Green Tea Antioxidant Epigallocatechin Gallate (EGCG) May Increase Levels of p53 – Natural Anti-Cancer Protein

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An antioxidant found in green tea may increase levels of p53, a natural anti-cancer protein, known as the “guardian of the genome” for its ability to repair DNA damage or destroy cancerous cells.

Published today in Nature Communications, a study of the direct interaction between p53 and the green tea compound, epigallocatechin gallate (EGCG), points to a new target for cancer drug discovery.

“Both p53 and EGCG molecules are extremely interesting. Mutations in p53 are found in over 50% of human cancer, while EGCG is the major anti-oxidant in green tea, a popular beverage worldwide,” said Chunyu Wang, corresponding author and a professor of biological sciences at Rensselaer Polytechnic Institute.

“Now we find that there is a previously unknown, direct interaction between the two, which points to a new path for developing anti-cancer drugs. Our work helps to explain how EGCG is able to boost p53’s anti-cancer activity, opening the door to developing drugs with EGCG-like compounds.”

Wang, a member of the Rensselaer Center for Biotechnology and Interdisciplinary Studies, is an expert in using nuclear magnetic resonance spectroscopy to study specific mechanisms in Alzheimer’s disease and cancer, including p53, which he described as “arguably the most important protein in human cancer.”

P53 has several well-known anti-cancer functions, including halting cell growth to allow for DNA repair, activating DNA repair, and initiating programmed cell death—called apoptosis—if DNA damage cannot be repaired. One end of the protein, known as the N-terminal domain, has a flexible shape, and therefore, can potentially serve several functions depending on its interaction with multiple molecules.

EGCG is a natural antioxidant, which means it helps to undo the near constant damage caused by using oxygen metabolism. Found in abundance in green tea, EGCG is also packaged as an herbal supplement.

Wang’s team found that the interaction between EGCG and p53 preserves the protein from degradation. Typically, after being produced within the body, p53 is quickly degraded when the N-terminal domain interacts with a protein called MDM2. This regular cycle of production and degradation holds p53 levels at a low constant.

“Both EGCG and MDM2 bind at the same place on p53, the N-terminal domain, so EGCG competes with MDM2,” said Wang. “When EGCG binds with p53, the protein is not being degraded through MDM2, so the level of p53 will increase with the direct interaction with EGCG, and that means there is more p53 for anti-cancer function. This is a very important interaction.”

“EGCG Binds Intrinsically Disordered N-Terminal Domain of p53 and Disrupts p53-MDM2 Interaction” was published with support from multiple grants from the National Institutes of Health. At Rensselaer, Wang was joined in the research by Lauren Gandy, Weihua Jin, Lufeng Yan, Xinyue Liu, and Yuanyuan Xiao.

First author Jing Zhao is a former member of Wang’s lab, now on the faculty at China Agricultural University in Beijing, China. Co-first author Alan Blaney is an M.D.-Ph.D. student at Upstate Medical University.

Researchers also contributed from SUNY Upstate Medical Center; the University of Massachusetts, Amherst; New York University; the State University of New York at Binghamton; NYU Shanghai; and Merck Research Laboratories.


Tea is one of the most widely consumed beverages worldwide, and is available in various forms. Green tea is richer in antioxidants compared to other forms of tea. Tea is composed of polyphenols, caffeine, minerals, and trace amounts of vitamins, amino acids, and carbohydrates.

The composition of the tea varies depending on the fermentation process employed to produce it. The phytochemicals present in green tea are known to stimulate the central nervous system and maintain overall health in humans. Skin aging is a complex process mediated by intrinsic factors such as senescence, along with extrinsic damage induced by external factors such as chronic exposure to ultraviolet (UV) irradiation—

A process known as photoaging—Which can lead to erythema, edema, sunburn, hyperplasia, premature aging, and the development of non-melanoma and melanoma skin cancers. UV can cause skin damage either directly, through absorption of energy by biomolecules, or indirectly, by increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS).

Green tea phytochemicals are a potent source of exogenous antioxidant candidates that could nullify excess endogenous ROS and RNS inside the body, and thereby diminish the impact of photoaging. Several in vivo and in vitro studies suggest that green tea supplementation increases the collagen and elastin fiber content, and suppresses collagen degrading enzyme MMP-3 production in the skin, conferring an anti-wrinkle effect.

The precise mechanism behind the anti-photoaging effect of green tea has not been explored yet. Studies using the worm model have suggested that green tea mediated lifespan extension depends on the DAF-16 pathway. Apart from this, green tea has been reported to have stress resistance and neuroprotective properties. Its ROS scavenging activity makes it a potent stress mediator, as it can also regulate the stress induced by metal ions. It is known that tea polyphenols can induce the expression of different antioxidant enzymes and hinder the DNA oxidative damage.

Growing evidence suggests that green tea can also be used as a potential agent to mediate neurodegenerative diseases, including Alzheimer’s disease. EGCG, an abundant catechin in tea, was found to suppress the neurotoxicity induced by Aβ as it activates glycogen synthase kinase-3β (GSK-3β), along with inhibiting c-Abl/FE65—the cytoplasmic nonreceptor tyrosine kinase which is involved in the development of the nervous system and in nuclear translocation.

Additionally, green tea polyphenols induce autophagy, thereby revitalizing the overall health of the organism consuming it. Green tea was able to activate autophagy in HL-60 xenographs by increasing the activity of PI3 kinase and BECLIN-1. This manuscript describes the reported anti-photoaging, stress resistance, and neuroprotective and autophagy properties of one of the most widely known functional foods—green tea.

reference link : doi: 10.3390/nu11020474


Breast cancer is among the most common gynecological cancers worldwide, which, despite treatment advances, breast cancer-associated mortality is still the second leading cause of cancer-related death among women in the United States [1]. In addition to hormonal-based therapy and chemo- and radio-therapy, therapeutic strategies are focusing on dietary-derived compounds which are showing promising potential.

Such dietary factors are the polyphenols, members of a large family of phytochemical compounds which are present in almost all plant foods, particularly in fruit, vegetables, coffee and tea. The most extensively studied polyphenols in cancer research are the tea catechins, which among others, include (-)-epigallocatechin 3-gallate (EGCG) and (+)-catechin [2]. For instance, epidemiological studies have indicated an association between tea consumption and decreased risk of breast cancer [3, 4].

The anticancer activity of tea catechins has been attributed to alterations of various intracellular pathways of the cancer cell, almost all being primarily triggered by the antioxidant potency of the compounds. Reduction of highly required -by the cancer cell-intracellular reactive oxygen species (ROS) suppresses carcinogenesis [5].

This reduction is the result of the direct scavenging action of catechins on free radicals [6], as well as to the indirect increases of endogenous antioxidant enzymes, such as catalase and superoxide dismutase (SOD) [7]. These events gradually lead to stimulation of DNA repair and breast cancer cell apoptosis [8], and to inhibition of DNA methylation [9], angiogenesis [10] and metastasis [11].

Although among all catechins EGCG has been most extensively studied in cancer research and is currently being tested in 37 clinical trials, it has limitations as it can undergo self-oxidation [12] and trigger toxicity to normal tissues [13], in doses higher that 800mg/day [14].

On the other hand, (+)-catechin (cyanidanol-3) is more stable than EGCG and has the advantage that it can form complexes with one or two lysines, a property that can amplify (+)-catechin antioxidant potency over 400 times [15], and allow it to directly inhibit disease-associated enzymes such as xanthine oxidase, which is a superoxide-producing enzyme [16].

Complexed to lysine, (+)-catechin water solubility increases with an overall positive charge that allows the complex to enter mitochondria and exert its antioxidant effects predominantly at this site [17]. This is of particular importance, as our group has previously demonstrated that mitochondrial superoxide is a key driver of metastasis [18].

In support of this, (+)-catechin:lysine complexes have been demonstrated to prevent melanoma cell metastasis to lungs of mice [19] and also exert anti-migratory and pro-apoptotic effects on pancreatic, colorectal and breast cancer cells, without affecting normal cells [20].

In the light of these results, we sought to investigate and determine the anticancer and antimetastatic effects of (+)-catechin:lysine to experimental breast cancer, using in vitro MDA-MB231 cells and in vivo xenograft model of breast cancer, following orthotopic injections of MDA-MB231 cells to the mammary glands of immunocompromised mice.

Discussion

There is plenty of evidence that catechins can provide an additional tool for the treatment of cancer. Whether they could serve as a therapeutic regime on their own or be used complementarily to current therapies such as chemotherapy remains to be clarified by clinical studies ongoing. However, it has been well established that catechins have diverse modes of anticancer action based primarily on their ability to reduce intracellular ROS levels.

Cancer cells maintain large intracellular ROS pools due to their high metabolic rates and oncogene activation and account to their increased capacity to proliferate. While there is still a debate whether ROS scavenging or induction instead is the proper way to combat cancer [26], ROS in cancer cells are considered oncogenic as they trigger oncogenic DNA-damage, but can also promote anoikis resistance, thereby enabling metastasis [27]. Based on their antioxidant activity, catechins have been shown to exert anti-estrogenic [28], antiangiogenic [29] [30], anti-proliferative [8] and pro-apoptotic [31] effects in breast cancer models.

Cancer cell migration and invasion constitute key steps for the process of metastasis. Catechins have been shown to partially block all these events in various breast cancer in vitro models. For instance, EGCG was shown to suppress the EMT process and inhibit angiogenesis, cell migration and invasion in vitro [10].

While there is plenty of evidence of the in vitro antimetastatic effects of catechins, there is limited literature on in vivo antimetastatic ability of breast cancer cells, as also highlighted by a recent review on the topic [32]. In MDA-MB231 xenograft model, EGCG was shown to inhibit primary tumor growth but authors did not provide any evidence for metastasis inhibition [33].

Rather, we have previously shown that (+)-catechin:lysine 1:2 inhibits melanoma cell metastasis in mice but the cells were treated with the drug prior infusion to the bloodstream of mice, thus deviating from the pharmacological view of treatment. For breast cancer, the most relevant studies revealing antimetastatic actions of polyphenols were focusing on combined polyphenol supplements, such as green tea [11] and grape [34] extracts, rather than individual catechins alone.

Thus, any additional data regarding isolated catechin effects on breast cancer metastasis are pivotal for the understanding of their benefits against this process of cancer progression.

Since mitochondrial superoxide is viewed as the factor for metastasis initiation [18], investigation of anticancer properties of (+)-catechin moieties complexed to lysine residues are of increasing interest, as these can selectively remove mitochondrial superoxide [17]. The evidence of the effects of these complexes come mainly from our lab and collaborators.

Specifically, (+)-catechine:lysine complexes were shown to be more effective superoxide scavengers than EGCG, based on studies using DCFDA probes in human cervical cancer cells [19]. Similar results we obtained in MDA-MB231 cells by flow cytometric analysis using MitoSOX probes which are specific mitochondrial superoxide detectors, thus ensuring that the complex is acting within mitochondria (data not shown).

Recent data from collaborator’s lab indicated that (+)-catechin:lysine 1:2 is inhibiting migration of MDA-MB231 breast cancer cells, by mechanisms involving inhibition of JAK/STAT pathway and Wnt signaling, as well as promoting apoptosis. In that study, the concentration of (+)-catechin:lysine 1:2 that proved effective was the 1mmol/L dose for 24 hours.

Here, we present a complete scan of a range of concentrations and we demonstrate that our effective dose of (+)-catechin:lysine 1:2 is 5-to 10-fold less of that study whereby excluding any possible toxic effects of the compound to cells, and that this concentration proved reliable in terms that this can be reached in the plasma of mice orally-dosed with the complex. Inhibition of Wnt signaling and JAK/STAT pathway has been reported in response to catechin treatments [31, 35] and may represent a common effect of catechins in breast cancer cells.

We also demonstrate that (+)-catechin:lysine 1:2 inhibits MDA-MB231 cell invasion together with migration, in a dose-dependent manner, indicating potential markers responsible for these events, such as induction of the ER stress response and p38 signaling and reduced viability, as evident by reduced phosphorylation of Akt.

Similar results in breast cancer cells have been obtained in response to EGCG treatments [36, 37]. We have previously demonstrated that ER stress signaling can induce JNK and p38-induced apoptosis [38] and this could be likely the case here, an assumption that awaits verification. Further, the fact that in prolonged (72h) treatment with (+)-catechin:lysine 1:2 the GRP78 upregulation event is lost (Fig. 2C,2E) suggests that ER stress may be an initial event under the treatment, and later the cells have entered the apoptotic pathway, without necessity to upregulate GRP78.

In addition, under the in vitro treatments of the MDA-MB231 cells, we found that markers associated to metastasis were deregulated, as pyk2 phosphorylation and SNAIL levels were reduced. At the same time, p38 phosphorylation was greatly increased. Of note, pyk 2 (also called focal adhesion kinase 2; FAK2) is a ROS-sensitive cytoplasmic tyrosine kinase that plays a role in cellular adhesion and is conceived as a metastatic marker in various forms of human cancer [39], including breast cancer in humans [40].

Similarly, SNAIL, is a transcription factor that can be induced under DNA damage responses in breast cancer and trigger metastasis [41], although metastasis induction in MDA-MB231 cells is primarily driven by SLUG [42]. In addition, although the stress-inducible p38 MAPK has highly debatable effects on tumor progression [43], it has been shown that it can inhibit breast cancer metastasis [44].

Without having measured the direct effects of (+)-catechin:lysine 1:2 directly on DNA-damage parameters, all our in vitro data, taken together, allows the assumption to be made that the complex triggers p38-mediated (induced either by ER stress or DNA-damage) proliferation inhibition and decrease in metastatic potential of MDA-MB231 cells.

In attempts to validate the effects of (+)-catechin:lysine 1:2 on primary tumor growth following acute drug administration, we observed a robust increase of p53 and p38 phosphorylation levels, associated with decrease of the viability marker p-Akt and larger necrotic areas at the core of the tumors. The results of chronic administration experiment with lower (+)-catechin:lysine 1:2 doses revealed that the necrotic pattern gives its place to apoptosis, with also apparent p53 increases. P53 is a tumor suppressor protein [8] which can mediate antimetastatic effects on breast cancer cells [45], also being able to inhibit EMT via inhibition of β-catenin pathway [46].

For instance, we previously demonstrated that (+)-catechin:lysine 1:2 inhibits Wnt signaling in MDA-MB231 cells [20], thus this could be attributed to the enhanced p53 levels reported here. The absence of an effect of the drug on lung metastasis, together with the unexpected findings of increased levels of the metastatic markers pyk2 and SNAIL, without however an effect on E-cadherin levels, prompts us to suspect that p53 plays a key role in these observations.

Interestingly, both pyk2 [47] and SNAIL [48] have been demonstrated to directly inhibit p53 to promote cancer progression and metastasis and we therefore presume that p53 elevations are a stress response signal to the tumors that causes pyk2 and SNAIL upregulation to overcome stress. SNAIL elevations, on the other hand could account for p21 inhibition as previously described [49] and could likely be the case for the relevant observation in the acute administration experiment.

Of note, inhibition of apoptosis is the best known oncogenic function of p21. Up to date research directions encompass a therapeutic scheme based on wild type p53 overexpression with p21 downregulation [50], and the results of our study demonstrate that (+)-catechin:lysine 1:2 follows that pattern. At this point, it is hard to predict whether is mutated or wild-type the p53 protein for which we are showing the elevations, and how this affects the overall response of the tumor.

The work described here provide the first attempt in characterizing the anticancer effects of (+)-catechin:lysine 1:2 in a xenograft model, revealing that although in vitro it suppresses metastatic processes and cell growth, in vivo metastasis inhibition is absent. It is important to note certain limitations of this study.

These include the selected dosing concentrations of the animals, the duration of the in vivo experiments and the time-points for lung metastases detection. Alterations of these parameters may have great impact on the results. Further, we did not test metastasis detection to other organs, such as liver and bone, sites whereas polyphenol treatment may preferably affect in MDA-MB231 xenograft models [34], rather than lungs. Lastly, in our study, we did not get into detail in assessing all metastatic markers, as the primary aim was to elucidate whether the complex exerts direct antitumor and antimetastatic activity in vivo. More accurate conclusion can be made upon a complete scan of metastasis markers, DNA-damage assays and analysis of MAPK signaling using inhibitors. So far, however, we can conclude that a p53-mediated pyk2/SNAIL/p21 axis explains the positiveness of outcomes with regards to primary tumor and also the lack of metastasis inhibition by (+)-catechin:lysine 1:2 and we point out research focus on catechins towards that direction.

reference link: https://www.biorxiv.org/content/10.1101/2021.02.01.429090v1.full


More information: Nature Communications (2021). DOI: 10.1038/s41467-021-21258-5

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