Research ArticlesGrape and wine polyphenols down-regulate the expression of signal transduction genes and inhibit the growth of estrogen receptor–negative MDA-MB231 tumors in nu/nu mouse xenografts
Introduction
Breast cancer is a common form of malignancy affecting women around the world. Several genetic and environmental factors have been identified as causative factors for the development of breast cancer. Genetic disposition with the presence of genes, such as the BRCA1 and BRCA2, mutations in the tumor suppressor gene p53, hormonal status, environmental pollutants with hormonal activity (xenoestrogens), and a sedentary lifestyle are some of the factors known to influence the development of breast cancer. In general, breast cancer is categorized into the estrogen receptor (ER)–positive type and the ER-negative type, based on the prevalence of ERs within the cell. Estrogen receptor–positive cancers are treated by conventional procedures including surgery, radiation chemotherapy, and estrogen analogues. However, because of their estrogen independent nature, ER-negative breast cancer does not respond to estrogen analogues, and a more rigorous conventional form of cancer therapy is needed. Therefore, dietary strategies that may prevent the development of breast cancer, and especially the ER–negative breast cancer, is of great interest.
Epidemiological studies suggest a strong association between consumption of fruits and vegetables and cancer prevention [1]. However, such studies suggesting a positive correlation between polyphenols and cancer prevention are largely unavailable or inconclusive. Several preclinical animal studies have demonstrated the chemopreventive activity of flavonoids such as green tea catechins, quercetin, and anthocyanins. For example, (−) epigallocatechin-3-gallate given as an intraperitoneal injection at a concentration of 1 mg/d as an aqueous solution in 100 μL water resulted in rapid regression of MCF-7 tumors in nude mice [2]. Also, green tea extract has been shown to inhibit the growth of MDA-MB231 xenografts in severe combined immunodeficiency mice by greater than 10-fold at day 35, at a concentration of 2.5 g/L administered through water [3]. Ferguson et al [4] showed that a polyphenolic extract and a proanthocyanin fraction from cranberry are able to inhibit U87 (human glioblastoma) tumor cell growth in vivo, suggesting that they have a potential role in cancer cell growth. Immunocompromised male mice injected intraperitoneally with polyphenols (250 mg/kg) or proanthocyanidins (100 mg/kg) every second day up to day 21 significantly inhibited tumor growth up to 60%. Red wine polyphenols (50 mg/kg body weight) administered with the diet to F344 rats for 16 weeks inhibited colon carcinogenesis induced by azoxymethane or dimethylhydrazine [5]. In another study, Chen et al [6] demonstrated the breast cancer-preventive action of grape juice with a nude mouse model using MCF-7aro, an aromatase-transfected MCF-7 cell line. In mice fed by gavage with 0.5 mL of grape juice/d for 5 weeks, the tumor size was reduced by 70% compared with the tumor size in the animals that were not fed with grape juice. Proliferation of breast cancer is mostly estrogen-dependent, and polyphenols may influence estrogen metabolism in the body because of their ability to inhibit the enzymes responsible for detoxification of hormones [7]. Furthermore, it has been suggested that polyphenols may act as estrogen agonists or antagonists in different contexts [8], [9], [10]. Thus, several factors may play a role in determining the effect of polyphenols on breast tumor growth.
Long-term exposure to higher levels of estrogen has been associated with breast cancer cell proliferation [11]. Estrogen has been shown to cause an increased expression of oncogenes such as c-myc, ras, and bcl-2 in animals and cultured cells, resulting in tumorigenesis and abrupt cellular proliferation [12], [13], [14]. The effects of estrogen are associated largely with the way in which it is metabolized. Estrogen metabolism depends on 3 factors as follows: a woman's genetic makeup, lifestyle and diet, and environment. In premenopausal women, the ovaries produce the estrogen estradiol (E2), which is converted into estrone (E1). Before conjugation and excretion into the bile or urine, estrone and estradiol are hydroxylated at either the carbon-16 or the carbon-2 positions by cytochrome P-450–dependent enzymes. Prospective and case-control studies suggest that women with higher urinary 2-hydroxy estrone/16-hydroxy estrone ratios are less likely to be diagnosed with breast cancer [15], [16], [17]. This is because the 16α-hydroxyestrone (16-OHE) metabolite has a higher affinity for the ER and promotes cell proliferation [18], whereas the 2-hydroxyestrone (2-OHE) metabolite has a low affinity for the ER and reduces tumor growth and angiogenesis [19]. Dietary modifications can shift estrogen metabolism predominantly through the C2 hydroxylation metabolic pathway. Increasing the amount of cruciferous vegetables in the diet has been shown to increase the C2:C16 ratio. Consumption of 10 g/d of Brassica vegetable was associated with a statistically significant increase in 2:16 ratio in urine samples [20]. As well, isoflavones have also been shown to increase the C2:C16 ratio in urine [21], [22]. These results suggest that bioactive components may be able to exert their effects at the biochemical and molecular level.
Several researchers have studied the molecular mechanisms of polyphenol action in vitro and in vivo using a variety of proteomic and/or genomic approaches. Array-based technology is one of the latest methods that enable one to study gene expression changes [23] on a larger scale. This is a more comprehensive approach to studying pathways and mechanisms that are involved in cancer prevention. Guo et al [24] have shown that epigallocatechin gallate treatment induces changes in expression of a large number of genes involved in proliferation control, cell cycle control, and apoptosis in different cancer cell lines by greater than 2-fold. Banerjee et al [25] showed that resveratrol suppression of dimethylbenzanthracene-induced mammary carcinogenesis was correlated with the down-regulation of NFκB, COX-2 (cyclooxygenase-2), and matrix metalloprotease 9 (MMP-9) expression. The effect of quercetin on the expression of 4000 human genes in Caco-2 cells was studied by Erk et al [26] to elucidate possible mechanisms involved in its mode of action. Their results indicated that quercetin (5 μmol/L) down-regulated the expression of cell cycle genes, for example, cyclin-dependent kinase 6 (CDC6), cyclin-dependent kinase 4 (CDK4), and cyclin D1, and up-regulated the expression of several tumor suppressor genes. This correlated with the down-regulation of cell proliferation and induction of cell cycle arrest in Caco-2 cells. In addition, quercetin also modulated genes involved in signal transduction pathways such as the β catenin/T-cell factor signaling and mitogen-activated protein (MAP) kinase signaling [26]. At the in vivo level, suppression of tumor growth by quercetin and resveratrol has been shown to be associated with down-regulation of MAP kinase and NFκB signaling pathways [25], [26].
In previous studies, we have shown that a hydrophobic polyphenol fraction isolated from Merlot wine selectively showed cytotoxicity toward ER-positive breast cancer cells (MCF-7 [Michigan Cancer Foundation-7]) [27]. Further studies have shown that treatment of MCF-7 cells with the polyphenol fraction caused a sustained increase in cytosolic calcium levels. Such an increase in cytosolic calcium was not noticeable in the normal MCF-10A cells. A prolonged increase in cytosolic calcium also disrupted the mitochondrial membrane potential in MCF-7 cells. These cells showed the disruption of membrane structure, eventually resulting in necrosis [28]. Based on these results, it was hypothesized that the growth inhibitory effects of grape polyphenols may also involve modulation of gene expression and the proliferation of human breast cancer cells under in vivo conditions may be inhibited by dietary intervention with polyphenols. In this study, we have used the athymic mouse model system to evaluate the changes in gene expression that occur in tumors developing from transplanted ER-negative MDA-MB 231 cells in response to polyphenols. These results show that grape polyphenols down-regulate several genes that have been reported to be activated during the development of breast cancer. The inhibition of tumor growth by grape polyphenols suggests that a diet rich in such components may help prevent the development of breast cancer in humans.
Section snippets
Chemicals and reagents
Merlot grapes were obtained from Vineland Research Station, St Catharines, Ontario, Canada; Merlot wine (2002 season) from Niagara Peninsula (Ancient Coast, Niagara) (http://www.ancientcoast.com), was purchased from the Liquor Control Board of Ontario (LCBO), Ontario, Canada. Merlot wine was chosen because the cell culture studies were performed with this wine, which was prepared in the laboratory from Merlot grapes grown at Vineland Research Station. The AIN 93G purified diet was purchased
Anthocyanin profile of grape and wine by LC-MS
To analyze and quantify the potential differences in the anthocyanin composition of grapes, wine, and 60% to 80% wine extracts, these extracts were subjected to HPLC-electrospray ionization-MS analysis. Table 1 shows the anthocyanin profile of the grape and wine from these analyses. Results indicated that pure glycosides were present below detectable levels in the 60% to 80% wine fraction. Malvidin-3-O-glucoside appeared to be the major component of grape and wine extracts, whereas this was not
Discussion
Although the inhibition of carcinogenesis by dietary polyphenols has been studied extensively, the molecular mechanisms of action and their applicability to human cancer prevention are less well understood. Furthermore, the relevance of many of the in vitro studies is uncertain because of a much higher concentration of polyphenols used in comparison to the tissue levels that may be attainable via dietary intake, and therefore, the in vivo efficacy should be demonstrated. In addition, several
Acknowledgment
This study was supported by financial assistance from the Breast Cancer Society of Canada (Sarnia, Ontario, Canada) and the Food System Biotechnology Center, University of Guelph (Ontario, Canada). We thank Marcus Litman, Ontario Veterinary College (Ontario, Canada) for helping with the injections of the cells and Annette Morrison, Central Animal Facility (Guelph, Ontario, Canada) for helping with gavaging and maintenance of the animals.
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