Background

Aromatase is a member of the cytochrome P450 enzyme family and a product of the CYP 19A1 gene [1]. This membrane-bound protein (aromatase) is the rate limiting enzyme in the conversion of androstenedione to estrone (E1) and of testosterone to estradiol (E2) (Figure 1). Aromatase consists of two components: the hemoprotein aromatase cytochrome P450 encoded by the CYP19A1 gene and expressed only in steroidogenic cells, and the flavoprotein NADPH-cytochrome P450 reductase, expressed ubiquitously in many cell types [2–4]. The enzyme (aromatase) is localized in the endoplasmic reticulum of a cell, and catalyzes three hydroxylation reactions that convert androstenedione to E1 and testosterone to E2 [5, 6]. The enzyme activity is increased by alcohol, age, obesity, insulin and gonadotropins [7]. The CYP19A1 gene is highly expressed in the human placenta and in the granulosa cells of the ovarian follicles. However, many nonglandular tissues including liver, muscle, brain, bone, cartilage, blood vessels, breast (both normal and carcinogenic) and adipose tissues have lower level of CYP 19A1 expression under the control of tissue-specific promoters [8]. Inhibition of aromatase enzyme activity has been shown to reduce estrogen production throughout the body and aromatase inhibitors (AIs) are being used clinically to retard the development and progression of hormone-responsive breast cancer [6, 7].

Figure 1
figure 1

Schematic diagram of the reaction catalyzed by aromatase enzyme.

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The aromatase gene and tissue-specific promoter expression

Human aromatase is a 58 kDa protein which was first purified from placental microsomes in 1980s [9]. Only recently has the crystal structure of human placental aromatase been described [5]. Aromatase is encoded by a single copy of the CYP19A1 gene which is ~123 kb long, located on the short arm of the chromosome 15 (15q21), and is transcribed from the telomere to the centromere [2, 10–12]. The coding region spans 30 kb and includes nine translated exons (II-X) with two alternative polyadenylation sites [2]. The ATG translation initiation site is located on the exon II. There are a number of alternative non-coding first exons (I.1, I.2, I.3, I.4, I.5, I.6, I.7, and PII) which are expressed in tissue-specific manner, lie upstream to the coding region and are spliced to a common acceptor sites in exon 2 [13–68]. In addition, many natural products that have been used traditionally for nutritional or medicinal purposes as botanical dietary supplements (BDS) may also afford as AIs with reduced side effects [61, 69, 70]. Because many natural products are associated with low toxicity, they are potentially excellent candidates for use as chemopreventive agents [71–73]. Epidemiological evidence suggests that women living in Asia, where diets have traditionally included soybean products, report fewer postmenopausal symptoms and experience fewer breast cancers than women in Western countries [74–77]. More specifically, Asian women have a 3-fold lower breast cancer risk than women in the United States, independent of body weight [78]. Furthermore, serum concentrations of E2 are 40% lower in Asian women compared with their Caucasian counterparts [79]. Thus, environmental and dietary factors may explain at least some of the discrepancy in breast cancer risk between Asian and western populations [74, 75]. Despite the known AIs, there is still a need of searching for new AIs from natural products for future drug development [68]

Among the natural products tested as AIs, phytoestrogens, such as flavones and isoflavones are able to bind ER and induce estrogen action [77]. The binding characteristics and the structural requirements necessary for the inhibition of human aromatase by flavones and isoflavones were obtained by using computer modeling and confirmed by site-directed mutagenesis [80–82]. It was found that these compounds bind to the active site of aromatase in an orientation in which their rings A and C mimic rings D and C of the androgen substrate, respectively [80]. Until now ~ 300 natural products, most of them are phytoestrogens, have been evaluated for their ability to inhibit aromatase using noncellular (mostly using human microsome as a source of aromatase enzyme), cell-based, and in vivo aromatase inhibition assays [61, 83–85]; however, only a few studies (biochanin A from red clover, genistein from soybean, quercetin, isoliquiritigenin from licorice, resveratrol from grape peel and extracts of grape seeds, Figure 3) have been reported for their effect on aromatase promoter I.4, I.3/II activity [86–91]. The exact mechanisms how these plant products adapted to inhibit aromatase gene expression or enzyme activity is not fully understood.

Figure 3
figure 3

The chemical structures of biochanin A, genistein, quercetin, epicatechin, isoliquiritigenin, and resveratrol.

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Biochanin A (5, 7-dihydroxy-4'-methoxyisoflavone) is an isoflavone extracted from red clover (Trifolium pretense) by Pope et al. [92]. The first evidence that red clover has estrogenic activity were reported by Bennets et al. [93] after observing breeding problems of sheep grazing on red clover pastures which have been attributed to the isoflavone and coumestrol content of red clover. Serious fertility disturbances indicating estrogenic stimulation of cattle fed with red clover silage were reported [94–96]. Although biochanin A was moderately active in inhibiting microsomal aromatase activity (IC50: 5-10 μM) but was strongly active when tested in JEG-3 cells (human placental choriocarcinoma cell line). However, it did not inhibit aromatase activity in granulosa-luteal cells, and human preadipocyte cells and was also inactive in trout ovarian aromatase assay [61]. Interestingly, in MCF-7 cells (ER-positive breast cancer cells) biochanin A exhibited a dual action. It inhibited aromatase activity at low concentrations, but was estrogenic at high concentrations [97]. Furthermore, in SK-BR3 cells (ER-negative breast cancer cells) biochanin A was reported to inhibit aromatase enzyme activity and reduce mRNA expression. By using a luciferase reporter gene assay it was demonstrated that this phytochemical (biochanin A) was able to suppress the activation of breast-specific promoter I.3/II [88]. However, it is not known whether this inhibition is mediated through a PGE-2 or cAMP dependent PKA mechanisms. When genistein (a major metabolite of biochanin A) was tested in the same model, it was also found to suppress promoter I.3/II activation and showed an inhibition of aromatase enzyme activity [88]. Therefore, the inhibitory effect of biochanin A on aromatase promoter activation was suggested by the authors to be due to its metabolic conversion to genistein rather than its direct effect [88].

Genistein is a major phytoestrogen isolated from soybean, a potential nutraceutical, geared for women suffering from perimenopausal symptoms [98–101]. Genistein is also found in a number of other plants such as fava beans, lupin, kudzu, and psoralea [102]. Genistein is believed to be a chemopreventive agent against various types of cancers, including prostate, cervix, brain, breast, esophagus and colon [103]. Genistein was shown to increase aromatase activity in human adrenocortical carcinoma (H295R) cells and in isolated rat ovarian follicles [104, 105]. Dietary genistein, which produced circulating concentrations consistent with human exposures, did not act as an aromatase inhibitor; rather, dietary intake of genistein negated the inhibitory effect of an aromatase inhibitor letrozole (a 3rd generation aromatase inhibitor), by stimulating the growth of aromatase-expressing estrogen-dependent breast tumors [106]. This study raises concerns about the consumption of genistein-containing products by postmenopausal women with advanced breast cancer who may be treated with letrozole. Genistein suppressed promoter I.3/II transactivity in SK-BR-3 cells (an ER-negative breast cancer cell line), however, in HepG2 cells, genistein was found to induce promoter-specific aromatase mRNA expression with significant increases in promoters I.3 and II [89]. In addition, the phosphorylated forms of PKCα, p38, MEK and ERK1/2 kinases were also induced in HepG2 cells by genistein [89]. There are also some reports of a weak inhibition of aromatase enzyme activity by genistein as well [80, 107] and a decrease in the transcription of Cyp19 mRNA in human granulosa luteal cells [108].

Quercetin is one of the most abundant flavonols found in plants. Quercetin was found to inhibit human aromatase activity in placental microsomes [109]. When tested in cellular systems utilizing adrenocortical carcinoma cells, preadipocyte cells, or in co-culture experiments, it exhibited either a mild or no effect [86, 110, 111]. In the primary culture of human granulosa-luteal cells quercetin was able to reduce aromatase mRNA expression in a dose-dependent manner after an exposure period of 48 h [108]. In another study, H295R human adrenocortical carcinoma cells were exposed to quercetin for 24 h and an increase in aromatase enzyme activity was observed at lower concentration, while a decrease in the enzyme activity was observed at higher concentrations [105]. Quercetin increased p II and I.3-specific aromatase transcripts about 2.6-and 2-fold in H295R cells after 24 h exposure probably by enhancing intracellular cAMP levels [105].

Isoliquiritigenin, a flavonoid from licorice (Glycyrrhiza glabra), was found to be an inhibitor of aromatase enzyme activity in vitro [90]. Moreover, this compound was able to block MCF-7aro cells(MCF-7 cells stably transfected with CYP19) growth and when added in diet inhibited significantly the xenograft growth in ovariectomized athymic mice transplanted with MCF-7aro cells [90]. Isoliquiritigenin also inhibited aromatase mRNA expression and suppressed the activity of CYP19 promoters I.3 and II [90] in MCF-7 cells. Furthermore, binding of C/EBP to PII promoter of CYP19 was suppressed by isoliquiritigenin [90]. This study indicated that isoliquirititigenin has the potential to be used as a tissue-specific aromatase inhibitor in breast cancer.

The aromatase inhibitory activity of grapes and grape seed extracts (GSE) has been studied by many investigators [61, 83, 91]. The active chemicals found in grapes and red wine are procyanidin dimers that are also present in high concentrations in grape seeds [87]. GSE is composed of about 74-78% of proanthocyanidins and <6% of free flavanol monomers such as catechin, epicatechin, and their gallic acid esters [87]. Through the suppression of the expression of CREB-1 and glucocorticoid receptor (GR), grape seed extracts (GSE) has been found to decrease the expression of aromatase in MCF-7 and SK-BR-3 cells by suppressing the activity of promoters I.3/II, and I.4 in a dose-dependent manner [87]. The GSE (IH636) is in phase I clinical trials for the prevention of breast cancer in postmenopausal women who have an increased risk of breast cancer development [61].

The grape peel contains resveratrol, a polyphenolic compound which has structural similarity with estrogen [91]. This nonflavonoid phytoestrogen inhibited aromatase activity in MCF-7aro cells. In SK-BR-3 cells resveratrol significantly reduced aromatase mRNA and protein expression in a dose-dependent manner [91]. Moreover, this compound was able to repress the transactivation of CYP19 promoters I.3 and II in SK-BR-3 cells [91], which indicate that resveratrol could be able to reduce localized estrogen production in breast cancer cells.

Future directions

The expected direct outcome of aromatase inhibition is the maintenance of low levels of estrogen in the breast and surrounding adipose tissue. Understanding the molecular mechanism by which aromatase promoters I.4 and I.3/II are regulated is clinically significant and useful for develo** new drugs. Although only a few plant products have been documented to mediate their effects through aromatase promoters, there are many more potent natural products (such as white button mushroom (Agaricus bisporus) which is in phase I trials [83]) which could be potential candidates for future study. Moreover, accumulating evidence suggests that beside transcription factors and co-regulators there are many other factors such as cyclooxygenases (COX) which are involved in tissue-specific aromatase promoter regulation [112, 113]. Selective COX inhibitors from natural products can be used to suppress CYP19A1 gene expression. Studies also indicate that CYP 19A1 regulations are also under epigenetic control, including DNA methylation, and histone modification, which can add a new layer of complexity in the regulation of the aromatase gene [114]. DNA methylation generally occurs in gene promoters where the CpG rich dinucleotides are located. However, DNA methylation of CpG-poor promoter regions has also been shown as a mechanism of mediating tissue-specific gene transcription through the inhibition of transcription factor binding [115, 116]. Aromatase promoter I.3/II has six CpG dinucleotides subjected to methylation of cytosines and can be considered as CpG-poor promoter. However, in human skin fibroblasts hypermethylation of almost all six CpG sites resulted in markedly reduced aromatase promoter I.3/II activity, whereas hypomethylation of only two of the six sites led to increased promoter activity associated with an increase in cAMP [14]. In contrast to these studies, in breast adipose fibroblasts (BAF) promoter I.4 and I.3/II derived mRNA were not dependent on the CpG methylation status within respective aromatase promoters [114]. Further, DNA methylation is catalyzed by DNA methyl transferases (DNMTs). Inhibition of DNA methylation by 5-aza-2'-deoxycytidine, which is also a specific DNMT inhibitor, increased CYP19 mRNA expression in BAFs and breast cell lines [114]. These studies indicate that disruption in epigenetic regulation may give rise to increase in aromatase levels in the breast [114]. There are many synthetic chemicals that are undergoing clinical trials to be used as epigenetic drugs (epidrugs) for breast cancer treatment [117]. The major problems of these drugs are the unwanted side effects. Many natural products have the potential to be used as better epidrugs than synthetic epidrugs. One of the best examples is (-) - epigallocatechin-3-gallate from green tea which is used as demethylating agents for breast cancer patients [118–120]. Therefore extensive investigations in natural products seem promising or necessary.

Conclusions

Aromatase is a well-established molecular target and the AIs are proving to be an effective new class of agent for the chemoprevention of breast cancer. Regulation of aromatase expression in human tissues is a complex phenomenon, involving alternative promoter sites that provide tissue specific control. The promoters I.3 and II are the major promoters directing aromatase expression in breast cancer. The drugs that can selectively inhibit aromatase expression may be useful to obviate side effects induced by the nonselective AIs. Although many synthetic chemicals are used to inhibit tissue-specific inactivation of aromatase promoters I.3 and II, in the literature only a few natural products (we have included six of them) have been reported with such activities. More studies on natural products are necessary to find an appropriate tissue-specific AI.

Author's information

Shabana I. Khan is the Senior Scientist at the National Center for Natural Products Research and Associate Professor of the Department of Pharmacognosy at the University of Mississippi, University, MS 38677, USA. Jian** Zhao is the Associate Research Scientist at the National Center for Natural Products Research at the University of Mississippi, University, MS 38677, USA. Ikhlas A. Khan is the Assistant Director of the National Center for Natural Products Research and Professor of Pharmacognosy, School of Pharmacy of the University of Mississippi, University, MS 38677, USA. Larry A. Walker is the Director of the National Center for Natural Products Research at the University of Mississippi, and Associate Director for Basic Research Oxford, University of Mississippi Cancer Institute and the Professor of Pharmacology, School of Pharmacy of the University of Mississippi, University, MS 38677, USA, Asok K. Dasmahapatra is the Research Scientist at the National Center for Natural Products Research and Assistant Professor of the Department of Pharmacology, School of Pharmacy of the University of Mississippi, University, MS 38677, USA.