Introduction
After water, tea is the most commonly consumed beverage worldwide (1). Even in Western countries, such as Germany, tea consumption is increasing. In 2015, an average consumption of 28 liters per capita was registered, corresponding to approximately 20,000 tons of total domestic tea consumption. Since 2005, the share of green tea (2005: 20%) against black tea (2005: 80%) has increased to 30% (2). Similar to black tea, green tea is made from the leaves of the tea plant Camellia sinensis (1).
Recently, matcha tea (MT) has been gaining in popularity. The substrate concentrations are higher than that in other green teas because the leaves are ground with a ceramic mill to a fine powder that is later dissolved in hot water and completely consumed. Fujioka et al published a study in 2016 in which they observed that the protective effect of MT against oxygen radicals is significantly higher than the effect of normal tea leaves due to increased catechin levels (3).
Research on polyphenols in green tea (GT) has revealed considerable health benefits (4). The polyphenol family includes the flavonoids which, in turn, incorporate flavanols and catechins. Approximately 30% by mass of the dry substance of fresh tea leaves is made up of phenolic compounds. In GT, 90% of these polyphenolic compounds are catechins. The most abundant catechin in GT is epigallocatechin gallate (EGCG) (5). Epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC) are also catechins found in GT. However, these only make up 3–6% (EGC, ECG) and 1–3% (EC) of the dry mass. Other components of the tea leaves by percentage of dry mass are proteins (15%), amino acids (4%), caffeine (4%), raw fibers (26%), lipids (7%), other carbohydrates (7%), pigments, such as chlorophyll and carotenoids (2%) and minerals (5%) (6). A cup of GT (100 ml) contains approximately 20–100 mg of EGCG. The US Department of Agriculture states that 200 mg of EGCG are contained in 100 g of boiled GT (7,8).
It was demonstrated in various studies that EGCG and quercetin exhibit anticancer activity. Besides steroid receptor (9) and PPARγ receptor (10) interaction, other assumed mechanisms of action include interaction with the PI3K/Akt/mTOR signaling pathway (11), VEGF (12), the the 67-kDa laminin receptor (67-LR) (13), p53, Bax protein (14) and Bcl-2 (15).
In the present study, we analyzed the effects of GT and MT, as well as their components EGCG and quercetin, on MCF-7 and MDA-MB-231 breast carcinoma cells.
Discussion
This study confirmed that EGCG and quercetin are contained in GT and that the single substances, as well as GT itself, have an anticarcinogenic effect on both ER/PR receptor-positive and -negative breast cancer cells, suggesting estrogen receptor-independent pathways.
EGCG is the most abundant anticarcinogenic catechin in GT (4). In the German breast cancer guideline of 2012, EGCG has been previously described as a complementary drug used by patients that should not be applied outside of clinical trials (16). In the ATP luminescence test, a correlative (|r|=0.97) concentration-dependent inhibition of cell viability was observed in both cell lines. At the highest concentration tested, the effect of EGCG corresponded with that of tamoxifen. Wang et al performed the same ATP assay with MCF-7 cells and EGCG, testing EGCG concentrations between 4.5 and 54 µg/ml. These researchers results showed an inhibition of cell viability even at low concentrations after 24 h. In the MTT assay, Wang et al reported viability values after 48 h that are comparable to those of our study (18). In MDA-MB-231 cells, Thangapazham et al (17) described lower viability values in the MTT assay with EGCG at different concentrations after 48 h. The ED50 was reached at EGCG 50 µg/ml; however, in this study, the ED50 was not achieved by any of the tested concentrations. In the studies of both Wang et al and Thangapazham et al, the measurements were also carried out after 24, 48 and 72 h, and even 96 h, as reported by Thangapazham et al. All of the results showed that the cell viability was decreased further over time. An explanation for the difference in viability values, despite identical test methods and identical EGCG concentrations, could be the pH value. As early as 1997, Zhu et al reported that catechins are considerably more stable at acidic pH values than at alkaline pH values (19). Our pH measurements always showed alkaline pH values, suggesting instability of the EGCG molecules. In the investigations of Wang et al and Thangapazham et al, no information was available on the pH values of the cell cultures. Therefore, it is possible that they worked at lower pH values, leading to higher stability of the catechins and increased inhibition of cellular viability.
Not every EGCG concentration in the BrdU test in MCF-7 cells reached the ED50. However, in MDA-MB-231 cells, the mixture of EGCG and quercetin exhibited a stronger effect than the two single substances alone. An explanation could be the influence of the two substances on the PI3K/Akt/mTOR signaling pathway and thus on endothelial growth factor (VEGF). Van Aller et al (20) demonstrated that EGCG has an inhibitory effect on this signaling pathway in MDA-MB-231 cells. Bruning confirmed the inhibitory effect of quercetin on mTOR (21). Gu et al observed that the oral administration of EGCG in mice resulted in a VEGF-mediated reduction of tumor volume compared with the control group (22). Wang et al demonstrated in 2014, using a xenograft prostate tumor mouse model, that the combination of EGCG and quercetin resulted in greater bioavailability and lower methylation of the catechins. Tumor size could be reduced compared with that in the control groups. They also found that the quercetin and EGCG combination led to a significant reduction in phosphorylated AKT (pAKT) (23). Scandlyn et al combined EGCG with tamoxifen, provoking a reduction of mTOR by 78% in receptor-negative tumors in vivo (24).
Our results showed no significant differences between the effects of EGCG on estrogen receptor-negative MDA-MB-231 and ER-positive MCF-7 cells; therefore, it can be assumed that the effect is not, or at least not exclusively, generated via the estrogen receptor. Tachibana et al (26) and Umeda et al (25) found that EGCG exerts its anticarcinogenic effect on its binding behavior with the surface the 67-kDa laminin receptor (67-LR). The 67-LR is important in the metastasis of tumor cells (27). Mittal et al showed that EGCG inhibits telomerase in MCF-7 cells, which is increased in more than 90% of all cases in tumors (28). Roy et al (29) described increased expression of p53 and the proapoptotic protein Bax, as well as reduced expression of the anti-apoptotic protein Bcl-2 in estrogen receptor-negative breast cancer cells under EGCG influence. Moreover, although MDA-MB-231 cells are triple negative, these cells express ERβ. Therefore, tamoxifen treatment is possible (30).
In the tests performed using GT and MT extracts, a reduction in cellular viability, vitality, and proliferation was observed (Fig. 2A and B). It is assumed that EGCG is responsible for most of the anticancer effects of GT (4,31). As demonstrated in our results, Wang et al observed significantly higher viability values in the MTT assay than the ATP measurements (18). An explanation could be offered by the studies of Bruggisser et al (32) and Peng et al (48), who observed that antioxidants and flavonoids result in a reduced reaction of MTT to the dye formazan, even without the presence of living cells. The amount of formazan formed is measured photometrically, and false-high results are measured.
The flavonoid quercetin was detected by HPLC in GT. In almost all tests performed, inhibition of cell viability was demonstrated, except for the MCF-7 cells in the MTT assay, in which all results were above 100% cell viability but without a statistical significance. In a study by Scambia et al, quercetin inhibited the growth of MCF-7, as well as that of MDA-MB-231 cells, in a dose-dependent manner by binding to the estrogen-binding domain type II (EBS II) (33). Lee et al described that the induction of apoptosis of MCF-7 cells is caused by the formation of free oxygen radicals that activate the pro-apoptotic AMPKα1/ASK1/p38 signaling pathways (34). Duo et al recognized the apoptotic effect of quercetin in the upregulation of Bax protein and reduction of Bcl-2 protein (35). Similar to EGCG, quercetin also has an inhibitory effect on the mTOR signal cascade (36).
As already mentioned, EGCG is a very sensitive molecule. It is assumed that EGCG changes its structure under cell culture conditions by auto-oxidation and dimerization. Sang et al observed increased instability of the EGCG molecule in normal tap water, attributable to its iron constituents (37). Other factors that influence the molecular stability are pH, temperature and the concentration of EGCG (20,37). Sang et al demonstrated that 20 µmol/l of EGCG in RPMI-1640 medium (named after the Roswell Park Memorial Institute), similar to the DMEM used in our work, lost integrity after 1 h at 37°C. At high concentrations, EGCG tends to change the stereoconformation and become GCG (gallocatechingallate) and the molecules half-life is prolonged. At lower concentrations, dimer formation occurs, resulting in molecular instability. Inconsistent amounts of time passed between the preparation and measurement may contribute to the heterogeneous results in the literature on the amount of EGCG in a cup of tea, presuming that some of the EGCG may have already decayed after the preparation of the extracts at the time of the determination of the quantity (37).
Importantly, this study was performed in vitro; therefore, the bioavailability of EGCG in humans was not considered. Yang et al found in a patient study (n=18) that the consumption of 3 g of decaffeinated GT (EGCG content: 73 mg/g) resulted in a plasma concentration of no more than 326 ng/ml. However, an increase from 1.5 to 3 g resulted in a triple EGCG plasma concentration; a further increase at a dose of 4.5 g could not be observed. Thus, it appears that a saturation of EGCG uptake is achieved around 3 g (38). Chow et al tested the bioavailability of EGCG by administering EGCG capsules. They measured the plasma levels of 439 ng/ml after administering 800 mg of EGCG or 388 ng/ml after the administering 800 mg of uncontaminated EGCG (polyphenol E) (39). These results show that the bioavailability of EGCG in plasma is very low.
One way to increase EGCG bioavailability is described by Landis-Piwowar et al, who demonstrated in vitro as well as in animal experiments in vivo that a prodrug of EGCG, in which the hydroxyl groups were protected by peracetic acid groups before methylation, leads to greater bioavailability and results in higher concentrations in breast cancer cells (40). Moreover, in 2007, they found that the methylation of EGCG by catechol O-methyltransferase (COMT) leads to decreased anticarcinogenic activity of the catechins (41). This observation was also consistent with the work of Wu et al who demonstrated that predominantly, carriers of the COMT-L allele, a low-activity form of COMT, benefited from the anti-carcinogenic effects of the catechins; however, in carriers of the highly active COMT-H allele, there was no difference in breast cancer risk between tea consumers and non-consumers (42). Landis-Piwowars et al demonstrated that, in MDA-MB-231 cells expressing the COMT-H form, the effect of catechins can be increased using COMT inhibitors (43).
In conclusion, based on our results and the findings reported in the literature, the anti-carcinogenic activity by GT and its components EGCG and quercetin can be assumed.
A problem in the therapeutic setting of EGCG is the low oral bioavailability. In animal studies (e.g., in athymic mice), the possibility of intravenous EGCG administration to avoid the first-pass effect could be tested (44). Another approach would be to administer an EGCG prodrug, as described by Landis-Piwowar et al, leading to better bioavailability (40).
However, more studies are crucial to determine the potential toxicity of EGCG at higher doses, as hepatotoxic effects at high concentrations are described. The drug interactions of EGCG and associated risks, e.g., effects of the pharmacokinetics of concomitant medication by influencing enzymes such as the cytochrome P450 system, need further evaluation (45). It is also worth including Her-2 positive cell lines in further studies. But for this study, we focused on ERα/ERβ-positive cells.
EGCG and tamoxifen could represent a promising combination in breast cancer therapy as synergistic anti-proliferative and cytotoxic effects in both MCF-7 and MDA-MB-231 cells were observed (46,47). Scandlyn et al observed an anti-proliferative effect of EGCG in combination with low tamoxifen doses (75 µg/kg) in animals with triple-negative breast cancer (24). To better understand this effect, active compounds of the two substances and pathways associated should be investigated more intensively. Therefore, studies regarding the influence on apoptosis and neovascularization should be considered. To understand the mode of action of EGCG, it would be interesting to further investigate the role of the 67-kDa laminin receptor (25,27).