This study was designed to determine the combinatorial effect of polyphenols at dietary concentrations on established mammary tumors. Most investigations on environmental and dietary chemopreventives focus on breast cancer initiation and not on established mammary tumors. Studies conducted with chemically induced as well as spontaneous carcinogenic transgenic mouse models have demonstrated the use of red wine solids, grape juice, or grape seed extract in the prevention of mammary tumor initiation [9–13
]. However, many studies on the potential anticancer properties of grape polyphenols have been conducted using concentrations too high to be achieved using dietary consumption. Moreover, a majority of these reports investigates the effect of individual polyphenols. Previous studies in rodents including breast cancer models have demonstrated the cancer-preventive efficacy of individual treatments of resveratrol, quercetin, or catechins at pharmacological concentrations [28–31, 35–37
]. Because the majority of dietary compounds contain a mixture of polyphenols, it is important to understand their combinatorial effect at dietary concentrations.
The MDA-MB-231 low metastatic human breast cancer cell line was used to represent an aggressive breast cancer cell line that will illustrate the utility of using red wine polyphenols as preventives/therapeutics for advanced breast cancer. This cell line has been used in vitro
to demonstrate the inhibitory effect of red wine polyphenols at low concentrations [6
]. Moreover, our previously published data using this cell line has demonstrated that resveratrol can have biphasic concentration-dependent effects on cell functions relevant to breast cancer metastasis such as migration, invasion, and actin cytoskeletal changes as well as Akt and focal adhesion kinase activities that regulate cancer cell survival and invasion [38–40
Data presented herein illustrate the utility of using a combination of grape polyphenols as breast cancer chemopreventives and therapeutics. The grape polyphenol concentrations used in the in vitro
studies fall within the range of 0.1 to 10 µM that may be accumulated in the circulation following consumption of products rich in grapes such as red wine [5,19–21,38–42
]. However, caution must be used with the interpretation of in vitro
data because the compounds that were added to the tissue culture cells are relatively stable in their aglycone forms compared to the dietary polyphenols that accumulate in the blood following oral administration.
Our in vitro
data demonstrate that, individually, resveratrol, quercetin, or catechin at a dietary concentration of 0.5 µM does not exert a significant inhibitory effect on ERα (-) ERβ (+) MDA-MB-231 breast cancer cell proliferation or on cell cycle progression. Interestingly, combined resveratrol, quercetin, and catechin each at 0.5 µM exerted a synergistic effect to result in cell cycle arrest and reduced cell proliferation. Decreased cell proliferation and G2
/M phase arrest in response to a similar combination of polyphenols have been reported for breast cancer cells but not for normal mammary epithelial cells, indicating the utility of grape polyphenols as breast cancer therapeutics [6,23,43
]. The subtle but statistically significant increase in cell numbers in response to low concentrations of quercetin has been previously reported for other cell types [44
]. As demonstrated previously [24,45–47
], resveratrol and quercetin were potent inhibitors of cell proliferation at high concentrations. The effect of catechin on cell proliferation and cell cycle progression was modest even at 20 µM. Therefore, catechin may not have contributed to the anticancer properties of grape polyphenols at the concentrations tested. However, because catechin is the major monomeric polyphenol in red wine [31,48
], catechin was included in the in vivo
study on the effect of grape polyphenols on breast cancer progression.
The in vivo
effect of combined dietary treatment of resveratrol, quercetin, and catechin was quantified by intravital image analysis of fluorescently tagged breast tumor growth in nude mice. We and others have demonstrated the utility of such noninvasive imaging modalities for in situ
tumor analysis from the time of tumor cell inoculation to the formation of distant metastases from fluorescently tagged cancer cells in rodent models [34,49,50
In mice with an average bodyweight of 20 g, 5 mg/kg bodyweight of each compound in a 100-µl gavage volume equates to 4.38 µM resveratrol, 3.3 µM quercetin, and 3.48 µM catechin. These concentrations are found in dietary components rich in grape polyphenols such as red wine [14
]. A growing debate on the cancer-preventive properties of natural compounds is that dietary consumption is insufficient to achieve cancer inhibitory concentrations at target tissues [17–21
]. However, resveratrol, quercetin, and catechins are all considered viable chemopreventives because they are absorbed and metabolized rapidly in vivo
and can be detected in plasma and urine samples in the intact form in humans and rodent models [5,17–21
]. Data on bioavailability of grape polyphenols following consumption in animal models vary depending on the route of administration, animal models, and method of detection. For example, administration of as little as 50 µg/kg body weight of resveratrol per day for 3 months to rats achieved serum levels as high as 8.0 µM [39
]. However, others have shown that administration of 20 mg/kg resveratrol to rats, mice, or rabbits demonstrated a very short half-life (10 minutes) and reached circulating levels of less than 1 µM very rapidly [20
]. Similarly, following 20 mg/kg body weight administration of quercetin to mice, at concentrations that inhibited metastatic melanoma growth, the quercetin levels in the plasma decreased to ~ 1 µM after 120 minutes [21
]. Therefore, the combined polyphenols that demonstrated reduced mammary tumor growth in this study at 5 mg/kg body weight must be effective at the cellular level at very low concentrations.
As has been reported previously for this cell line [32,51
], MDA-MB-231 cells did not form significant metastases during the 4-month study period. Isolated lungs from mice that received only vehicle control demonstrated more micrometastases (2–10 cells) compared to mice treated with polyphenols. However, these differences were not statistically significant due to the small sample size of the mice that developed metastases. Therefore, we were unable to perform a comprehensive analysis of the effect of resveratrol, quercetin, and catechin on breast cancer metastasis. Resveratrol at similar concentrations has been shown to reduce lung metastasis in other systems [52,53
]. A recent study demonstrated that grape polyphenols, including resveratrol, epicatechin, and epigallocatechin, can inhibit cancer cell invasion [54
]. The contribution of combined grape polyphenols on prevention of metastasis or progression of established breast cancers will be a focus of future investigations using more efficient metastatic breast cancer cell lines. Overall, the data presented indicate a preventive/therapeutic role for dietary grape polyphenols in primary breast cancer. However, more animal and clinical studies are needed before dietary recommendations of grape products for breast cancer patients, for those at risk, or for survivors of breast cancer can be made.