM are shown for two separate experiments with duplicates for each experiment (n = 4). An asterisk indicates statistical significance (P < 0.05) when compared to vehicle

Fig. 3

Effect of grape polyphenols on breast cancer cell migration. Quiescent MDA-MB-231 (a) or MDA-MB-435 (b) cells were placed on the top well of Transwell chambers in serum-free, phenol red-free media and the number of cells that migrated through the membrane of the top well in response to various treatments was quantified relative to control. Data are quantified from analysis of 25 microscopic fields/treatment (n = 3 ± SEM). The bottom well contained the following for 8 h: vehicle, 0.5 or 5 μM resveratrol, quercetin, catechin or a combination of 0.5 or 5 μM each (RQC). An asterisk indicates statistical significance (P < 0.05) when compared to vehicle

Fig. 4

Effect of grape polyphenols on the growth of MDA-MB-435 mammary fat pad tumors. MDA-MB-435 cells (10⁶) in Matrigel were inoculated at the mammary fat pad of nude mice. One week following injection, mice were fed vehicle or a combination of 5 mg/kg BW Res, 5 mg/kg BW Quer, and 5 mg/kg BW Cat (RQC) three times a week by oral gavage. Whole body fluorescence images were acquired two times a week. a Average relative tumor area as a function of days following injection. Relative tumor area was calculated as the area of fluorescence, measured by fluorescence intensity on each day of imaging as a function of the fluorescence intensity of the same tumor on day 1. b GFP-MDA-MB-435 tumors following vehicle or RQC diets at day 77. Representative digital images and mammary tumor area as quantified from digital images acquired on day 77, made relative to same tumor image on day 1. Asterisk denotes statistical significance at P < 0.05

Fig. 5

Effect of grape polyphenols on lung metastasis. Following necropsy, lungs were excised from mice with GFP-MDA-MB-435 mammary tumors that received either vehicle or RQC diets and analyzed for metastases by fluorescence microscopy followed by quantitative image analysis. a Green fluorescence image of a representative lung demonstrating analysis of traced fluorescence area. b Lung metastatic efficiency expressed as average area of fluorescence from lungs of vehicle or RQC treated mice ±SEM (n = 8). c Average number of fluorescent metastatic foci/lung for vehicle or RQC treated mice. Asterisk denotes statistical significance at P < 0.05

Fig. 6

Effect of grape polyphenols on bone and liver metastasis. Following necropsy, femurs and livers were excised from mice with GFP-MDA-MB-435 mammary tumors that received either vehicle or RQC diets and analyzed for metastases by fluorescence microscopy followed by quantitative image analysis. a Green fluorescence image of a representative femur from vehicle treated mouse. b Average number of fluorescent metastatic foci/femur for vehicle or RQC treated mice. c Green fluorescence image of a representative liver from vehicle treated mouse. d Average number of fluorescent metastatic foci/liver for vehicle or RQC treated mice. Asterisk denotes statistical significance at P < 0.05