Combined resveratrol, quercetin, and catechin treatment reduces breast tumor growth in a nude mouse model - PubMed (original) (raw)

Combined resveratrol, quercetin, and catechin treatment reduces breast tumor growth in a nude mouse model

Alexander Schlachterman et al. Transl Oncol. 2008 Mar.

Abstract

Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 microM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 microM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression.

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Figures

Figure 1

Figure 1

Structures of red wine polyphenols. Structures of the stilbene resveratrol, flavan-3-ol catechin, and flavonol quercetin are compared with estrogen. These polyphenols can act as antioxidants and may have estrogenic/antiestrogenic activity due to similarity in distance between terminal hydroxyl groups of estrogen that can interact with estrogen receptors.

Figure 2

Figure 2

Schematic design of the animal experiments. Six-week-old female nude mice were acclimated for 2 weeks and GFP-MDA-MB-231 xenografts were inoculated at the mammary fat pad at 8 weeks of age (day 0). On day 1, mice were administered vehicle, 0.5, 5, or 25 mg/kg each of resveratrol, quercetin, and catechin thrice a week for 117 days. Fluorescence image analysis of tumors commenced on day 0 and continued twice a week until the end of the study at 118 days (32 weeks).

Figure 3

Figure 3

Effect of grape polyphenols on MDA-MB-231 cell proliferation. MDA-MB-231 cells at a density of 5 x 104 in 5% charcoal-stripped FBS and phenol red-free media were plated on six-well plates. After 4 hours and every 48 hours for 96 hours, cells were treated with vehicle (Veh), 0.5, 5, or 20 µM resveratrol (Res), quercetin (Quer), or catechin (Cat), or a combination of 0.5, 5, or 20 µM each Res, Quer, and Cat (RQC). Cells were fixed and nuclei were stained with PI. (A) Representative micrographs of cells stained with PI. (B) Cell proliferation as analyzed by quantification of intact (nonapoptotic) nuclei. Percent viable cells ± SEM for 10 microscopic fields per triplicate treatments (N = 30) were calculated.

Figure 4

Figure 4

Effect of grape polyphenols on MDA-MB-231 cell cycle progression. MDA-MB-231 cells at a density of 5 x 105 in 5% charcoal-stripped FBS and phenol red-free media were plated on 10-cm plates. After 4 hours and every 48 hours for 96 hours, cells were treated with vehicle (Veh), or with 0.5 µM each resveratrol (Res), quercetin (Quer), or catechin (Cat), or a combination of 0.5 µM each Res, Quer, and Cat (RQC). Cells were fixed and nuclei were stained with PI. Percentage of cells at each cell cycle stage was quantified by flow cytometry of PI-stained cells. (A) Representative histograms (FL-2A) for cells treated with vehicle or polyphenols. (B) Average percentage of cells at each cell cycle stage for vehicle or polyphenol treatment. Results shown are for N = 3 ± S.D. Asterisks indicate statistical significance (P < .05) when compared to Veh controls.

Figure 5

Figure 5

Effect of grape polyphenols on mouse body weight. Mice were weighed weekly. Initial and final body weights (BW) are shown. Data are represented as weight ± SEM for 10 mice per treatment group.

Figure 6

Figure 6

Effect of grape polyphenols on mammary tumor growth. Mice were administered with vehicle (10% ethanol in oil) or with 0.5, 5 or 25 mg/kg resveratrol, quercetin, and catechin (RQC) thrice a week. GFP-MDA-MB-231 xenografts (∼ 2 mm2) were inoculated in the right mammary fat pad of female athymic nude mice. Tumor growth was analyzed from digital fluorescent images and made relative to each image acquired on day 0 immediately following inoculation. (A) Average relative tumor growth as analyzed by the increase in pixel intensity for 10 mice per experimental group. Regions of green fluorescence from unsaturated digital images were outlined to define tumor area. Tumor area (in pixels) and average intensity were acquired using ImageJ software (NIH). (B) Representative digital images of primary GFP mammary tumors from mice treated with dietary polyphenol treatments on days 0 and 118. (C) Quantification of mammary tumor growth from digital images acquired twice a week for 118 days. Relative tumor area from mice treated with dietary treatments on day 118. Asterisks denote a statistically significant difference (P < .05) from control.

Figure 6

Figure 6

Effect of grape polyphenols on mammary tumor growth. Mice were administered with vehicle (10% ethanol in oil) or with 0.5, 5 or 25 mg/kg resveratrol, quercetin, and catechin (RQC) thrice a week. GFP-MDA-MB-231 xenografts (∼ 2 mm2) were inoculated in the right mammary fat pad of female athymic nude mice. Tumor growth was analyzed from digital fluorescent images and made relative to each image acquired on day 0 immediately following inoculation. (A) Average relative tumor growth as analyzed by the increase in pixel intensity for 10 mice per experimental group. Regions of green fluorescence from unsaturated digital images were outlined to define tumor area. Tumor area (in pixels) and average intensity were acquired using ImageJ software (NIH). (B) Representative digital images of primary GFP mammary tumors from mice treated with dietary polyphenol treatments on days 0 and 118. (C) Quantification of mammary tumor growth from digital images acquired twice a week for 118 days. Relative tumor area from mice treated with dietary treatments on day 118. Asterisks denote a statistically significant difference (P < .05) from control.

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